Question: The locus rs647161 is associated with colorectal carcinoma.

Title: Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease Content: The gene encoding apolipoprotein E (APOE) on chromosome 19 is the only confirmed susceptibility locus for late-onset Alzheimer's disease. To identify other risk loci, we conducted a large genome-wide association study of 2,032 individuals from France with Alzheimer's disease (cases) and 5,328 controls. Markers outside APOE with suggestive evidence of association (P < 10−5) were examined in collections from Belgium, Finland, Italy and Spain totaling 3,978 Alzheimer's disease cases and 3,297 controls. Two loci gave replicated evidence of association: one within CLU (also called APOJ), encoding clusterin or apolipoprotein J, on chromosome 8 (rs11136000, OR = 0.86, 95% CI 0.81–0.90, P = 7.5 × 10−9 for combined data) and the other within CR1, encoding the complement component (3b/4b) receptor 1, on chromosome 1 (rs6656401, OR = 1.21, 95% CI 1.14–1.29, P = 3.7 × 10−9 for combined data). Previous biological studies support roles of CLU and CR1 in the clearance of β amyloid (Aβ) peptide, the principal constituent of amyloid plaques, which are one of the major brain lesions of individuals with Alzheimer's disease.

Title: Three common functional polymorphisms in microRNA encoding genes in the susceptibility to hepatocellular carcinoma: a systematic review and meta-analysis. Content: Emerging evidences have shown that common genetic polymorphisms in microRNAs may be associated with the development of hepatocellular carcinoma (HCC); but individually published studies and previous meta-analyses revealed inconclusive results. The aims of this review and meta-analysis are to assess whether common single-nucleotide polymorphisms (SNPs) in the genes encoding the microRNAs are associated with susceptibility to HCC development and clinicopathologic characteristics of hepatitis B virus (HBV) related HCC. A computerized search was performed in PubMed, Embase, Web of Science and China BioMedicine (CBM) databases to identify relevant articles published before January 1st 2013. Ten case-control studies were assessed with a total of 3437 cases and 3437 healthy controls. Three common functional SNPs in miRNA-encoding genes were found, including miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913) and miR-499 T>C (rs3746444). This meta-analysis revealed that the miR-146a C variant was associated with a decrease in HCC risk, especially among Asian and male populations; while the miR-196a-2 T variant was associated with susceptibility to HCC among Caucasian populations. However, we failed to find any significant correlations between the miR-499 C polymorphism and HCC risks. When further stratification on HBV status was conducted, a similar trend of association between the three SNPs and the HBV-related HCC risks was observed, but these results were not statistically significant due to small sample sizes. The current meta-analysis demonstrates that SNPs contained in the genes encoding miR-146a and miR-196a-2 may play a major role in genetic susceptibility to HCC.

Title: Genome-wide association analyses in East Asians identify new susceptibility loci for colorectal cancer Content: To identify new genetic factors for colorectal cancer (CRC), we conducted a genome-wide association study in east Asians. By analyzing genome-wide data in 2,098 cases and 5,749 controls, we selected 64 promising SNPs for replication in an independent set of samples, including up to 5,358 cases and 5,922 controls. We identified four SNPs with association P values of 8.58 × 10(-7) to 3.77 × 10(-10) in the combined analysis of all east Asian samples. Three of the four were replicated in a study conducted in 26,060 individuals of European descent, with combined P values of 1.22 × 10(-10) for rs647161 (5q31.1), 6.64 × 10(-9) for rs2423279 (20p12.3) and 3.06 × 10(-8) for rs10774214 (12p13.32 near the CCND2 gene), derived from meta-analysis of data from both east Asian and European-ancestry populations. This study identified three new CRC susceptibility loci and provides additional insight into the genetics and biology of CRC.

Title: Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer Content: Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n=14), when compared with matched normal prostate (n=7). An expanded patient cohort (tumor n=149, matched normal n=30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio=2.005, 95% confidence interval (1.109, 3.625), P=0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.

Title: Occurrence of chromosome 9 and p53 alterations in multifocal dysplasia and carcinoma in situ of human urinary bladder Content: To define the genetic changes of flat urothelial lesions, carcinoma in situ (CIS) and moderate dysplasias (DII) were investigated for alterations in the two chromosomal regions most frequently involved in bladder cancer. Overall, 33 CIS and 16 DII from 21 patients were used to microdissect urothelium. Dual color fluorescence in situ hybridization (FISH) using gene locus probes of 9q22 (FACC), 9p21 (CDK), 17p13 (p53), and related centromeric probes was applied on interphase nuclei. In parallel, preamplified DNA of these samples was used for loss of heterozygosity (LOH) analyses with eight microsatellite markers on chromosomes 9p, 9q and 17p, and for sequencing of exons 5-9 of p53. Data indicated nearly identical deletion frequencies for chromosomes 9 and 17 for CIS (chromosome 9, 86%; p53, 84%). DII showed a lower deletion rate in comparison with CIS (chromosome 9, 75%; p53, 53%). A very high correlation between the results of FISH and LOH analyses was found. p53 mutations were detected in 12 of 15 patients (CIS, 72%; DII, 67%). In three of 16 patients with multifocal tumors, oligoclonal lesions were identified by LOH analyses, a finding further supported by sequencing of p53, by which two different p53 deletions were detected in two cases. In conclusion, data from microdissected flat urothelial lesions indicate that chromosome 9 deletions cannot be regarded as indicators of papillary growth, because they are found frequently in both types of flat lesions of the urothelium: those associated with papillary tumors and those that are not. The similar distribution and lower amount of genetic changes in DII render DII a possible precursor lesion of CIS.

Title: hClock gene expression in human colorectal carcinoma. Content: In this study, we aimed to investigate changes in the expression of human Clock (hClock), a gene at the core of the circadian gene family, in colorectal carcinomas (CRCs) and to discuss the possible effects. Previous studies have revealed that the disruption of circadian rhythms is one of the endogenous factors that contribute to the initiation and development of CRCs. However, the underlying molecular changes to the circadian genes associated with CRCs have not been explored. Immunofluorescence and quantitative polymerase chain reaction (qPCR) analysis of the hCLOCK protein and gene expression were performed in 30 cases of CRC. The hCLOCK protein was expressed in all specimens obtained from 30 CRC patients. Higher levels of hCLOCK expression were observed in human CRC tissues compared with the paired non-cancerous tissues. hCLOCK expression was significantly higher in poorly differentiated, or late-stage, Dukes' grade tumors and in 64.3% of tumor cases with lymph node metastasis. The hClock gene was expressed in all specimens. A significantly higher expression of hClock was found in human CRC cases compared with paired non-cancerous tissues. There was a strong positive linear correlation between hClock gene expression and protein expression in human CRCs. A strong positive linear correlation was also found between hClock gene expression and ARNT, HIF-1α and VEGF expression in human CRCs. There was no significant correlation between hClock and Bak, Bax, Bid, tumor necrosis factor receptor I (TNFR I) and TNFR II. The circadian gene hClock was stably expressed in human colorectal mucosa and was important in regulating the expression of downstream clock-controlled genes. hCLOCK may interact with HIF-1α/ARNT and activate VEGF to stimulate tumor angiogenesis and metastasis.

Title: High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma. Content: The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) can be inactivated by multiple genetic mechanisms. We analyzed 29 invasive primary head and neck squamous cell carcinomas (HNSCC) for p16 inactivation with immunohistochemistry utilizing a new monoclonal antibody (mAb), DCS-50. p16 staining of the primary lesions was correlated with genetic analysis including: (a) detailed microsatellite analysis of markers at the p16 locus to detect homozygous deletion; (b) sequence analysis of p16; and (c) Southern blot analysis to determine the methylation status of the 5' CpG island of p16. Twenty-four of 29 (83%) head and neck squamous cell carcinoma tumors displayed an absence of p16 nuclear staining using immunohistochemistry. Of these 24 tumors, we found that 16 (67%) harbored homozygous deletions, 5 (21%) were methylated, 1 displayed a rearrangement at the p16 locus, and 1 displayed a frameshift mutation in exon 1. These data suggest that: (a) inactivation of the p16 tumor suppressor gene is a frequent event in squamous cell carcinomas of the head and neck; (b) p16 is inactivated by several distinct and exclusive events including homozygous deletion, point mutation, and promoter methylation; and (c) immunohistochemical analysis for expression of the p16 gene product is an accurate and relatively simple method for evaluating p16 gene inactivation.

Title: Association of hypomethylation of LINE-1 repetitive element in blood leukocyte DNA with an increased risk of hepatocellular carcinoma Content: Global DNA hypomethylation has been associated with increased risk for cancers of the colorectum, bladder, breast, head and neck, and testicular germ cells. The aim of this study was to examine whether global hypomethylation in blood leukocyte DNA is associated with the risk of hepatocellular carcinoma (HCC). A total of 315 HCC cases and 356 age-, sex- and HBsAg status-matched controls were included. Global methylation in blood leukocyte DNA was estimated by analyzing long interspersed element-1 (LINE-1) repeats using bisulfite-polymerase chain reaction (PCR) and pyrosequencing. We observed that the median methylation level in HCC cases (percentage of 5-methylcytosine (5mC)=77.7%) was significantly lower than that in controls (79.5% 5mC) (P=0.004, Wilcoxon rank-sum test). The odds ratios (ORs) of HCC for individuals in the third, second, and first (lowest) quartiles of LINE-1 methylation were 1.1 (95% confidence interval (CI) 0.7–1.8), 1.4 (95% CI 0.8–2.2), and 2.6 (95% CI 1.7–4.1) (P for trend <0.001), respectively, compared to individuals in the fourth (highest) quartile. A 1.9-fold (95% CI 1.4–2.6) increased risk of HCC was observed among individuals with LINE-1 methylation below the median compared to individuals with higher (>median) LINE-1 methylation. Our results demonstrate for the first time that individuals with global hypomethylation measured in LINE-1 repeats in blood leukocyte DNA have an increased risk for HCC. Our data provide the evidence that global hypomethylation detected in the easily obtainable DNA source of blood leukocytes may help identify individuals at risk of HCC.

Title: CpG island methylation in premalignant stages of gastric carcinoma. Content: There are limited reports on methylation analysis of the premalignant lesions of gastric carcinoma thus far. This is despite the fact that gastric carcinoma is one of the tumors with a high frequency of CpG island hypermethylation. To determine the frequency and timing of hypermethylation during multistep gastric carcinogenesis, non-neoplastic gastric mucosa (n = 118), adenomas (n = 61), and carcinomas (n = 64) were analyzed for their p16, human Mut L homologue 1 (hMLH1), death-associated protein (DAP)-kinase, thromobospondin-1 (THBS1), and tissue inhibitor of metalloproteinase 3 (TIMP-3) methylation status using methylation-specific PCR. Three different classes of methylation behaviors were found in the five tested genes. DAP-kinase was methylated at a similar frequency in all four stages, whereas hMLH1 and p16 were methylated in cancer samples (20.3% and 42.2%, respectively) more frequently than in intestinal metaplasia (6.3% and 2.1%, respectively) or adenomas (9.8% and 11.5%, respectively). However, hMLH1 and p16 were not methylated in chronic gastritis. THBS-1 and TIMP-3 were methylated in all stages but showed a marked increase in hypermethylation frequency from chronic gastritis (10.1% and 14.5%, respectively) to intestinal metaplasia (34.7% and 36.7%, respectively; P < 0.05) and from adenomas (28.3% and 26.7%, respectively) to carcinomas (48.4% and 57.4%, respectively: P < 0.05). The hMLH1, THBS1, and TIMP-3 hypermethylation frequencies were similar in both intestinal metaplasia and adenomas, but the p16 hypermethylation frequency tended to be higher in adenomas (11.5%) than in intestinal metaplasia (2.1%; P = 0.073). The average number of methylated genes was 0.6, 1.1, 1.1, and 2.0 per five genes per sample in chronic gastritis, intestinal metaplasia, adenomas, and carcinomas, respectively. This shows a marked increase in methylated genes from non-metaplastic mucosa to intestinal metaplasia (P = 0.001) as well as from premalignant lesions to carcinomas (P = 0.002). These results suggest that CpG island hypermethylation occur early in multistep gastric carcinogenesis and tend to accumulate along the multistep carcinogenesis.

Title: ARID1A mutations in endometriosis-associated ovarian carcinomas. Content: BACKGROUND Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described. METHODS We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI–SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas. RESULTS ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions. CONCLUSIONS These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital–University of British Columbia Hospital Foundation.).

Title: Lack of Absent in Melanoma 2 (AIM2) expression in tumor cells is closely associated with poor survival in colorectal cancer patients. Content: Functional studies on colorectal cancer cells indicated a protective role of the interferon-inducible dsDNA sensor Absent in Melanoma 2 (AIM2) in cancer progression. Given that a high mutation rate and lack of AIM2 expression was previously detected in a subset of colorectal cancers, we here investigated the association of AIM2 expression in tumor cells and patient prognosis (5-year follow-up). A tissue microarray analysis of 476 matched tissue pairs (colorectal tumor and adjacent normal colon epithelium) was performed by two independent observers. Samples from 62 patients were excluded because of missing follow-up information or due to neo-adjuvant therapy before tissue sampling. Out of the remaining 414 tissue pairs, 279 (67.4%) displayed reduced AIM2 expression in cancer cells when compared to epithelial cells of their normal counterpart. Thirty-eight patients (9.18%) had completely lost AIM2 expression in tumor cells. After adjustment for sex, age, cancer stage, tumor site, tumor grade and chemotherapy, complete lack of AIM2 expression was associated with an up to 3-fold increase in overall mortality (HR=2.40; 95% CI=1.44-3.99) and disease specific mortality (HR=3.14; 95% CI=1.75-5.65) in comparison to AIM2-positive tumor samples. Our results demonstrate that lack of AIM2 expression is closely associated with poor outcome in colorectal cancer. The data thus strongly substantiate a protective role of AIM2 against progression of colorectal tumors. Further studies are required to assess whether lack of AIM2 expression may be used as a biomarker for the identification of colorectal cancer patients with poor prognosis.

Title: NEAT1: A novel cancer‐related long non‐coding RNA Content: Aberrant overexpression of the long non-coding RNA NEAT1 (nuclear paraspeckle assembly transcript 1) has been documented in different types of solid tumours, such as lung cancer, oesophageal cancer, colorectal cancer and hepatocellular carcinoma, in which its high levels are associated with poor prognosis. In contrast, NEAT1 is downregulated in acute promyelocytic leukaemia where it promotes leucocyte differentiation. In this review, we provide an overview of current evidence concerning the oncogenic role and potential clinical utilities of NEAT1. Further investigations are warranted to elucidate the upstream and downstream mechanisms of NEAT1 overexpression.

