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1.  Wiring miRNAs to pathways: a topological approach to integrate miRNA and mRNA expression profiles 
Nucleic Acids Research  2014;42(11):e96.
The production rate of gene expression data is nothing less than astounding. However, with the benefit of hindsight we can assert that, since we completely ignored the non-coding part of the transcriptome, we spent the last decade to study cell mechanisms having few data in our hands. In this scenario, microRNAs, which are key post-trascriptional regulators, deserve special attention. Given the state of knowledge about their biogenesis, mechanisms of action and the numerous experimentally validated target genes, miRNAs are also gradually appearing in the formal pathway representations such as KEGG and Reactome maps. However, the number of miRNAs annotated in pathway maps are very few and pathway analyses exploiting this new regulatory layer are still lacking. To fill these gaps, we present ‘micrographite’ a new pipeline to perform topological pathway analysis integrating gene and miRNA expression profiles. Here, micrographite is used to study and dissect the epithelial ovarian cancer gene and miRNA transcriptome defining and validating a new regulatory circuit related to ovarian cancer histotype specificity.
PMCID: PMC4066781  PMID: 24803669
2.  Multifactorial ERβ and NOTCH1 control of squamous differentiation and cancer 
The Journal of Clinical Investigation  2014;124(5):2260-2276.
Downmodulation or loss-of-function mutations of the gene encoding NOTCH1 are associated with dysfunctional squamous cell differentiation and development of squamous cell carcinoma (SCC) in skin and internal organs. While NOTCH1 receptor activation has been well characterized, little is known about how NOTCH1 gene transcription is regulated. Using bioinformatics and functional screening approaches, we identified several regulators of the NOTCH1 gene in keratinocytes, with the transcription factors DLX5 and EGR3 and estrogen receptor β (ERβ) directly controlling its expression in differentiation. DLX5 and ERG3 are required for RNA polymerase II (PolII) recruitment to the NOTCH1 locus, while ERβ controls NOTCH1 transcription through RNA PolII pause release. Expression of several identified NOTCH1 regulators, including ERβ, is frequently compromised in skin, head and neck, and lung SCCs and SCC-derived cell lines. Furthermore, a keratinocyte ERβ–dependent program of gene expression is subverted in SCCs from various body sites, and there are consistent differences in mutation and gene-expression signatures of head and neck and lung SCCs in female versus male patients. Experimentally increased ERβ expression or treatment with ERβ agonists inhibited proliferation of SCC cells and promoted NOTCH1 expression and squamous differentiation both in vitro and in mouse xenotransplants. Our data identify a link between transcriptional control of NOTCH1 expression and the estrogen response in keratinocytes, with implications for differentiation therapy of squamous cancer.
PMCID: PMC4002597  PMID: 24743148
3.  p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis 
p63 is up-regulated in melanoma and prevents nuclear accumulation of p53.
The role of apoptosis in melanoma pathogenesis and chemoresistance is poorly characterized. Mutations in TP53 occur infrequently, yet the TP53 apoptotic pathway is often abrogated. This may result from alterations in TP53 family members, including the TP53 homologue TP63. Here we demonstrate that TP63 has an antiapoptotic role in melanoma and