Title: The Association between Single-Nucleotide Polymorphisms of ORAI1 Gene and Breast Cancer in a Taiwanese Population Content: BACKGROUND Breast cancer is the most common malignancy in women. There is increasing evidence suggesting that ORAI1, components of store-operated calcium channel, play a pivotal role in breast cancer progression and metastasis. METHODS A total of 384 female patients with breast cancer were included in this study. We selected five representative tagging ORAI1 SNPs from HapMap database with minimum allele frequency (MAF) >10%. Genotyping was performed using TaqMan allelic discrimination assay. Chi-square (χ²) test was used to analyze statistical differences among control and patient groups in genotype and allelic frequencies. RESULTS Two of the ORAI1 SNPs (rs12320939 and rs12313273) were associated with estrogen receptors positive in breast cancer patients under the recessive model. When the Bonferroni correction was performed, the significance still existed. In addition, rs12320939 also associated with the lymph nodal involvement. CONCLUSION We showed that genetic polymorphisms of ORAI1 associated strongly with lymph nodal involvement and estrogen receptors (ERs) positive breast cancer patients in a Taiwanese population.

Title: PD-L1 expression in human cancers and its association with clinical outcomes Content: PD-L1 is an immunoinhibitory molecule that suppresses the activation of T cells, leading to the progression of tumors. Overexpression of PD-L1 in cancers such as gastric cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, and bladder cancer is associated with poor clinical outcomes. In contrast, PD-L1 expression correlates with better clinical outcomes in breast cancer and merkel cell carcinoma. The prognostic value of PD-L1 expression in lung cancer, colorectal cancer, and melanoma is controversial. Blocking antibodies that target PD-1 and PD-L1 have achieved remarkable response rates in cancer patients who have PD-L1-overexpressing tumors. However, using PD-L1 as an exclusive predictive biomarker for cancer immunotherapy is questionable due to the low accuracy of PD-L1 immunohistochemistry staining. Factors that affect the accuracy of PD-L1 immunohistochemistry staining are as follows. First, antibodies used in different studies have different sensitivity. Second, in different studies, the cut-off value of PD-L1 staining positivity is different. Third, PD-L1 expression in tumors is not uniform, and sampling time and location may affect the results of PD-L1 staining. Therefore, better understanding of tumor microenvironment and use of other biomarkers such as gene marker and combined index are necessary to better identify patients who will benefit from PD-1/PD-L1 checkpoint blockade therapy.

Title: LncRNA NEAT1/let-7a-5p axis regulates the cisplatin resistance in nasopharyngeal carcinoma by targeting Rsf-1 and modulating the Ras-MAPK pathway. Content: The long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) was reported to be upregulated and be involved in oncogenic growth and drug resistance in nasopharyngeal carcinoma (NPC). However, the exact roles of NEAT1 and its underlying mechanisms in the drug resistance of NPC remain largely unclear. In this study, the expressions of NEAT1, let-72-5p and Rsf-1 mRNA were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of NEAT1 and let-72-5p on cell proliferation and cisplatin resistance of NPC cells were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-20-deoxyuridine (EdU) assay. Western blot analysis was performed to detect the protein levels of Rsf-1, Ras, p-Raf1, Raf1, p-MEK1, MEK1, p-ERK1/2 and ERK1/2. Xenograft tumor assay was done to elucidate the role of NEAT1 involved in NPC tumor growth in vivo. We found that NEAT1 was upregulated and let-7a-5p was downregulated in NPC tissues, as well as NPC cell lines. Inhibition of NEAT1 markedly repressed the cisplatin resistance of NPC cells. NEAT1 was demonstrated to interact with let-7a-5p. Besides, a negative correlation between NEAT1 and let-7a-5p expression was observed in NPC tissues. Rsf-1 was confirmed as a target of let-7a-5p. NEAT1 remarkably reversed the inhibitory effect of let-7q-5p on the cisplatin resistance of NPC cells in vitro. Additionally, NEAT1 knockdown inhibited the Ras-MAPK pathway in NPC cells. NEAT1 knockdown suppressed tumor growth in the presence of cisplatin in vivo. Overall, these findings suggest that NEAT1/let-7a-5p axis regulates the cisplatin resistance in NPC by targeting Rsf-1 and modulating the Ras-MAPK signaling pathway.

Title: Retinoblastoma regulatory pathway in lung cancer. Content: Lung cancer is the leading cause of cancer related deaths accounting for more deaths than breast, colon and prostate cancers combined. The Rb-p16 regulatory pathway plays an essential role in tumor suppression in the lung epithelium. This is evidenced by the nearly universal alterations in Rb-p16 pathway components in lung cancer, and the increased incidence of pulmonary carcinomas in persons with germline Rb mutations. Interestingly, p16 loss and Rb mutations preferentially occur in phenotypically distinct lung cancer subtypes. Analysis of human tumors has identified progressive preneoplastic lesions that accumulate molecular alterations in an orderly sequence. Epigenetic p16 gene modifications represent an early event in lung cancer progression. This review summarizes the human studies that demonstrate a critical role for the Rb-p16 tumor suppressor pathway in lung carcinogenesis, and discusses how these findings in combination with genetically engineered mouse models have significantly contributed to our understanding of lung cancer pathogenesis.

Title: Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation. Content: Although 75% of endometrial cancers are treated at an early stage, 15% to 20% of these recur. We performed an integrated analysis of genome-wide expression and copy-number data for primary endometrial carcinomas with extensive clinical and histopathological data to detect features predictive of recurrent disease. Unsupervised analysis of the expression data distinguished 2 major clusters with strikingly different phenotypes, including significant differences in disease-free survival. To identify possible mechanisms for these differences, we performed a global genomic survey of amplifications, deletions, and loss of heterozygosity, which identified 11 significantly amplified and 13 significantly deleted regions. Amplifications of 3q26.32 harboring the oncogene PIK3CA were associated with poor prognosis and segregated with the aggressive transcriptional cluster. Moreover, samples with PIK3CA amplification carried signatures associated with in vitro activation of PI3 kinase (PI3K), a signature that was shared by aggressive tumors without PIK3CA amplification. Tumors with loss of PTEN expression or PIK3CA overexpression that did not have PIK3CA amplification also shared the PI3K activation signature, high protein expression of the PI3K pathway member STMN1, and an aggressive phenotype in test and validation datasets. However, mutations of PTEN or PIK3CA were not associated with the same expression profile or aggressive phenotype. STMN1 expression had independent prognostic value. The results affirm the utility of systematic characterization of the cancer genome in clinically annotated specimens and suggest the particular importance of the PI3K pathway in patients who have aggressive endometrial cancer.

Title: Chromosome 7 gain and DNA hypermethylation at the HOXA10 locus are associated with expression of a stem cell related HOX-signature in glioblastoma Content: BACKGROUND HOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma. RESULTS We observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively. CONCLUSIONS Based on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

Title: Elevated expression of miR-24-3p is a potentially adverse prognostic factor in colorectal adenocarcinoma. Content: OBJECTIVES Several miRNAs are aberrantly expressed in cancer. miR-24-3p is involved in cancer-related cellular processes, including cell cycle control, cell growth, proliferation, and apoptosis. In this study, we examined the potential diagnostic and prognostic significance of miR-24-3p expression in colorectal adenocarcinoma. DESIGN AND METHODS Total RNA was isolated from 182 colorectal adenocarcinoma specimens and 86 paired non-cancerous colorectal mucosae. After polyadenylation of 2μg total RNA and reverse transcription into first-strand cDNA using an oligo-dT-adapter primer, miR-24-3p expression was quantified using an in-house-developed reverse-transcription real-time quantitative PCR method, based on the SYBR Green chemistry. SNORD43 (RNU43) was used as a reference gene. RESULTS miR-24-3p levels do not significantly differ between colorectal adenocarcinoma and non-cancerous colorectal mucosae. Thus, miR-24-3p expression cannot be used for diagnostic purposes. However, high miR-24-3p expression predicts poor disease-free survival (DFS) and overall survival (OS) of colorectal adenocarcinoma patients. Multivariate Cox regression analysis confirmed that miR-24-3p overexpression is a significant predictor of relapse in colorectal adenocarcinoma and that its prognostic significance is independent of other established prognostic factors and treatment of patients. Of note, miR-24-3p overexpression retains its rather unfavorable prognostic value in the subgroup of patients with advanced yet locally restricted colorectal adenocarcinoma (T3) and in those without distant metastasis (M0). Moreover, miR-24-3p overexpression is a potentially unfavorable prognosticator for patients who were not treated with radiotherapy. CONCLUSIONS Strong expression of miR-24-3p predicts poor DFS and OS of colorectal adenocarcinoma patients, independently of clinicopathological parameters that are currently used for prognosis in this human malignancy.

Title: NDRG1 promotes growth of hepatocellular carcinoma cells by directly interacting with GSK-3β and Nur77 to prevent β-catenin degradation Content: The N-myc downstream regulated gene 1 (NDRG1) is significantly associated with advanced tumor stages and poor survival of hepatocellular carcinoma (HCC), thereby implicating it as a potential target for HCC treatment. We aim to further understand its biological roles in hepatocarcinogenesis, as a means to exploit it for therapeutic purposes. By screening using the ProtoArray® Human Protein Microarrays, we identified glycogen synthase kinase 3β (GSK-3β) and the orphan nuclear receptor (Nur77) as potential interaction partners of NDRG1. These interactions were confirmed in HCC cell lines in vitro by co-immunoprecipitation; and co-localizations of NDRG1 with GSK-3β and Nur77 were observed by immunofluorescence staining. Additionally, high levels of NDRG1 competitively bind to GSK-3β and Nur77 to allow β-catenin to escape degradation, with consequent elevated levels of downstream oncogenic genes. In vivo, we consistently observed that NDRG1 suppression in HCC xenografts decreased β-catenin levels and its downstream target Cyclin D1, with concomitant tumor growth inhibition. Clinically, the over-expression of NDRG1 in HCC patient samples is positively correlated with GSK-3β-9ser (| R | = 0.28, p = 0.01), Nur77 (| R | = 0.42, p < 0.001), and β-catenin (| R |= 0.32, p = 0.003) expressions. In conclusion, we identified GSK-3β and Nur77 as novel interaction partners of NDRG1. These protein-protein interactions regulate the turnover of β-catenin and subsequent downstream signaling mediated by β-catenin in HCC cells, and provides potential targets for future therapeutic interventions.

Title: Mutated KRAS results in overexpression of DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase, in rectal carcinomas. Content: Mutations of the KRAS oncogene are predictive for resistance to treatment with antibodies against the epithelial growth factor receptor in patients with colorectal cancer. Overcoming this therapeutic dilemma could potentially be achieved by the introduction of drugs that inhibit signaling pathways that are activated by KRAS mutations. To identify comprehensively such signaling pathways, we profiled pretreatment biopsies and normal mucosa from 65 patients with locally advanced rectal cancer-30 of which carried mutated KRAS-using global gene expression microarrays. By comparing all tumor tissues exclusively to matched normal mucosa, we could improve assay sensitivity, and identified a total of 22,297 features that were differentially expressed (adjusted P-value <0.05) between normal mucosa and cancer, including several novel potential rectal cancer genes. We then used this comprehensive description of the rectal cancer transcriptome as the baseline for identifying KRAS-dependent alterations. The presence of activating KRAS mutations is significantly correlated to an upregulation of 13 genes (adjusted P-value <0.05), among them DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase. Inhibition of the expression of both genes has previously been shown using the MEK1-inhibitor PD98059 and the antibacterial compound Novobiocin, respectively. These findings suggest a potential approach to overcome resistance to treatment with antibodies against the epithelial growth factor receptor in patients with KRAS-mutant rectal carcinomas.

Title: Tumor-associated macrophages correlate with the clinicopathological features and poor outcomes via inducing epithelial to mesenchymal transition in oral squamous cell carcinoma Content: BACKGROUND Both tumor-associated macrophages (TAMs) and the epithelial to mesenchymal transition (EMT) of cancer cells play key roles in promoting tumor progression. However, whether TAMs could induce EMT in the progression of oral squamous cell carcinoma (OSCC) remains undefined. RESULTS Here we detected the expression of macrophages markers CD68 and CD163, epithelial marker E-cadherin and mesenchymal marker vimentin in 127 OSCC patients by using semi-quantitative immunohistochemistry. CD68 and CD163 expression was not confined to the infiltrating TAMs, but also detected in cancer cells. The high number of CD68-positive macrophages was correlated with poor overall survival. Meanwhile, the expression of CD163 both in macrophages and in cancer cells was associated with poor overall survival and had a significant prognostic impact in OSCC. Importantly, the expression of CD163 in cancer cells had a significant relationship with E-cadherin and vimentin. Furthermore, the incubation of TAMs conditioned medium resulted in a fibroblast-like appearance of cancer cells (HN4, HN6 and SCC9) together with the decreased/increased expression of E-cadherin/ vimentin, which were correlated with the enhanced ability of migration and invasion. CONCLUSIONS Our results indicate that TAMs could promote the EMT of cancer cells, thereby leading to the progression of oral cancer.

Title: Variants at 6q21 implicate PRDM1 in the etiology of therapy-induced second malignancies after Hodgkin's lymphoma Content: Survivors of pediatric Hodgkin's lymphoma are at risk for radiation therapy–induced second malignant neoplasms (SMNs). We identified two variants at chromosome 6q21 associated with SMNs in survivors of Hodgkin's lymphoma treated with radiation therapy as children but not as adults. The variants comprise a risk locus associated with decreased basal expression of PRDM1 (encoding PR domain containing 1, with ZNF domain) and impaired induction of the PRDM1 protein after radiation exposure. These data suggest a new gene-exposure interaction that may implicate PRDM1 in the etiology of radiation therapy-induced SMNs.

Title: Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients. Content: PURPOSE AND EXPERIMENTAL DESIGN Using real-time quantitative methylation-specific PCR (RTQ-MSP), we quantified methylated p16INK4a sequences and determined the fractional concentrations of circulating tumor DNA in plasma, serum, and peripheral blood cells collected preoperatively, intraoperatively, and postoperatively from 49 patients with hepatocellular carcinoma (HCC). RESULTS RTQ-MSP was sufficiently sensitive to detect down to 10 genome-equivalents of methylated p16INK4a sequences. Quantitative MSP data were expressed in terms of the methylation index, which was the percentage of bisulfite converted unmethylated and methylated p16INK4a sequences that consisted of methylated p16INK4a sequences. Quantities of methylated p16INK4a sequences were detected in peripheral circulation of 80% (23 of 29) of HCC patients. No significant difference was seen in the detectability and concentrations of methylated p16INK4a sequences (range: 10-4046 genome-equivalents/ml) between preoperative plasma and serum samples from HCC patients. Preoperatively, the p16INK4a methylation indices ranged from 0.2 to 100% and from 0.012 to 0.075% in the patients' plasma and buffy coat samples, respectively. After surgical resection, the median p16INK4a methylation indices in plasma and buffy coat concordantly decreased 12- and 15-fold, respectively. These results demonstrated the clinical usefulness and effectiveness of peripheral blood RTQ-MSP for detecting and monitoring HCC after treatment. Furthermore, none of the intraoperative plasma samples and only two of the intraoperative buffy coat samples were p16INK4a methylation positive. CONCLUSIONS Quantification of epigenetic changes in peripheral blood by RTQ-MSP is useful for the detection and monitoring of HCC.

Title: Circulating plasma microRNAs as a screening method for detection of colorectal adenomas. Content: BACKGROUND MicroRNAs (miRNAs) are small non-coding RNA molecules. Reduced or increased levels of specific miRNAs are observed in colon and other cancers, supporting their role in carcinogenesis. Detection of colorectal polyps is the cornerstone of the Bowel Cancer Screening Programme in the UK. However, uptake of screening nationally remains under 60%. We aimed to see whether circulating plasma miRNAs can be used to screen for patients with colorectal polyps, adenomas, or both. METHODS Blood samples were taken from patients from the Bowel Cancer Screening Programme (asymptomatic but faecal occult blood testing [FOBt] positive). Plasma RNA was extracted, target miRNAs (19a, 98, 146b, 186, 191, 222*, 331-5p, 452, 625, 664, 1247) were identified on pooled case miRNA assay cards, and miRNA fraction was quantified by quantitative RT-PCR assay. Results were compared with endoscopy reports and with histology of any polyps identified and removed. Analysis was done with Excel (2011) and SPSS (version 20) software. FINDINGS 210 patients were included (117 with polyps, 12 with cancer, 81 healthy controls [FOBt positive]). The miRNA panel showed significant differences in expression (on t testing) for patients compared with controls for those with polyps, cancer, or both (miR-19a, p=0·0184; miR-98, p=0·0206; miR-146b, p=0·0029; miR-186, p=0·0006; miR-62,5 p=0·0008), polyps (miR-19a, p=0·0233; miR-98, p=0·0224; miR-146b, p=0·003; miR-186, p=0·0004; miR-625, p=0·001), adenomas (miR-19a, p=0·0339; miR-98, p=0·0266; miR-146b, p=0·0045; miR-186, p=0·0008; miR-625, p=0·0049), multiple adenomas (both sides of colon; miR-146b, p=0·0194; miR-186, p=0·0226; miR-625, p=0·0013), and right-sided adenomas (miR-98, p=0·031; miR-146b, p=0·0076; miR-186, p=0·0041; miR-331-5p, p=0·0142; miR-625, p=0·0049). Receiver operating characteristic analysis showed sensitivity of 60% or more, and specificity of 86% or more for men with polyps, men with adenomas, all patients with haemorrhoids or diverticulosis and polyps, and all patients with haemorrhoids or diverticulosis and adenomas. INTERPRETATION The target miRNAs that we identified showed significant differences in expression levels for patients with polyps and patients with adenomas from controls. Use of this panel has potential as a screening test. FUNDING Bowel Disease Research Foundation.

Title: Genetic Variation in the Interleukin-28B Gene Is Associated with Spontaneous Clearance and Progression of Hepatitis C Virus in Moroccan Patients Content: BACKGROUND Genetic variation in the IL28B gene has been strongly associated with treatment outcomes, spontaneous clearance and progression of the hepatitis C virus infection (HCV). The aim of the present study was to investigate the role of polymorphisms at this locus with progression and outcome of HCV infection in a Moroccan population. METHODS We analyzed a cohort of 438 individuals among them 232 patients with persistent HCV infection, of whom 115 patients had mild chronic hepatitis and 117 had advanced liver disease (cirrhosis and hepatocellular carcinoma), 68 individuals who had naturally cleared HCV and 138 healthy subjects. The IL28B SNPs rs12979860 and rs8099917 were genotyped using a TaqMan 5' allelic discrimination assay. RESULTS The protective rs12979860-C and rs8099917-T alleles were more common in subjects with spontaneous clearance (77.9% vs 55.2%; p = 0.00001 and 95.6% vs 83.2%; p = 0.0025, respectively). Individuals with clearance were 4.69 (95% CI, 1.99-11.07) times more likely to have the C/C genotype for rs12979860 polymorphism (p = 0.0017) and 3.55 (95% CI, 0.19-66.89) times more likely to have the T/T genotype at rs8099917. Patients with advanced liver disease carried the rs12979860-T/T genotype more frequently than patients with mild chronic hepatitis C (OR = 1.89; 95% CI, 0.99-3.61; p = 0.0532) and this risk was even more pronounced when we compared them with healthy controls (OR = 4.27; 95% CI, 2.08-8.76; p = 0.0005). The rs8099917-G allele was also associated with advanced liver disease (OR = 2.34; 95% CI, 1.40-3.93; p = 0.0100). CONCLUSIONS In the Moroccan population, polymorphisms near the IL28B gene play a role both in spontaneous clearance and progression of HCV infection.

Title: Trans-ancestry mutational landscape of hepatocellular carcinoma genomes Content: Diverse epidemiological factors are associated with hepatocellular carcinoma (HCC) prevalence in different populations. However, the global landscape of the genetic changes in HCC genomes underpinning different epidemiological and ancestral backgrounds still remains uncharted. Here a collection of data from 503 liver cancer genomes from different populations uncovered 30 candidate driver genes and 11 core pathway modules. Furthermore, a collaboration of two large-scale cancer genome projects comparatively analyzed the trans-ancestry substitution signatures in 608 liver cancer cases and identified unique mutational signatures that predominantly contribute to Asian cases. This work elucidates previously unexplored ancestry-associated mutational processes in HCC development. A combination of hotspot TERT promoter mutation, TERT focal amplification and viral genome integration occurs in more than 68% of cases, implicating TERT as a central and ancestry-independent node of hepatocarcinogenesis. Newly identified alterations in genes encoding metabolic enzymes, chromatin remodelers and a high proportion of mTOR pathway activations offer potential therapeutic and diagnostic opportunities.

Title: Association of aspirin and NSAID use with risk of colorectal cancer according to genetic variants. Content: IMPORTANCE Use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with lower risk of colorectal cancer. OBJECTIVE To identify common genetic markers that may confer differential benefit from aspirin or NSAID chemoprevention, we tested gene × environment interactions between regular use of aspirin and/or NSAIDs and single-nucleotide polymorphisms (SNPs) in relation to risk of colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS Case-control study using data from 5 case-control and 5 cohort studies initiated between 1976 and 2003 across the United States, Canada, Australia, and Germany and including colorectal cancer cases (n=8634) and matched controls (n=8553) ascertained between 1976 and 2011. Participants were all of European descent. EXPOSURES Genome-wide SNP data and information on regular use of aspirin and/or NSAIDs and other risk factors. MAIN OUTCOMES AND MEASURES Colorectal cancer. RESULTS Regular use of aspirin and/or NSAIDs was associated with lower risk of colorectal cancer (prevalence, 28% vs 38%; odds ratio [OR], 0.69 [95% CI, 0.64-0.74]; P = 6.2 × 10(-28)) compared with nonregular use. In the conventional logistic regression analysis, the SNP rs2965667 at chromosome 12p12.3 near the MGST1 gene showed a genome-wide significant interaction with aspirin and/or NSAID use (P = 4.6 × 10(-9) for interaction). Aspirin and/or NSAID use was associated with a lower risk of colorectal cancer among individuals with rs2965667-TT genotype (prevalence, 28% vs 38%; OR, 0.66 [95% CI, 0.61-0.70]; P = 7.7 × 10(-33)) but with a higher risk among those with rare (4%) TA or AA genotypes (prevalence, 35% vs 29%; OR, 1.89 [95% CI, 1.27-2.81]; P = .002). In case-only interaction analysis, the SNP rs16973225 at chromosome 15q25.2 near the IL16 gene showed a genome-wide significant interaction with use of aspirin and/or NSAIDs (P = 8.2 × 10(-9) for interaction). Regular use was associated with a lower risk of colorectal cancer among individuals with rs16973225-AA genotype (prevalence, 28% vs 38%; OR, 0.66 [95% CI, 0.62-0.71]; P = 1.9 × 10(-30)) but was not associated with risk of colorectal cancer among those with less common (9%) AC or CC genotypes (prevalence, 36% vs 39%; OR, 0.97 [95% CI, 0.78-1.20]; P = .76). CONCLUSIONS AND RELEVANCE In this genome-wide investigation of gene × environment interactions, use of aspirin and/or NSAIDs was associated with lower risk of colorectal cancer, and this association differed according to genetic variation at 2 SNPs at chromosomes 12 and 15. Validation of these findings in additional populations may facilitate targeted colorectal cancer prevention strategies.

Title: NRAGE promotes the malignant phenotype of hepatocellular carcinoma. Content: Hepatocellular carcinoma (HCC) is a fatal disease, primarily due to the limited effective therapies available for patients with advanced or recurrent stages of the disease. Therefore, in order to improve patient prognosis, it is important to identify an informative biomarker for HCC progression, as well as a molecular target for therapy. Neurotrophin receptor-interacting melanoma antigen-encoding protein (NRAGE), a member of the type II melanoma-associated antigen family, mediates apoptosis and cell death through interactions with a wide range of proteins, and is implicated as a tumor suppressor or oncoprotein depending on cell type. However, the role of NRAGE in HCC is currently unknown, therefore, the present study aimed to identify the underlying function of NRAGE in HCC tumorigenesis. Resected tumor and non-cancerous liver tissues from 151 patients with HCC, alongside HCC cell lines, were analyzed by polymerase chain reaction and immunohistochemical techniques to determine NRAGE expression levels, as well as the expression levels of potential genes encoding interacting proteins. It was demonstrated that the expression levels of NRAGE mRNA correlated significantly with those of apoptosis-antagonizing transcription factor (AATF), and were not affected by cirrhosis in non-cancerous liver tissues when compared to elevated levels in HCC tissues. The expression patterns of NRAGE protein and mRNA were consistent among 30 representative specimen pairs. Furthermore, increased NRAGE expression in patients with HCC correlated significantly with a shorter disease-specific survival time, and was identified as an independent prognostic factor via multivariate analysis (hazard ratio, 2.23; 95% confidence interval, 1.06-3.83; P=0.020). Therefore, the results of the present study indicated that increased NRAGE expression affects HCC progression via its interaction with AATF, and may represent a novel biomarker and molecular target for the treatment of HCC.

Title: IL6 derived from cancer-associated fibroblasts promotes chemoresistance via CXCR7 in esophageal squamous cell carcinoma Content: Various factors and cellular components in the tumor microenvironment are key drivers associated with drug resistance in many cancers. Here, we analyzed the factors and molecular mechanisms involved in chemoresistance in patients with esophageal squamous cell carcinoma (ESCC). We found that interleukin 6 (IL6) derived mainly from cancer-associated fibroblasts played the most important role in chemoresistance by upregulating C-X-C motif chemokine receptor 7 (CXCR7) expression through signal transducer and activator of transcription 3/nuclear factor-κB pathway. CXCR7 knockdown resulted in the inhibition of IL6-induced proliferation and chemoresistance. In addition, CXCR7 silencing significantly decreased gene expression associated with stemness, chemoresistance and epithelial–mesenchymal transition and suppressed the proliferation ability of ESCC cells in three-dimensional culture systems and angiogenesis assay. In clinical samples, ESCC patients with high expression of CXCR7 and IL6 presented a significantly worse overall survival and progression-free survival upon receiving cisplatin after operation. These results suggest that the IL6–CXCR7 axis may provide a promising target for the treatment of ESCC.

Title: A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome. Content: Loss of imprinting at IGF2, generally through an H19-independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith-Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island (KvDMR1) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2. Reverse transcription-PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1-associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and/or its associated antisense RNA (KvLQT1-AS) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains.

Title: CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. Content: Cancer stem cells drive tumor formation and metastasis, but how they acquire metastatic traits is not well understood. Here, we show that all colorectal cancer stem cells (CR-CSCs) express CD44v6, which is required for their migration and generation of metastatic tumors. CD44v6 expression is low in primary tumors but demarcated clonogenic CR-CSC populations. Cytokines hepatocyte growth factor (HGF), osteopontin (OPN), and stromal-derived factor 1α (SDF-1), secreted from tumor associated cells, increase CD44v6 expression in CR-CSCs by activating the Wnt/β-catenin pathway, which promotes migration and metastasis. CD44v6(-) progenitor cells do not give rise to metastatic lesions but, when treated with cytokines, acquire CD44v6 expression and metastatic capacity. Importantly, phosphatidylinositol 3-kinase (PI3K) inhibition selectively killed CD44v6 CR-CSCs and reduced metastatic growth. In patient cohorts, low levels of CD44v6 predict increased probability of survival. Thus, the metastatic process in colorectal cancer is initiated by CSCs through the expression of CD44v6, which is both a functional biomarker and therapeutic target.

Title: Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences. Content: Cyclin D1 expression is deregulated by chromosome translocation in mantle cell lymphoma and a subset of multiple myeloma. The molecular mechanisms involved in long-distance gene deregulation remain obscure, although changes in acetylated histones and methylated CpG dinucleotides may be important. The patterns of DNA methylation and histone acetylation were determined at the cyclin D1 locus on chromosome 11q13 in B-cell malignancies. The cyclin D1 promoter was hypomethylated and hyperacetylated in expressing cell lines and patient samples, and methylated and hypoacetylated in nonexpressing cell lines. Domains of hyperacetylated histones and hypomethylated DNA extended over 120 kb upstream of the cyclin D1 gene. Interestingly, hypomethylated DNA and hyperacetylated histones were also located at the cyclin D1 promoter but not the upstream major translocation cluster region in cyclin D1-nonexpressing, nontumorigenic B and T cells. RNA polymerase II binding was demonstrated both at the cyclin D1 promoter and 3' immunoglobulin heavy-chain regulatory regions only in malignant B-cell lines with deregulated cyclin D1 expression. Our results suggest a model where RNA polymerase II bound at IgH regulatory sequences can activate the cyclin D1 promoter by either long-range polymerase transfer or tracking.

Title: Cancer-associated fibroblasts promote hepatocellular carcinoma metastasis through chemokine-activated hedgehog and TGF-β pathways. Content: Fibroblasts are rich in the surrounding microenvironment of hepatocellular carcinoma (HCC) because most HCCs occur in fibrotic or cirrhotic livers. However, the role of cancer-associated fibroblasts (CAFs) in HCC metastasis remains obscure. Here, we reported that CAFs promote the migration and invasion of HCC cells in vitro and facilitate the HCC metastasis to the bone, brain and lung in NOD/SCID mice. The RayBio human chemokine antibody array revealed that CAFs secret higher levels of CCL2, CCL5, CCL7 and CXCL16 than peri-tumor fibroblasts. CCL2 and CCL5 increase the migration but not the invasion of HCC cells, while CCL7 and CXCL16 promote both migration and invasion of HCC cells. Moreover, CCL2 and CCL5 stimulate the activation of the hedgehog (Hh) pathway, while CCL7 and CXCL16 enhance the activity of the transforming growth factor-β (TGF-β) pathway in HCC cells. The neutralizing antibodies of chemokines notably attenuate the effect of CAFs on HCC metastasis and compromised the activation of Hh and TGF-β pathways in HCC cells. In summary, CAF-secreted CCL2, CCL5, CCL7 and CXCL16 promote HCC metastasis through the coordinate activation of Hh and TGF-β pathways in HCC cells.

Title: Aberrant expression and altered cellular localization of desmosomal and hemidesmosomal proteins are associated with aggressive clinicopathological features of oral squamous cell carcinoma Content: Disruption of cell adhesion plays a central role in dedifferentiation, invasion, and metastasis of various cancers. The desmosome and hemidesmosome are anchoring junctions that control cell-cell and cell-matrix adhesion, respectively. To clarify their contributions in mediating the biological properties of oral cancer, we immunohistochemically examined the expression of desmoglein 1 (DSG1), DSG2, DSG3, desmocollin 2 (DSC2), integrin beta 4 (ITGB4), laminin gamma chain 2 (LAMC2), and collagen type 17 alpha 1 (COL17A1) in 51 cases of oral squamous cell carcinoma. On normal oral epithelial cells, DSG1, DSG3, DSC2, and COL17A1 were expressed on the plasma membrane, while ITGB4 and mature LAMC2 were present at the basement membrane. In cancer, the expression of DSG1, DSG3, DSC2, and COL17A1 decreased and internalized to the cytoplasm. Cytoplasmic expression of DSG2, ITGB4, and LAMC2 was induced in the cancer cells facing to the stroma. We scored immunohistochemical expression and correlated this to clinicopathological parameters including histologic differentiation, pattern of invasion, and presence of lymph node metastasis. Decrease of DSG3 and DSC2 expression correlated with a more aggressive cancer phenotype: less differentiated and more invasive histologic features and a higher incidence of nodal metastasis. Lower COL17A1 and higher LAMC2 expression were also associated with a more aggressive phenotype. The present study demonstrates that aberrant expression and altered cellular localization of desmosomal and hemidesmosomal proteins are associated with aggressive clinicopathological features of oral cancer. This reinforces the notion that disturbance of the keratin-associated anchoring junctions confers aggressive features to cancer cells.

Title: KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer Content: BACKGROUND Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas. METHODS We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally. RESULTS Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity. CONCLUSION These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.

Title: Poor prognosis in carcinoma is associated with a gene expression signature of aberrant PTEN tumor suppressor pathway activity. Content: Pathway-specific therapy is the future of cancer management. The oncogenic phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in solid tumors; however, currently, no reliable test for PI3K pathway activation exists for human tumors. Taking advantage of the observation that loss of PTEN, the negative regulator of PI3K, results in robust activation of this pathway, we developed and validated a microarray gene expression signature for immunohistochemistry (IHC)-detectable PTEN loss in breast cancer (BC). The most significant signature gene was PTEN itself, indicating that PTEN mRNA levels are the primary determinant of PTEN protein levels in BC. Some PTEN IHC-positive BCs exhibited the signature of PTEN loss, which was associated to moderately reduced PTEN mRNA levels cooperating with specific types of PIK3CA mutations and/or amplification of HER2. This demonstrates that the signature is more sensitive than PTEN IHC for identifying tumors with pathway activation. In independent data sets of breast, prostate, and bladder carcinoma, prediction of pathway activity by the signature correlated significantly to poor patient outcome. Stathmin, encoded by the signature gene STMN1, was an accurate IHC marker of the signature and had prognostic significance in BC. Stathmin was also pathway-pharmacodynamic in vitro and in vivo. Thus, the signature or its components such as stathmin may be clinically useful tests for stratification of patients for anti-PI3K pathway therapy and monitoring therapeutic efficacy. This study indicates that aberrant PI3K pathway signaling is strongly associated with metastasis and poor survival across carcinoma types, highlighting the enormous potential impact on patient survival that pathway inhibition could achieve.

Title: 15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer. Content: Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.

Title: Overexpression of the circadian clock gene Bmal1 increases sensitivity to oxaliplatin in colorectal cancer. Content: PURPOSE The circadian clock gene Bmal1 is involved in cancer cell proliferation and DNA damage sensitivity. The aim of this study was to explore the effect of Bmal1 on oxaliplatin sensitivity and to determine its clinical significance in colorectal cancer. EXPERIMENTAL DESIGN Three colorectal cancer cell lines, HCT116, THC8307 and HT29, were used. The Bmal1-mediated control of colorectal cancer cell proliferation was tested in vitro and in vivo. MTT and colony formation assays were performed to determine the sensitivity of colorectal cancer cells to oxaliplatin. Flow cytometry was used to examine changes in the cell-cycle distribution and apoptosis rate. Proteins expressed downstream of Bmal1 upon its overexpression were determined by Western blotting. Immunohistochemistry was used to analyze Bmal1 expression in 82 archived colorectal cancer tumors from patients treated with oxaliplatin-based regimens. RESULTS Bmal1 overexpression inhibited colorectal cancer cell proliferation and increased colorectal cancer sensitivity to oxaliplatin in three colorectal cancer cell lines and HCT116 cells model in vivo. Furthermore, the overall survival of patients with colorectal cancer with high Bmal1 levels in their primary tumors was significantly longer than that of patients with low Bmal1 levels (27 vs. 19 months; P = 0.043). The progression-free survival of patients with high Bmal1 expression was also significantly longer than that of patients with low Bmal1 expression (11 vs. 5 months; P = 0.015). Mechanistically, the effect of Bmal1 was associated with its ability to regulate G2-M arrest by activating the ATM pathway. CONCLUSION Bmal1 shows the potential as a novel prognostic biomarker and may represent a new therapeutic target in colorectal cancer.

Title: Genetic variants rs1994016 and rs3825807 in ADAMTS7 affect its mRNA expression in atherosclerotic occlusive peripheral arterial disease Content: AIM Peripheral artery disease (PAD) is a vascular disease affecting peripheral circulation. Recently, genome-wide association studies revealed a relationship between single nucleotide polymorphisms (SNPs) in ADAMTS7 (a disintegrin and metalloprotease with thrombospondin motif 7) and atherosclerosis. In this study, we aimed to determine ADAMTS7 expression in peripheral blood mononuclear cells (PBMCs) and the frequency of ADAMTS7 rs1994016 and rs3825807 polymorphisms in a sample of Turkish patients with PAD, and to evaluate the association of matrix metalloproteinase (MMP) levels with PAD development. METHODS In this case-control study, ADAMTS7mRNA and protein expression was determined using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, respectively, and rs1994016 and rs3825807 variants in ADAMTS7 were determined by real-time PCR in 115 PAD patients and 116 healthy controls. Plasma levels of nine MMPs were determined using a multiplex immunoassay system. RESULTS ADAMTS7mRNA levels were significantly higher in PAD patients than in controls (t=-2.75, P=.007). There was no significant difference in the frequencies of rs1994016 and rs3825807 between PAD patients and controls (P>.05). In PAD patients, ADAMTS7mRNA levels were significantly increased for the CC genotype of rs1994016 (t=-2.31, P=.026) and TT genotype of rs3825807 (t=-2.23, P=.032). Furthermore, plasma levels of MMP-1, MMP-3, MMP-7, MMP-10, MMP-12, and MMP-13 were significantly higher in PAD patients than in controls (P<.05). CONCLUSION This is the first report of the relationship between PAD and ADAMTS7 expression and the effects of the rs1994016 and rs3825807 variants on PAD development. ADAMTS7 may be associated with PAD development.

Title: Reduced NF1 expression confers resistance to EGFR inhibition in lung cancer. Content: Activating mutations in the EGF receptor (EGFR) are associated with clinical responsiveness to EGFR tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib. However, resistance eventually arises, often due to a second EGFR mutation, most commonly T790M. Through a genome-wide siRNA screen in a human lung cancer cell line and analyses of murine mutant EGFR-driven lung adenocarcinomas, we found that erlotinib resistance was associated with reduced expression of neurofibromin, the RAS GTPase-activating protein encoded by the NF1 gene. Erlotinib failed to fully inhibit RAS-ERK signaling when neurofibromin levels were reduced. Treatment of neurofibromin-deficient lung cancers with a MAP-ERK kinase (MEK) inhibitor restored sensitivity to erlotinib. Low levels of NF1 expression were associated with primary and acquired resistance of lung adenocarcinomas to EGFR TKIs in patients. These findings identify a subgroup of patients with EGFR-mutant lung adenocarcinoma who might benefit from combination therapy with EGFR and MEK inhibitors.

Title: Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization. Content: Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.

Title: Genetic Variants at Newly Identified Lipid Loci Are Associated with Coronary Heart Disease in a Chinese Han Population Content: BACKGROUND Recent genome-wide association studies (GWAS) have mapped several novel loci influencing blood lipid levels in Caucasians. We sought to explore whether the genetic variants at newly identified lipid-associated loci were associated with CHD susceptibility in a Chinese Han population. METHODOLOGY/PRINCIPAL FINDINGS We conducted a two-stage case-control study in a Chinese Han population. The first-stage, consisting of 1,376 CHD cases and 1,376 sex and age- frequency matched controls, examined 5 novel lipid-associated single-nucleotide polymorphisms (SNPs) identified from GWAS among Caucasians in relation to CHD risk in Chinese. We then validated significant SNPs in the second-stage, consisting of 1,269 cases and 2,745 controls. We also tested associations between SNPs within the five novel loci and blood lipid levels in 4,121 controls. We identified two novel SNPs (rs599839 in CELSR2-PSRC1-SORT1 and rs16996148 in NCAN-CILP2) that were significantly associated with reduced CHD risk in Chinese (odds ratios (95% confidence intervals) in the dominant model 0.76 (0.61-0.90; P = 0.001), 0.67 (0.57-0.77; P = 3.4×10(-8)), respectively). Multiple linear regression analyses using dominant model showed that rs599839 was significantly associated with decreased LDL levels (P = 0.022) and rs16996148 was significantly associated with increased LDL and HDL levels (P = 2.9×10(-4) and 0.001, respectively). CONCLUSIONS/SIGNIFICANCE We identified two novel SNPs (rs599839 and rs16996148) at newly identified lipid-associated loci that were significantly associated with CHD susceptibility in a Chinese Han population.

Title: Expression of circadian genes correlates with liver metastasis and outcomes in colorectal cancer. Content: Circadian rhythms are daily oscillations in various biological processes, generated by the feedback loops of eight core circadian genes: Period1 (Per1), Period2 (Per2), Period3 (Per3), Cryptochrome1 (Cry1), Cryptochrome2 (Cry2), Clock, Bmal1 and Casein Kinase I ε (CKIε). Recent studies have suggested that circadian genes participate in the growth and development of various cancers. This study examined the relations of circadian gene expression to clinicopathological factors and outcomes in patients with colorectal cancer. We studied surgical specimens of cancer tissue and adjacent normal mucosa obtained from 202 patients with untreated colorectal cancer. The relative expression levels of the circadian genes in the specimens were measured by quantitative real-time, reverse-transcription polymerase chain reaction. Expression of the Clock gene and the CKIε gene in cancer tissue were significantly higher compared to that in adjacent normal mucosa. Expression of the Per1 and Per3 genes in cancer tissue was significantly lower compared to that in adjacent normal mucosa. Analysis of the relations between clinicopathological features and expression of the eight circadian genes in cancer tissue showed that high expression of the Bmal1 gene and low expression of the Per1 gene correlated with liver metastasis. On analysis of the relations between outcomes and gene expression, high expression of the Per2 gene was associated with significantly better outcomes than low expression of the Per2 gene. Overexpression of the Bmal1 gene and reduced expression of the Per1 gene may thus be useful predictors of liver metastasis. Moreover, reduced expression of the Per2 gene may be a predictor of outcomes in patients with colorectal cancer.

Title: A long non-coding RNA signature to improve prognosis prediction of colorectal cancer Content: Increasing evidence suggests long non-coding RNAs (lncRNAs) are frequently aberrantly expressed in cancers, however, few related lncRNA signatures have been established for prediction of cancer prognosis. We aimed to develop a lncRNA signature to improve prognosis prediction of colorectal cancer (CRC). Using a lncRNA-mining approach, we performed lncRNA expression profiling in large CRC cohorts from Gene Expression Ominus (GEO), including GSE39582 test series(N=436), internal validation series (N=117); and two independent validation series GSE14333 (N=197) and GSE17536(N=145). We established a set of six lncRNAs that were significantly correlated with the disease free survival (DFS) in the test series. Based on this six-lncRNA signature, the test series patients could be classified into high-risk and low-risk subgroups with significantly different DFS (HR=2.670; P<0.0001). The prognostic value of this six-lncRNA signature was confirmed in the internal validation series and another two independent CRC sets. Gene set enrichment analysis (GSEA) analysis suggested that risk score positively correlated with several cancer metastasis related pathways. Functional experiments demonstrated three dysregulated lncRNAs, AK123657, BX648207 and BX649059 were required for efficient invasion and proliferation suppression in CRC cell lines. Our results might provide an efficient classification tool for clinical prognosis evaluation of CRC.

Title: Colorectal Cancers from Distinct Ancestral Populations Show Variations in BRAF Mutation Frequency Content: It has been demonstrated for some cancers that the frequency of somatic oncogenic mutations may vary in ancestral populations. To determine whether key driver alterations might occur at different frequencies in colorectal cancer, we applied a high-throughput genotyping platform (OncoMap) to query 385 mutations across 33 known cancer genes in colorectal cancer DNA from 83 Asian, 149 Black and 195 White patients. We found that Asian patients had fewer canonical oncogenic mutations in the genes tested (60% vs Black 79% (P = 0.011) and White 77% (P = 0.015)), and that BRAF mutations occurred at a higher frequency in White patients (17% vs Asian 4% (P = 0.004) and Black 7% (P = 0.014)). These results suggest that the use of genomic approaches to elucidate the different ancestral determinants harbored by patient populations may help to more precisely and effectively treat colorectal cancer.

Title: Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria Content: Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) was originally identified as a positive regulator of drug detoxifying enzyme gene expression during exposure to environmental electrophiles. Currently, Nrf2 is known to regulate the expression of hundreds of cytoprotective genes to counteract endogenously or exogenously generated oxidative stress. Furthermore, when activated in human tumors by somatic mutations, Nrf2 confers growth advantages and chemoresistance by regulating genes involved in various processes such as the pentose phosphate pathway and nucleotide synthesis in addition to antioxidant proteins. Interestingly, increasing evidence shows that Nrf2 is associated with mitochondrial biogenesis during environmental stresses in certain tissues such as the heart. Furthermore, SKN-1, a functional homolog of Nrf2 in C. elegans, is activated by mitochondrial reactive oxygen species and extends life span by promoting mitochondrial homeostasis (i.e., mitohormesis). Similarly, Nrf2 activation was recently observed in the heart of surfeit locus protein 1 (Surf1) -/- mice in which cellular respiration was decreased due to cytochrome c oxidase defects. In this review, we critically examine the relationship between Nrf2 and mitochondria and argue that the Nrf2 stress pathway intimately communicates with mitochondria to maintain cellular homeostasis during oxidative stress.

Title: Vitamin D and prevention of colorectal cancer. Content: BACKGROUND Inadequate photosynthesis or oral intake of Vitamin D are associated with high incidence rates of colorectal cancer, but the dose-response relationship has not been adequately studied. METHODS Dose-response gradients from observational studies of Vitamin D intake and serum 25-hydroxyvitamin D were plotted as trend lines. The point on each linear trend line corresponding to an odds ratio of 0.50 provided the prediagnostic Vitamin D intake or 25-hydroxyvitamin D concentration associated with 50% lower risk compared to <100IU/day Vitamin D or <13ng/ml serum 25-hydroxyvitamin D. Medians of these values were determined. RESULTS Overall, individuals with >or=1000IU/day oral Vitamin D (p<0.0001) or >or=33ng/ml (82nmol/l) serum 25-hydroxyvitamin D (p<0.01) had 50% lower incidence of colorectal cancer compared to reference values. CONCLUSIONS Intake of 1000IU/day of Vitamin D, half the safe upper intake established by the National Academy of Sciences, was associated with 50% lower risk. Serum 25-hydroxyvitamin D of 33ng/ml, which is known to be safe, also was associated with 50% lower risk. Prompt public health action is needed to increase intake of Vitamin D(3) to 1000IU/day, and to raise 25-hydroxyvitamin D by encouraging a modest duration of sunlight exposure.

Title: Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion Content: Fibroblasts often constitute the majority of the stromal cells within a breast carcinoma, yet the functional contributions of these cells to tumorigenesis are poorly understood. Using a coimplantation tumor xenograft model, we demonstrate that carcinoma-associated fibroblasts (CAFs) extracted from human breast carcinomas promote the growth of admixed breast carcinoma cells significantly more than do normal mammary fibroblasts derived from the same patients. The CAFs, which exhibit the traits of myofibroblasts, play a central role in promoting the growth of tumor cells through their ability to secrete stromal cell-derived factor 1 (SDF-1); CAFs promote angiogenesis by recruiting endothelial progenitor cells (EPCs) into carcinomas, an effect mediated in part by SDF-1. CAF-secreted SDF-1 also stimulates tumor growth directly, acting through the cognate receptor, CXCR4, which is expressed by carcinoma cells. Our findings indicate that fibroblasts within invasive breast carcinomas contribute to tumor promotion in large part through the secretion of SDF-1.

Title: Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes Content: Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

Title: Shared and distinct genetic variants in type 1 diabetes and celiac disease. Content: BACKGROUND Two inflammatory disorders, type 1 diabetes and celiac disease, cosegregate in populations, suggesting a common genetic origin. Since both diseases are associated with the HLA class II genes on chromosome 6p21, we tested whether non-HLA loci are shared. METHODS We evaluated the association between type 1 diabetes and eight loci related to the risk of celiac disease by genotyping and statistical analyses of DNA samples from 8064 patients with type 1 diabetes, 9339 control subjects, and 2828 families providing 3064 parent-child trios (consisting of an affected child and both biologic parents). We also investigated 18 loci associated with type 1 diabetes in 2560 patients with celiac disease and 9339 control subjects. RESULTS Three celiac disease loci--RGS1 on chromosome 1q31, IL18RAP on chromosome 2q12, and TAGAP on chromosome 6q25--were associated with type 1 diabetes (P<1.00x10(-4)). The 32-bp insertion-deletion variant on chromosome 3p21 was newly identified as a type 1 diabetes locus (P=1.81x10(-8)) and was also associated with celiac disease, along with PTPN2 on chromosome 18p11 and CTLA4 on chromosome 2q33, bringing the total number of loci with evidence of a shared association to seven, including SH2B3 on chromosome 12q24. The effects of the IL18RAP and TAGAP alleles confer protection in type 1 diabetes and susceptibility in celiac disease. Loci with distinct effects in the two diseases included INS on chromosome 11p15, IL2RA on chromosome 10p15, and PTPN22 on chromosome 1p13 in type 1 diabetes and IL12A on 3q25 and LPP on 3q28 in celiac disease. CONCLUSIONS A genetic susceptibility to both type 1 diabetes and celiac disease shares common alleles. These data suggest that common biologic mechanisms, such as autoimmunity-related tissue damage and intolerance to dietary antigens, may be etiologic features of both diseases.

Title: Up-regulated NRIP2 in colorectal cancer initiating cells modulates the Wnt pathway by targeting RORβ Content: BACKGROUND Colorectal cancer remains one of the most common malignant tumors worldwide. Colorectal cancer initiating cells (CCICs) are a small subpopulation responsible for malignant behaviors of colorectal cancer. Aberrant activation of the Wnt pathways regulates the self-renewal of CCIC. However, the underlying mechanism(s) remain poorly understood. METHODS Via retroviral library screening, we identified Nuclear Receptor-Interacting Protein 2 (NRIP2) as a novel interactor of the Wnt pathway from enriched colorectal cancer colosphere cells. The expression levels of NRIP2 and retinoic acid-related orphan receptor β (RORβ) were further examined by FISH, qRT-PCR, IHC and Western blot. NRIP2 overexpressed and knockdown colorectal cancer cells were produced to study the role of NRIP2 in Wnt pathway. We also verified the binding between NRIP2 and RORβ and investigated the effect of RORβ on CCICs both in vitro and in vivo. Genechip-scanning speculated downstream target HBP1. Western blot, ChIP and luciferase reporter were carried to investigate the interaction between NRIP2, RORβ, and HBP1. RESULTS NRIP2 was significantly up-regulated in CCICs from both cell lines and primary colorectal cancer tissues. Reinforced expression of NRIP2 increased Wnt activity, while silencing of NRIP2 attenuated Wnt activity. The transcription factor RORβ was a key target through which NRIP2 regulated Wnt pathway activity. RORβ was a transcriptional enhancer of inhibitor HBP1 of the Wnt pathway. NRIP2 prevented RORβ to bind with downstream HBP1 promoter regions and reduced the transcription of HBP1. This, in turn, attenuated the HBP1-dependent inhibition of TCF4-mediated transcription. CONCLUSIONS NRIP2 is a novel interactor of the Wnt pathway in colorectal cancer initiating cells. interactions between NRIP2, RORβ, and HBP1 mediate a new mechanism for CCIC self-renewal via the Wnt activity.

Title: Gene expression analysis by cDNA microarray in oral squamous cell carcinoma. Content: BACKGROUND Oral squamous cell carcinoma (OSCC) is common type of human cancer, but little is known about the molecular mechanisms deciding on this malignancy. Comprehensive gene expression profiling is essential for understanding OSCC. METHODS cDNA microarray was used to analyze expression patterns of 16 617 genes in nine OSCC patients. RESULTS Forty-seven genes with altered expression among all cases were extracted. The ontology of these 47 genes was classified into 10 categories. To validate the microarray data, the expression of genes, including TGFBI, FADD and DUSP1 was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). By hierarchical clustering analysis, the nine cases were divided into two clusters. CONCLUSIONS The 47 genes are suggested as having a functional significance in oral squamous cell carcinogenesis. It is also suggested that the gene expression patterns by hierarchical clustering analysis can represent degrees of differentiation. The postoperative recovery was uneventful and patients free from tumor after surgery. In the future, on the occasion when the time comes that the number of cases accumulated for microarray increases and each case is observed more over a long-term, these data of 5-year survival rate will be added. Thereby, it will become possible to represent the malignancy of OSCC by these gene expression patterns.

Title: Deleted in liver cancer-1 (DLC-1): a tumor suppressor not just for liver. Content: Deleted in liver cancer 1 (DLC-1), as its name implied, was originally isolated as a potential tumor suppressor gene often deleted in hepatocellular carcinoma. Further studies have indicated that down-expression of DLC-1 either by genomic deletion or DNA methylation is associated with a variety of cancer types including lung, breast, prostate, kidney, colon, uterus, ovary, and stomach. Re-expression of DLC-1 in cancer cells regulates the structure of actin cytoskeleton and focal adhesions and significantly inhibits cell growth, supporting its role as a tumor suppressor. This tumor suppressive function relies on DLC-1's RhoGTPase activating protein (RhoGAP) activity and steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain, as well as its focal adhesion localization, which is recruited by the Src Homology 2 (SH2) domains of tensins in a phosphotyrosine-independent fashion. Therefore, the expression and subcellular localization of DLC-1 could be a useful molecular marker for cancer prognosis, whereas DLC-1 and its downstream signaling molecules might be therapeutic targets for the treatment of cancer.

Title: Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors. Content: Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects.

Title: Loss of imprinting in colorectal cancer linked to hypomethylation of H19 and IGF2. Content: Epigenetic alterations in human cancers include global DNA hypomethylation,gene hypomethylation and promoter hypermethylation, and loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2). A mechanism for LOI described previously is hypermethylation of a differentially methylated region (DMR) upstream of the H19 gene, allowing activation of the normally silent maternal allele of IGF2. Here we show that this mechanism does not apply to colorectal cancers, which show hypomethylation of the H19 DMR as well as a DMR upstream of exon 3 of IGF2. This hypomethylation is found in both colorectal cancers and normal mucosa from the same patients, and in cell lines with somatic cell knockout of DNA methyltransferases DNMT1 and DNMT3B. These data suggest that hypomethylation is a mechanism for LOI, that the popular IGF2-H19 enhancer competition model for IGF2 imprinting does not apply to the human colon, and that an alternative model for LOI would involve a transcriptional repressor acting on the normally silent maternal allele of IGF2.

Title: LIN28B enhanced tumorigenesis in an autochthonous KRASG12V-driven lung carcinoma mouse model Content: LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.

Title: Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Content: PD 0332991 is a highly specific inhibitor of cyclin-dependent kinase 4 (Cdk4) (IC50, 0.011 micromol/L) and Cdk6 (IC50, 0.016 micromol/L), having no activity against a panel of 36 additional protein kinases. It is a potent antiproliferative agent against retinoblastoma (Rb)-positive tumor cells in vitro, inducing an exclusive G1 arrest, with a concomitant reduction of phospho-Ser780/Ser795 on the Rb protein. Oral administration of PD 0332991 to mice bearing the Colo-205 human colon carcinoma produces marked tumor regression. Therapeutic doses of PD 0332991 cause elimination of phospho-Rb and the proliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control of E2F. The results indicate that inhibition of Cdk4/6 alone is sufficient to cause tumor regression and a net reduction in tumor burden in some tumors.

Title: Premature chromosome condensation: evidence for in vivo cell fusion in human malignant tumours. Content: Premature chromosome condensation (PCC) was studied in direct preparations of tissues from patients with haematological diseases and carcinomas of various histological types. PCC was found in 6 out of 166 malignancies (128 haematological cases, 35 carcinomas and 3 malignant effusions) analysed with the GTG-technique. Chromosome analysis revealed S-phase and G1-phase PCC in each case; the frequency of PCC varied between 1, 4 and 8.6% of the metaphases analysed. It is suggested that PCC chromosomes, which represent cell fusion in vivo, are not very rare in naturally-occurring human malignancies, and that cell fusion may affect the malignant phenotype. In conjunction with other factors they may also explain the heterogeneity of tumour cell populations.

Title: JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation Content: Jumonji domain-containing 6 (JMJD6) is a member of the Jumonji C domain-containing family of proteins. Compared to other members of the family, the cellular activity of JMJD6 is still not clearly defined and its biological function is still largely unexplored. Here we report that JMJD6 is physically associated with the tumor suppressor p53. We demonstrated that JMJD6 acts as an α-ketoglutarate- and Fe(II)-dependent lysyl hydroxylase to catalyze p53 hydroxylation. We found that p53 indeed exists as a hydroxylated protein in vivo and that the hydroxylation occurs mainly on lysine 382 of p53. We showed that JMJD6 antagonizes p53 acetylation, promotes the association of p53 with its negative regulator MDMX, and represses transcriptional activity of p53. Depletion of JMJD6 enhances p53 transcriptional activity, arrests cells in the G1 phase, promotes cell apoptosis, and sensitizes cells to DNA damaging agent-induced cell death. Importantly, knockdown of JMJD6 represses p53-dependent colon cell proliferation and tumorigenesis in vivo, and significantly, the expression of JMJD6 is markedly up-regulated in various types of human cancer especially in colon cancer, and high nuclear JMJD6 protein is strongly correlated with aggressive clinical behaviors of colon adenocarcinomas. Our results reveal a novel posttranslational modification for p53 and support the pursuit of JMJD6 as a potential biomarker for colon cancer aggressiveness and a potential target for colon cancer intervention.

Title: Oncofetal long noncoding RNA PVT1 promotes proliferation and stem cell-like property of hepatocellular carcinoma cells by stabilizing NOP2. Content: UNLABELLED Many protein-coding oncofetal genes are highly expressed in murine and human fetal liver and silenced in adult liver. The protein products of these hepatic oncofetal genes have been used as clinical markers for the recurrence of hepatocellular carcinoma (HCC) and as therapeutic targets for HCC. Herein we examined the expression profiles of long noncoding RNAs (lncRNAs) found in fetal and adult liver in mice. Many fetal hepatic lncRNAs were identified; one of these, lncRNA-mPvt1, is an oncofetal RNA that was found to promote cell proliferation, cell cycling, and the expression of stem cell-like properties of murine cells. Interestingly, we found that human lncRNA-hPVT1 was up-regulated in HCC tissues and that patients with higher lncRNA-hPVT1 expression had a poor clinical prognosis. The protumorigenic effects of lncRNA-hPVT1 on cell proliferation, cell cycling, and stem cell-like properties of HCC cells were confirmed both in vitro and in vivo by gain-of-function and loss-of-function experiments. Moreover, mRNA expression profile data showed that lncRNA-hPVT1 up-regulated a series of cell cycle genes in SMMC-7721 cells. By RNA pulldown and mass spectrum experiments, we identified NOP2 as an RNA-binding protein that binds to lncRNA-hPVT1. We confirmed that lncRNA-hPVT1 up-regulated NOP2 by enhancing the stability of NOP2 proteins and that lncRNA-hPVT1 function depends on the presence of NOP2. CONCLUSION Our study demonstrates that the expression of many lncRNAs is up-regulated in early liver development and that the fetal liver can be used to search for new diagnostic markers for HCC. LncRNA-hPVT1 promotes cell proliferation, cell cycling, and the acquisition of stem cell-like properties in HCC cells by stabilizing NOP2 protein. Regulation of the lncRNA-hPVT1/NOP2 pathway may have beneficial effects on the treatment of HCC.

Title: Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts. Content: Activated fibroblasts are associated with many different tumors. Myofibroblasts, activated fibroblasts, and perivascular mesenchymal cells such as pericytes play a role in cancer progression. Many studies suggest that myofibroblasts facilitate tumor growth and cancer progression. The source for myofibroblasts and other activated fibroblasts within the tumors is still debated. Although de novo activation of quiescent fibroblasts into alpha-smooth muscle actin (alpha SMA)-positive myofibroblasts is one likely source, epithelial to mesenchymal transition and bone marrow recruitment are also evolving as possible mechanisms for the emergence of a heterogeneous population of carcinoma-associated fibroblasts. Here, we show that transforming growth factor-beta1 could induce proliferating endothelial cells to undergo a phenotypic conversion into fibroblast-like cells. Such endothelial to mesenchymal transition (EndMT) is associated with the emergence of mesenchymal marker fibroblast-specific protein-1 (FSP1) and down-regulation of CD31/PECAM. Additionally, we show EndMT in tumors using the B16F10 melanoma model and the Rip-Tag2 spontaneous pancreatic carcinoma model. Crossing Tie2-Cre mice with R26Rosa-lox-Stop-lox-LacZ mice allows for irreversible tagging of endothelial cells. We provide unequivocal evidence for EndMT at the invasive front of the tumors in these transgenic mice. Collectively, our results show that EndMT is a unique mechanism for the accumulation of carcinoma-associated fibroblasts and suggest that antiangiogenic treatment of tumors may have a direct effect in decreasing activated fibroblasts that likely facilitate cancer progression.

Title: P19 embryonal carcinoma cells. Content: P19 cells are a line of pluripotent embryonal carcinoma able to grow continuously in serum-supplemented media. The differentiation of these cells can be controlled by nontoxic drugs. Retinoic acid effectively induces the development of neurons, astroglia and microglia--cell types normally derived from the neuroectoderm. Aggregates of P19 cells exposed to dimethyl sulfoxide differentiate into endodermal and mesodermal derivatives including cardiac and skeletal muscle. P19 cells can be effectively transfected with DNA encoding recombinant genes and stable lines expressing these genes can be readily isolated. These manipulations make P19 cells suitable material for investigating the molecular mechanisms governing developmental decision made by differentiating pluripotent cells.

Title: Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Content: Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.

Title: DNMT1 Inhibition Reprograms Pancreatic Cancer Stem Cells via Upregulation of the miR-17-92 Cluster. Content: Pancreatic ductal adenocarcinoma (PDAC) and other carcinomas are hierarchically organized, with cancer stem cells (CSC) residing at the top of the hierarchy, where they drive tumor progression, metastasis, and chemoresistance. As CSC and non-CSC share an identical genetic background, we hypothesize that differences in epigenetics account for the striking functional differences between these two cell populations. Epigenetic mechanisms, such as DNA methylation, play an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the role of DNA methylation in pancreatic CSC is obscure. In this study, we investigated the genome-wide DNA methylation profile of PDAC CSC, and we determined the importance of DNA methyltransferases for CSC maintenance and tumorigenicity. Using high-throughput methylation analysis, we discovered that sorted CSCs have a higher level of DNA methylation, regardless of the heterogeneity or polyclonality of the CSC populations present in the tumors analyzed. Mechanistically, CSC expressed higher DNMT1 levels than non-CSC. Pharmacologic or genetic targeting of DNMT1 in CSCs reduced their self-renewal and in vivo tumorigenic potential, defining DNMT1 as a candidate CSC therapeutic target. The inhibitory effect we observed was mediated in part through epigenetic reactivation of previously silenced miRNAs, in particular the miR-17-92 cluster. Together, our findings indicate that DNA methylation plays an important role in CSC biology and also provide a rationale to develop epigenetic modulators to target CSC plasticity and improve the poor outcome of PDAC patients. Cancer Res; 76(15); 4546-58. ©2016 AACR.

Title: Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers Content: Human tumors show a high level of genetic heterogeneity, but the processes that influence the timing and route of metastatic dissemination of the subclones are unknown. Here we have used whole-exome sequencing of 103 matched benign, malignant and metastatic skin tumors from genetically heterogeneous mice to demonstrate that most metastases disseminate synchronously from the primary tumor, supporting parallel rather than linear evolution as the predominant model of metastasis. Shared mutations between primary carcinomas and their matched metastases have the distinct A-to-T signature of the initiating carcinogen dimethylbenzanthracene, but non-shared mutations are primarily G-to-T, a signature associated with oxidative stress. The existence of carcinomas that either did or did not metastasize in the same host animal suggests that there are tumor-intrinsic factors that influence metastatic seeding. We also demonstrate the importance of germline polymorphisms in determining allele-specific mutations, and we identify somatic genetic alterations that are specifically related to initiation of carcinogenesis by Hras or Kras mutations. Mouse tumors that mimic the genetic heterogeneity of human cancers can aid our understanding of the clonal evolution of metastasis and provide a realistic model for the testing of novel therapies.

Title: A novel Ku70 function in colorectal homeostasis separate from nonhomologous end joining Content: Ku70, a known nonhomologous end-joining (NHEJ) factor, also functions in tumor suppression, although this molecular mechanism remains uncharacterized. Previously, we showed that mice deficient for DNA ligase IV (Lig4), another key NHEJ factor, succumbed to aggressive lymphoma in the absence of tumor suppressor p53. However, the tumor phenotype is abrogated by the introduction of a hypomorphic mutant p53R172P, which impaired p53-mediated apoptosis but not cell-cycle arrest. However, Lig4−/−p53R172P mice succumbed to severe diabetes. To further elucidate the role of NHEJ and p53-mediated apoptosis in vivo, we bred Ku70−/− p53R172P mice. Unexpectedly, these mice were free of diabetes, although 80% of the mutant mice had abnormally enlarged colons with pronounced inflammation. Remarkably, most of these mutant mice progressed to dysplasia, adenoma and adenocarcinoma; this is in contrast to the Lig4−/−p53R172P phenotype, strongly suggesting an NHEJ-independent function of Ku70. Significantly, our analyses of Ku70−/−p53R172P colonic epithelial cells show nuclear stabilization of β-catenin accompanied by higher expression of cyclin D1 and c-Myc in affected colon sections than in control samples. This is not due to the p53 mutation, as Ku70−/− mice share this phenotype. Our results not only unravel a novel function of Ku70 essential for colon homeostasis, but also establish an excellent in vivo model in which to study how chronic inflammation and abnormal cellular proliferation underlie tumorigenesis and tumor progression in the colon.

Title: Whole genomes redefine the mutational landscape of pancreatic cancer Content: Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Title: Molecular characterization of endometrial cancer: a correlative study assessing microsatellite instability, MLH1 hypermethylation, DNA mismatch repair protein expression, and PTEN, PIK3CA, KRAS, and BRAF mutation analysis. Content: Endometrial cancer is associated with numeric and structural chromosomal abnormalities, microsatellite instability (MSI), and alterations that activate oncogenes and inactivate tumor suppressor genes. The aim of this study was to characterize a set of endometrial cancers using multiple molecular genetic and immunohistochemical techniques. Ninety-six cases were examined for genomic alterations by MSI, MLH1 promoter hypermethylation, p53 and mismatch repair protein expression (MLH1, MSH2, MSH6, PMS2), and PTEN, PIK3CA, KRAS, and BRAF mutation analysis. At least 1 alteration was identified in 48 of 87 (55%) specimens tested for PTEN, making it the most common abnormality in this study. A PIK3CA alteration was observed in 16 (17%) specimens. Twenty-nine of 94 (31%) MSI tested tumors exhibited an MSI-H phenotype. Of the 29 MSI-H cases, 24 (83%) were positive for methylation of the MLH1 promoter region. Twenty-three (82%) of the 28 MSI-H cases with immunohistochemistry results showed loss of expression of MLH1/PMS2 (n=19), MSH2/MSH6 (n=2), or MSH6 only (n=2). Of the 19 MSI-H cases with loss of MLH1/PMS2 on immunohistochemistry, 18 were positive, and 1 was equivocal for MLH1 promoter hypermethylation. Twelve of 94 cases (13%) analyzed for KRAS mutations were found to have a mutation. No BRAF V600E mutations were indentified. This study provides a comprehensive molecular genetic analysis of commonly analyzed targets in a large cohort of endometrial cancers.

Title: Genetic variants and non-genetic factors predict circulating vitamin D levels in Hispanic and non-Hispanic White women: the Breast Cancer Health Disparities Study. Content: Genome-wide association studies (GWAS) have identified common polymorphisms in or near GC, CYP2R1, CYP24A1, and NADSYN1/DHCR7 genes to be associated with circulating levels of 25-hydroxyvitamin D [25(OH)D] in European populations. To replicate these GWAS findings, we examined six selected polymorphisms from these regions and their relation with circulating 25(OH)D levels in 1,605 Hispanic women (629 U.S. Hispanics and 976 Mexicans) and 354 non-Hispanic White (NHW) women. We also assessed the potential interactions between these variants and known non-genetic predictors of 25(OH)D levels, including body mass index (BMI), sunlight exposure and vitamin D intake from diet and supplements. The minor alleles of the two GC polymorphisms (rs7041 and rs2282679) were significantly associated with lower 25(OH)D levels in both Hispanic and NHW women. The CYP2R1 polymorphism, rs2060793, also was significantly associated with 25(OH)D levels in both groups. We found no significant associations for the polymorphisms in the CYP24A1. In Hispanic controls, 25(OH)D levels were significantly associated with the rs12785878T and rs1790349G haplotype in the NADSYN1/DHCR7 region. Significant interactions between GC rs2282679 and BMI and between rs12785878 and time spent in outdoor activities were observed. These results provide further support for the contribution of common genetic variants to individual variability in circulating 25(OH)D levels. The observed interactions between SNPs and non-genetic factors warrant confirmation.

Title: Heterogenic Loss of the Wild-Type BRCA Allele in Human Breast Tumorigenesis Content: For individuals genetically predisposed to breast and ovarian cancer through inheritance of a mutant BRCA allele, somatic loss of heterozygosity affecting the wild-type allele is considered obligatory for cancer initiation and/or progression. However, several lines of evidence suggest that phenotypic effects may result from BRCA haploinsufficiency. Archival fixed and embedded tissue specimens from women with germ line deleterious mutations in BRCA1 or BRCA2 were identified. After pathologic review, focal areas of normal breast epithelium, atypical ductal hyperplasia, ductal carcinoma-in-situ, and invasive ductal carcinoma were identified from 14 BRCA1-linked and 9 BRCA2-linked breast cancers. Ten BRCA-linked prophylactic mastectomy specimens and 12 BRCA-linked invasive ovarian carcinomas were also studied. Laser catapult microdissection was used to isolate cells from the various pathologic lesions and corresponding normal tissues. After DNA isolation, real-time polymerase chain reaction assays were used to quantitate the proportion of wild-type to mutant BRCA alleles in each tissue sample. Quantitative allelotyping of microdissected cells revealed a high level of heterogeneity in loss of heterozygosity within and between preinvasive lesions and invasive cancers from BRCA1 and BRCA2 heterozygotes with breast cancer. In contrast, all BRCA-associated ovarian cancers displayed complete loss of the wild-type BRCA allele. These data suggest that loss of the wild-type BRCA allele is not required for BRCA-linked breast tumorigenesis, which would have important implications for the genetic mechanism of BRCA tumor suppression and for the clinical management of this patient population.

Title: Inflammation-induced tumorigenesis in the colon is regulated by caspase-1 and NLRC4. Content: Chronic inflammation is a known risk factor for tumorigenesis, yet the precise mechanism of this association is currently unknown. The inflammasome, a multiprotein complex formed by NOD-like receptor (NLR) family members, has recently been shown to orchestrate multiple innate and adaptive immune responses, yet its potential role in inflammation-induced cancer has been little studied. Using the azoxymethane and dextran sodium sulfate colitis-associated colorectal cancer model, we show that caspase-1-deficient (Casp1(-/-)) mice have enhanced tumor formation. Surprisingly, the role of caspase-1 in tumorigenesis was not through regulation of colonic inflammation, but rather through regulation of colonic epithelial cell proliferation and apoptosis. Consequently, caspase-1-deficient mice demonstrate increased colonic epithelial cell proliferation in early stages of injury-induced tumor formation and reduced apoptosis in advanced tumors. We suggest a model in which the NLRC4 inflammasome is central to colonic inflammation-induced tumor formation through regulation of epithelial cell response to injury.

Title: Genetic variation and gastric cancer risk: a field synopsis and meta-analysis. Content: BACKGROUND Data on genetic susceptibility to sporadic gastric carcinoma have been published at a growing pace, but to date no comprehensive overview and quantitative summary has been available. METHODS We conducted a systematic review and meta-analysis of the evidence on the association between DNA variation and risk of developing stomach cancer. To assess result credibility, summary evidence was graded according to the Venice criteria and false positive report probability (FPRP) was calculated to further validate result noteworthiness. Meta-analysis was also conducted for subgroups, which were defined by ethnicity (Asian vs Caucasian), tumour histology (intestinal vs diffuse), tumour site (cardia vs non-cardia) and Helicobacter pylori infection status (positive vs negative). RESULTS Literature search identified 824 eligible studies comprising 2 530 706 subjects (cases: 261 386 (10.3%)) and investigating 2841 polymorphisms involving 952 distinct genes. Overall, we performed 456 primary and subgroup meta-analyses on 156 variants involving 101 genes. We identified 11 variants significantly associated with disease risk and assessed to have a high level of summary evidence: MUC1 rs2070803 at 1q22 (diffuse carcinoma subgroup), MTX1 rs2075570 at 1q22 (diffuse), PSCA rs2294008 at 8q24.2 (non-cardia), PRKAA1 rs13361707 5p13 (non-cardia), PLCE1 rs2274223 10q23 (cardia), TGFBR2 rs3087465 3p22 (Asian), PKLR rs3762272 1q22 (diffuse), PSCA rs2976392 (intestinal), GSTP1 rs1695 11q13 (Asian), CASP8 rs3834129 2q33 (mixed) and TNF rs1799724 6p21.3 (mixed), with the first nine variants characterised by a low FPRP. We also identified polymorphisms with lower quality significant associations (n=110). CONCLUSIONS We have identified several high-quality biomarkers of gastric cancer susceptibility. These data will form the backbone of an annually updated online resource that will be integral to the study of gastric carcinoma genetics and may inform future screening programmes.

Title: CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling via transactivation of GSK-3β and Axin2 expression Content: Caudal-related homeobox transcription factor 2 (CDX2), an intestine-specific nuclear transcription factor, has been strongly implicated in the tumourigenesis of various human cancers. However, the functional role of CDX2 in the development and progression of colorectal cancer (CRC) is not well known. In this study, CDX2 knockdown in colon cancer cells promoted cell proliferation in vitro, accelerated tumor formation in vivo, and induced a cell cycle transition from G0/G1 to S phase, whereas CDX2 overexpression inhibited cell proliferation. TOP/FOP-Flash reporter assay showed that CDX2 knockdown or CDX2 overexpression significantly increased or decreased Wnt signaling activity. Western blot assay showed that downstream targets of Wnt signaling, including β-catenin, cyclin D1 and c-myc, were up-regulated or down-regulated in CDX2-knockdown or CDX2-overexpressing colon cancer cells. In addition, suppression of Wnt signaling by XAV-939 led to a marked suppression of the cell proliferation enhanced by CDX2 knockdown, whereas activation of this signaling by CHIR-99021 significantly enhanced the cell proliferation inhibited by CDX2 overexpression. Dual-luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays further confirmed that CDX2 transcriptionally activates glycogen synthase kinase-3β (GSK-3β) and axis inhibition protein 2 (Axin2) expression by directly binding to the promoter of GSK-3β and the upstream enhancer of Axin2. In conclusion, these results indicated that CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling.

Title: Diverse tumorigenic pathways in ovarian serous carcinoma. Content: This study was undertaken to analyze genetic alterations in 108 sporadic serous ovarian neoplasms to elucidate ovarian serous carcinogenesis. Our results demonstrate that K-ras mutations occur in approximately 50% of serous borderline tumors (SBTs), non-invasive micropapillary serous carcinomas (MPSCs), and invasive micropapillary serous carcinomas, which represent a morphological continuum of tumor progression. Moreover, progressive increase in the degree of allelic imbalance of chromosomes 1p, 5q, 8p, 18q, 22q, and Xp was observed comparing serous borderline tumors to noninvasive and invasive micropapillary serous carcinomas. In contrast, high-grade (conventional serous carcinoma) tumors contained wild-type K-ras in all 23 cases studied and a high frequency of allelic imbalance even in small (early) primary tumors similar to that found in advanced stage tumors. Based on these findings, we propose a dualistic model for ovarian serous carcinogenesis. One pathway involves a stepwise progression from SBT to noninvasive and then invasive MPSC. The other pathway is characterized by rapid progression from the ovarian surface epithelium or inclusion cysts to a conventional (high-grade) serous carcinoma.

Title: CpG island methylator phenotype predicts progression of malignant melanoma. Content: PURPOSE The CpG island methylator phenotype (CIMP) may be associated with development of malignancy through coordinated inactivation of tumor suppressor and tumor-related genes (TRG) and methylation of multiple noncoding, methylated-in-tumor (MINT) loci. These epigenetic changes create a distinct CIMP pattern that has been linked to recurrence and survival in gastrointestinal cancers. Because epigenetic inactivation of TRGs also has been shown in malignant melanoma, we hypothesized the existence of a clinically significant CIMP in cutaneous melanoma progression. EXPERIMENTAL DESIGN The methylation status of the CpG island promoter region of TRGs related to melanoma pathophysiology (WIF1, TFPI2, RASSF1A, RARbeta2, SOCS1, and GATA4) and a panel of MINT loci (MINT1, MINT2, MINT3, MINT12, MINT17, MINT25, and MINT31) in primary and metastatic tumors of different clinical stages (n=122) was assessed. RESULTS Here, we show an increase in hypermethylation of the TRGs WIF1, TFPI2, RASSF1A, and SOCS1 with advancing clinical tumor stage. Furthermore, we find a significant positive association between the methylation status of MINT17, MINT31, and TRGs. The methylation status of MINT31 is associated with disease outcome in stage III melanoma. CONCLUSIONS These findings show the significance of a CIMP pattern that is associated with advancing clinical stage of malignant melanoma. Future prospective large-scale studies may determine if CIMP-positive primary melanomas are at high risk of metastasis or recurrence.

Title: 14q deletions are associated with trisomy 12, NOTCH1 mutations and unmutated IGHV genes in chronic lymphocytic leukemia and small lymphocytic lymphoma. Content: Deletions of the long arm of chromosome 14 [del(14q)] are rare but recurrently observed in mature B-cell neoplasms, particularly in chronic lymphocytic leukemia (CLL). To further characterize this aberration, we studied 81 cases with del(14q): 54 of CLL and 27 of small lymphocytic lymphoma (SLL), the largest reported series to date. Using karyotype and fluorescence in situ hybridization (FISH), the most frequent additional abnormality was trisomy 12 (tri12), observed in 28/79 (35%) cases, followed by del13q14 (12/79, 15%), delTP53 (11/80, 14%) delATM (5/79, 6%), and del6q21 (3/76, 4%). IGHV genes were unmutated in 41/53 (77%) patients, with a high frequency of IGHV1-69 (21/52, 40%). NOTCH1 gene was mutated in 14/45 (31%) patients. There was no significant difference in cytogenetic and molecular abnormalities between CLL and SLL. Investigations using FISH and SNP-array demonstrated the heterogeneous size of the 14q deletions. However, a group with the same del(14)(q24.1q32.33) was identified in 48% of cases. In this group, tri12 (P = 0.004) and NOTCH1 mutations (P = 0.02) were significantly more frequent than in the other patients. In CLL patients with del(14q), median treatment-free survival (TFS) was 27 months. In conclusion, del(14q) is associated with tri12 and with pejorative prognostic factors: unmutated IGHV genes (with over-representation of the IGHV1-69 repertoire), NOTCH1 mutations, and a short TFS.

Title: Common BRCA1 variants and susceptibility to breast and ovarian cancer in the general population. Content: Most multiple case families of young onset breast cancer and ovarian cancer are thought to be due to highly penetrant mutations in the predisposing genes BRCA1 and BRCA2. However, these mutations are uncommon in the population and they probably account for only a few percent of all breast cancer incidence. A much larger fraction of breast cancer might, in principle, be due to common variants which confer more modest individual risks. There are several common polymorphisms in the BRCA1 gene which generate amino acid substitutions. We have examined the frequency of four of these polymorphisms: Gln356Arg, Pro871Leu, Glu1038Gly and Ser1613Gly in large series of breast and ovarian cancer cases and matched controls. Due to strong linkage disequilibrium, these four sites generate only three haplotypes with a frequency > 1.3%. The most common haplotypes, defined by the alleles Gln356Pro871Glu1038Ser1613 and Gln356Leu871Gly1038Gly1613, have frequencies of 0.57 and 0.32 respectively, and these frequencies do not differ significantly between patient and control groups. Thus the most common polymorphisms of the BRCA1 gene do not make a significant contribution to breast or ovarian cancer risk. However, our data suggest that the Arg356 allele may have a different genotype distribution in breast cancer patients from that in controls (Arg356 homozygotes are more frequent in the control groups, P = 0.01), indicating that it may be protective against breast cancer. If this finding can be confirmed, it may provide an insight into the structural features of the BRCA1 protein that are important for its function.

Title: Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx. Content: A long-term cell culture epithelioid cell line was established from a recurrent squamous carcinoma of the nasopharynx of a Chinese male 17 1/2 years after radiation therapy. The cell line, designated NPC/HK1, has been passed 72 times over a period 1 year. The cells have been shown by light and electron microscopies to be of the squamous epithelial type. When they were transplanted subcutaneously into the back of athymic nude BALB/c (nu/nu) mice, tumors developed at the sites of inoculation, which on histological examination were shown to be well-differentiated squamous carcinomas, similar in morphology to the recurrent human tumor from which they were derived. Karyotypic analysis of cells from the cell line demonstrates an aneuploid human type with a modal chromosome number of 74 with both numerical and structural aberrations. Viral particles or Epstein-Barr viral nuclear antigen (EBNA) has not been demonstrated in the cells from the primary culture or several of the subcultures tested. The presence of EBNA in touch smears prepared from the biopsy tissue was inconclusive. Infection of the subcultured cells with EBV from P3HR1 and B95-8 cells was unsuccessful.

Title: The long non-coding RNA H19-derived miR-675 modulates human gastric cancer cell proliferation by targeting tumor suppressor RUNX1. Content: The lncRNA H19 has been recently shown to be upregulated and play important roles in gastric cancer tumorigenesis. However, the precise molecular mechanism of H19 and its mature product miR-675 in the carcinogenesis of gastric cancer remains unclear. In this study, we found that miR-675 was positively expressed with H19 and was a pivotal mediator in H19-induced gastric cancer cell growth promotion. Subsequently, the tumor suppressor Runt Domain Transcription Factor1 (RUNX1) was confirmed to be a direct target of miR-675 using a luciferase reporter assay and Western blotting analyses. A series of rescue assays indicated that RUNX1 mediated H19/miR-67-induced gastric cancer cell phenotypic changes. Moreover, the inverse relationship between the expression of RUNX1 and H19/miR-675 was also revealed in gastric cancer tissues and gastric cancer cell lines. Taken together, our study demonstrated that the novel pathway H19/miR-675/RUNX1 regulates gastric cancer development and may serve as a potential target for gastric cancer therapy.

Title: LncRNA HANR Promotes Tumorigenesis and Increase of Chemoresistance in Hepatocellular Carcinoma Content: Background/Aims: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third leading cause of cancer-related death. Critical roles for long non-coding RNAs (lncRNAs) have recently been demonstrated for a variety of cancers, including hepatocellular carcinoma. However, the effect and mechanism of lncRNAs in HCC tumorigenesis and chemoresistance have not been extensively characterized. Methods: In the current study, we have identified a HCC-expressed lncRNA termed as HANR (HCC associated long non-coding RNA). We identified HANR by microarray analysis and validated its up-regulated expression by quantitative PCR. RNA pull-down and pathway analyses were conducted to evaluate physical and functional interactions with HANR. In vivo experiments were performed to assess tumorigenesis and increase of chemoresistance. In addition, the HANR expression in HCC specimens was detected by FISH. Xenograft and orthotopic mice model was constructed to observe the effect of HANR on tumorigenesis and chemoresistance in vivo. Results: HANR was demonstrated to be up-regulated in HCC patients and HCC cell lines. Increased HANR expression in HCC predicted short survival of patients. Knock-down of HANR markedly retarded cell proliferation, suppressed HCC xenograft/orthotopic tumor growth, induced apoptosis and enhanced chemosensitivity to doxorubicin, while over-expression of HANR showed the opposite effects. It was found that HANR bind to GSKIP for regulating the phosphorylation of GSK3β in HCC. Conclusion: Our results demonstrate that HANR contributes to the development of HCC and is a promising therapeutic target for chemosensitization of HCC cells to doxorubicin, which may represent a promising therapeutic target in the future.

Title: Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer Content: Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case–control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10−5), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10−4) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10−9). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Title: Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers Content: ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

Title: Multipoint Kras oncogene mutations potentially indicate mucinous carcinoma on the entire spectrum of mucinous ovarian neoplasms Content: Kras mutation is a common phenomenon in many human neoplasms. We aimed to assess the Kras mutational status along the histological continuum from normal ovaries to the development of benign, borderline and malignant ovarian mucinous neoplasms. We analyzed 41 cases of malignant, 10 cases of borderline, 7 cases of benign mucinous ovarian tumors and 7 cases of normal ovarian tissue. The prevalence of Kras mutations in the normal ovary was 0.00% (n=0/7), while the prevalence in benign, borderline and malignant mucinous neoplasms was 57.14% (n=4/7), 90.00% (n=9/10) and 75.61% (n=31/41), respectively. Multiple Kras mutations were detected in 6 cases of mucinous carcinoma, including 5 double mutations with G13D/V14I (n=1), G12V/G13S (n=1), G12D/G13S (n=3) and one triple mutation with A11V/G13N/V14I (n=1). We identified six cases with 3 novel Kras mutations not previously described in the COSMIC database, which included A11V (n=3) and V14I (n=2) in mucinous carcinomas, and A11T (n=1) in a mucinous borderline tumor. In conclusion, Kras mutation appears to be one of the imperative events in the ovarian mucinous adenoma-borderline tumor-carcinoma sequence, as increased numbers of Kras mutations have been shown to be the strongest predictor of unequivocal malignancy in ovarian mucinous neoplasms.

Title: Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer. Content: PURPOSE To investigate hypoxia measured by pimonidazole binding, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CA-IX) expression, proliferation, and vascularity in liver metastases of colorectal cancer and to compare GLUT1 and CA-IX expression in corresponding primary tumors. METHODS AND MATERIALS Twenty-five patients with liver metastases of colorectal cancer, planned for metastasectomy, were included. The hypoxia marker pimonidazole and proliferation marker iododeoxyuridine were administered before surgery. After immunofluorescent staining of the frozen metastases, pimonidazole binding, vascularity, and proliferation were analyzed quantitatively. Thirteen paraffin-embedded primary tumors were stained immunohistochemically for GLUT1 and CA-IX expression, which was analyzed semiquantitatively in primary tumors and corresponding liver metastases. RESULTS In liver metastases, pimonidazole binding showed a pattern consistent with diffusion-limited hypoxia. The mean pimonidazole-positive fraction was 0.146; the mean distance from vessels to pimonidazole-positive areas was 80 microm. When expressed, often co-localization was observed between pimonidazole binding and GLUT1 or CA-IX expression, but microregional areas of mismatch were also observed. No correlation between the level of pimonidazole binding and GLUT1 or CA-IX expression was observed. In some patients, a large fraction (up to 30%) of proliferating cells was present in pimonidazole-stained areas. Expression of CA-IX in primary tumors and metastases showed a significant correlation, which was absent for GLUT1 expression. CONCLUSIONS Compared with other tumor types, liver metastases of colorectal cancer contain large amounts of hypoxic cells. The lack of correlation with pimonidazole binding brings into question the value of GLUT1 and CA-IX as endogenous markers of hypoxia.

Title: Tumor engraftment in nude mice and enrichment in stroma- related gene pathways predict poor survival and resistance to gemcitabine in patients with pancreatic cancer. Content: PURPOSE The goal of this study was to evaluate prospectively the engraftment rate, factors influencing engraftment, and predictability of clinical outcome of low-passage xenografts from patients with resectable pancreatic ductal adenocarcinoma (PDA) and to establish a bank of PDA xenografts. EXPERIMENTAL DESIGN Patients with resectable PDA scheduled for resection at the Johns Hopkins Hospital were eligible. Representative pieces of tumor were implanted in nude mice. The status of the SMAD4 gene and content of tumor-generating cells were determined by immunohistochemistry. Gene expression was carried out by using a U133 Plus 2.0 array. Patients were followed for progression and survival. RESULTS A total of 94 patients with PDA were resected, 69 tumors implanted in nude mice, and 42 (61%) engrafted. Engrafted carcinomas were more often SMAD4 mutant, and had a metastatic gene expression signature and worse prognosis. Tumors from patients resistant to gemcitabine were enriched in stroma-related gene pathways. Tumors sensitive to gemcitabine were enriched in cell cycle and pyrimidine gene pathways. The time to progression for patients who received treatment with gemcitabine for metastatic disease (n = 7) was double in patients with xenografts sensitive to gemcitabine. CONCLUSION A successful xenograft was generated in 61% of patients attempted, generating a pool of 42 PDA xenografts with significant biological information and annotated clinical data. Patients with PDA and SMAD4 inactivation have a better engraftment rate. Engraftment is a poor prognosis factor, and engrafted tumors have a metastatic gene expression signature. Tumors from gemcitabine-resistant patients were enriched in stromal pathways.

Title: The lncRNAs PCGEM1 and PRNCR1 are not implicated in castration resistant prostate cancer Content: Long noncoding RNAs (IncRNAs) are increasingly implicated in cancer biology, contributing to essential cancer cell functions such as proliferation, invasion, and metastasis. In prostate cancer, several lncRNAs have been nominated as critical actors in disease pathogenesis. Among these, expression of PCGEM1 and PRNCR1 has been identified as a possible component in disease progression through the coordination of androgen receptor (AR) signaling (Yang et al., Nature 2013, see ref. [1]). However, concerns regarding the robustness of these findings have been suggested. Here, we sought to evaluate whether PCGEM1 and PRNCR1 are associated with prostate cancer. Through a comprehensive analysis of RNA-sequencing data (RNA-seq), we find evidence that PCGEM1 but not PRNCR1 is associated with prostate cancer. We employ a large cohort of >230 high-risk prostate cancer patients with long-term outcomes data to show that, in contrast to prior reports, neither gene is associated with poor patient outcomes. We further observe no evidence that PCGEM1 nor PRNCR1 interact with AR, and neither gene is a component of AR signaling. Thus, we conclusively demonstrate that PCGEM1 and PRNCR1 are not prognostic lncRNAs in prostate cancer and we refute suggestions that these lncRNAs interact in AR signaling.

Title: Deregulated DNA polymerase β strengthens ionizing radiation-induced nucleotidic and chromosomal instabilities Content: DNA polymerase β (Pol β) is an error-prone enzyme which has been found to be overexpressed in several human tumors. By using a couple of recombinant CHO cells differing only from the exogenous expression of Pol β, we showed here that cells overexpressing Pol β are much more sensitive to IR treatments by increasing apoptosis. We also found that the surviving cells displayed an hypermutator phenotype which could be explained by different pathways involving Pol β, such as (i) an increased capacity to incorporate into DNA the mutagenic dGTP analog, 8-oxo-dGTP, one of the most abundant purine-derived nucleotides exposed to γ-irradiation, (ii) the induction of IR-induced DNA breaks and (iii) accumulation of chromosome aberrations induced by radiation. Alteration of Pol β expression in irradiated cells thus appears to strengthen both cell death and genetic changes associated with a malignant phenotype. These data provide new insights into the cellular response to radiations and the associated carcinogenic consequences.

Title: Identification of p18 INK4c as a tumor suppressor gene in glioblastoma multiforme. Content: Genomic alterations leading to aberrant activation of cyclin/cyclin-dependent kinase (cdk) complexes drive the pathogenesis of many common human tumor types. In the case of glioblastoma multiforme (GBM), these alterations are most commonly due to homozygous deletion of p16(INK4a) and less commonly due to genomic amplifications of individual genes encoding cyclins or cdks. Here, we describe deletion of the p18(INK4c) cdk inhibitor as a novel genetic alteration driving the pathogenesis of GBM. Deletions of p18(INK4c) often occurred in tumors also harboring homozygous deletions of p16(INK4a). Expression of p18(INK4c) was completely absent in 43% of GBM primary tumors studied by immunohistochemistry. Lentiviral reconstitution of p18(INK4c) expression at physiologic levels in p18(INK4c)-deficient but not p18(INK4c)-proficient GBM cells led to senescence-like G(1) cell cycle arrest. These studies identify p18(INK4c) as a GBM tumor suppressor gene, revealing an additional mechanism leading to aberrant activation of cyclin/cdk complexes in this terrible malignancy.

Title: A Teratocarcinoma-Like Human Embryonic Stem Cell (hESC) Line and Four hESC Lines Reveal Potentially Oncogenic Genomic Changes Content: The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis.

Title: An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Content: The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

Title: Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1. Content: The epithelial-mesenchymal transition (EMT) is required in the embryo for the formation of tissues for which cells originate far from their final destination. Carcinoma cells hijack this program for tumor dissemination. The relevance of the EMT in cancer is still debated because it is unclear how these migratory cells colonize distant tissues to form macrometastases. We show that the homeobox factor Prrx1 is an EMT inducer conferring migratory and invasive properties. The loss of Prrx1 is required for cancer cells to metastasize in vivo, which revert to the epithelial phenotype concomitant with the acquisition of stem cell properties. Thus, unlike the classical EMT transcription factors, Prrx1 uncouples EMT and stemness, and is a biomarker associated with patient survival and lack of metastasis.

Title: MUC1 oncoprotein activates the IκB kinase β complex and constitutive NF-κB signalling Content: Nuclear factor-κB (NF-κB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-κB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-κB p65. We show that MUC1 interacts with the high-molecular-weight IκB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKβ and IKKγ. Interaction of MUC1 with both IKKβ and IKKγ is necessary for IKKβ activation, resulting in phosphorylation and degradation of IκBα. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKβ–IKKγ in response to TNFα stimulation. TNFα-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKβ is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKβ and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKβ–NF-κB p65 pathway.

Title: Large scale replication and meta-analysis of variants on chromosome 4q25 associated with atrial fibrillation. Content: AIMS A recent genome-wide association study identified a haplotype block on chromosome 4q25 associated with atrial fibrillation (AF). We sought to replicate this association in four independent cohorts. METHODS AND RESULTS The Framingham Heart Study and Rotterdam Study are community-based longitudinal studies. The Vanderbilt AF Registry and German AF Network (AFNet) are case-control studies. Participants with AF (n = 3508) were more likely to be male and were older than referent participants (n = 12 173; Framingham 82 +/- 10 vs. 71 +/- 13 years; Rotterdam 73 +/- 8 vs. 69 +/- 9 years; Vanderbilt 54 +/- 14 vs. 57 +/- 14 years; AFNet 62 +/- 12 vs. 49 +/- 14 years). Single nucleotide polymorphism (SNP) rs2200733 was associated with AF in all four cohorts, with odds ratios (ORs) ranging from 1.37 in Rotterdam [95% confidence interval (CI) 1.18-1.59; P = 3.1 x 10(-5)] to 2.52 in AFNet (95% CI 2.22-2.8; P = 1.8 x 10(-49)). There also was a significant association between AF and rs10033464 in Framingham (OR 1.34; 95% CI 1.03-1.75; P = 0.031) and AFNet (OR 1.30; 95% CI 1.13-1.51; P = 0.0002), but not Vanderbilt (OR 1.16; 95% CI 0.86-1.56; P = 0.33). A meta-analysis of the current and prior AF studies revealed an OR of 1.90 (95% CI 1.60-2.26; P = 3.3 x 10(-13)) for rs2200733 and of 1.36 (95% CI 1.26-1.47; P = 6.7 x 10(-15)) for rs10033464. CONCLUSION The non-coding SNPs rs2200733 and rs10033464 are strongly associated with AF in four cohorts of European descent. These results confirm the significant relations between AF and intergenic variants on chromosome 4.

Title: Extracellular vesicles derived from renal cancer stem cells induce a pro-tumorigenic phenotype in mesenchymal stromal cells Content: Renal carcinomas have been shown to contain a population of cancer stem cells (CSCs) that present self-renewing capacity and support tumor growth and metastasis. CSCs were shown to secrete large amount of extracellular vesicles (EVs) that can transfer several molecules (proteins, lipids and nucleic acids) and induce epigenetic changes in target cells. Mesenchymal Stromal Cells (MSCs) are susceptible to tumor signalling and can be recruited to tumor regions. The precise role of MSCs in tumor development is still under debate since both pro- and anti-tumorigenic effects have been reported. In this study we analysed the participation of renal CSC-derived EVs in the interaction between tumor and MSCs. We found that CSC-derived EVs promoted persistent phenotypical changes in MSCs characterized by an increased expression of genes associated with cell migration (CXCR4, CXCR7), matrix remodeling (COL4A3), angiogenesis and tumor growth (IL-8, Osteopontin and Myeloperoxidase). EV-stimulated MSCs exhibited in vitro an enhancement of migration toward the tumor conditioned medium. Moreover, EV-stimulated MSCs enhanced migration of renal tumor cells and induced vessel-like formation. In vivo, EV-stimulated MSCs supported tumor development and vascularization, when co-injected with renal tumor cells. In conclusion, CSC-derived EVs induced phenotypical changes in MSCs that are associated with tumor growth.

Title: Polymorphisms in EGFR and IL28B are associated with spontaneous clearance in an HCV-infected iranian population Content: Although most hepatitis C virus (HCV)-infected individuals develop chronic infection, about 25% of them are able to clear the virus spontaneously without any therapeutic intervention. The aim of the present study was to identify genes associated with spontaneous HCV clearance in a population of Iranian patients. We genotyped 110 single-nucleotide polymorphisms (SNPs) in 59 selected—candidate—genes in a cohort of 107 HCV-infected participants who spontaneously cleared the infection and 176 participants whose infection persisted. Three out of the 110 SNPs were found to be associated with HCV outcome (P-values<0.03). rs11506105 in EGFR (epidermal growth factor receptor gene), and rs11881222 and rs12979860 in IL28B (interferon-λ3 gene). Multivariate logistic regression of the three markers showed that the A/A genotypes in both rs11506105 (EFGR) and rs11881222 (IL28B), and the C/C genotype in rs12979860 (IL28B) are associated with HCV clearance (recessive model: odds ratio (OR)=2.06, 95% confidence interval (95% CI)=1.09–3.88, P=0.025; OR=2.09, 95% CI=1.23–3.60, P=0.007; and OR=1.95, 95% CI=1.15–3.35, P=0.014 for rs11506105, rs12979860 and rs11881222, respectively). In conclusion, EGFR and IL28B SNPs are strong independent predictive markers of spontaneous viral clearance.

Title: Circulating microRNAs as biomarkers for hepatocellular carcinoma. Content: GOALS We investigated whether measurement of serum levels of the microRNAs (miRNAs) miR-16, miR-195, and miR-199a, alone or in combination with conventional serum markers, can help to differentiate hepatocellular carcinoma (HCC) from chronic liver diseases (CLDs). BACKGROUND Recent reports suggest a link between aberrant expression of miRNA, and HCC. STUDY This retrospective analysis was conducted using sera from 105 HCC patients, 107 CLD patients, and 71 normal control subjects. The miRNAs were measured using real-time reverse transcription-polymerase chain reaction. The conventional HCC markers α-fetoprotein (AFP), lens culinaris agglutinin-reactive AFP (AFP-L3%), and des-γ-carboxyprothrombin (DCP) were measured with commercial kits. RESULTS Serum levels of miR-16 and miR-199a were significantly lower in HCC than in CLD patients or control subjects (P<0.01). As a single marker, miR-16 had the highest sensitivity for HCC, followed by miR-199a, AFP, DCP, AFP-L3%, and miR-195. The combination of miR-16, AFP, AFP-L3%, and DCP yielded the optimal combination of sensitivity (92.4%) and specificity (78.5%) for HCC, overall and when analysis was restricted to patients with tumors size smaller than 3 cm. As a second-line HCC marker, miR-16 yielded positive HCC predictions in 18 of the 26 (69.2%) HCC patients with negative results on all 3 conventional markers, most of whom had tumors size smaller than 3 cm; miR-16 was falsely positive in only 12 of 96 (12.5%) CLD patients. CONCLUSIONS The addition of miR-16 to conventional serum markers improved sensitivity and specificity for HCC. Use of miR-16 for second-line testing in cases considered negative on the basis of conventional HCC markers should be explored in larger, prospective studies.

Title: p53 mutation in breast cancer. Correlation with cell kinetics and cell of origin. Content: AIM Several studies have investigated the expression of the cytokeratins (CKs), vimentin, the epithelial growth factor receptor (EGFR), the oestrogen receptor (ER), and the progesterone receptor (PgR), in breast cancer, but no study has directly compared p53 mutations with these phenotypic and differentiation markers in the same case. The present study was designed to provide some of this information. METHODS The expression of the p53 and bcl-2 proteins was evaluated by immunohistochemistry in relation to phenotypic characteristics and cellular kinetic parameters (mitotic index and apoptotic index) in 37 cases of ductal carcinoma in situ (DCIS) and 27 cases of infiltrating ductal carcinoma (IDC) of the breast. In addition, p53 gene mutation was examined by polymerase chain reaction single strand conformation polymorphism analysis (SSCP). RESULTS Thirteen cases (eight DCIS and five IDC) showed expression of CK8, CK14, CK18, vimentin, and EGFR, consistent with a stem cell phenotype, whereas 44 cases (27 DCIS and 17 IDC) showed expression of CK8 and CK1, weak or negative expression of CK18, but were negative for vimentin and EGFR, consistent with a luminal cell phenotype. DCIS and IDC cases with a stem cell phenotype were ER/PgR negative and intermediately or poorly differentiated. In contrast, the cases with luminal cell phenotype were ER/PgR positive and well or intermediately differentiated. In addition, intermediately or poorly differentiated cases with a stem cell phenotype showed higher proliferative activity (per cent of MIB-l positive cells) than did intermediately or well differentiated cases with a luminal cell phenotype. Both DCIS and IDC cases with a stem cell phenotype were p53 positive and bcl-2 negative by immunohistochemistry. In IDC, p53 expression was associated with a reduction of both mitotic index and apoptotic index compared with DCIS. Most of the tumours showing a more differentiated phenotype (luminal) were p53 negative and bcl-2 positive. In these cases, cell kinetic parameters increased from DCIS to IDC. These data suggest the existence of subsets of DCIS and IDC that, because of their phenotypic characteristics, could be derived from subpopulations of normal breast cells having different control mechanisms of cell proliferation and neoplastic progression. CONCLUSIONS These results are compatible with the hypothesis that the phenotype of the cell of origin constrains both tumour phenotype and the choice of genetic events; however, the occurrence of p53 mutants by chance during neoplastic transformation cannot be excluded.

Title: The novel immunosuppressive enzyme IL4I1 is expressed by neoplastic cells of several B-cell lymphomas and by tumor-associated macrophages Content: We previously reported a strong IL4I1 gene expression in primary mediastinal B-cell lymphoma (PMBL) and recently identified the protein as a secreted L-phenylalanine oxidase, physiologically expressed by myeloid cells, which inhibits T-cell proliferation in vitro. Here, we analyzed the pattern of IL4I1 protein expression in 315 human lymphoid and non-lymphoid malignancies. Besides PMBL, IL4I1 expression in tumors was very frequent. IL4I1 was detected in tumor-associated macrophages from most of the tumors and in neoplastic cells from follicular lymphoma, classic and nodular lymphocyte predominant Hodgkin lymphomas and small lymphocytic lymphoma, three of which are germinal center derived. IL4I1-positive tumor cells were also detected in rare cases of solid cancers, mainly mesothelioma. The enzymatic activity paralleled protein expression, suggesting that IL4I1 is functional in vivo. Depending on the tumor type, IL4I1 may impact on different infiltrating lymphocyte populations with consequences on tumor evolution. In the particular case of follicular lymphoma cells, which are susceptible to antitumor cytotoxic T cells killing but depend on interactions with local T helper cells for survival, a high level of IL4I1 expression seems associated with the absence of bone marrow involvement and a better outcome. These findings plead for an evaluation of IL4I1 as a prognosis factor.

Title: Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma Content: Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.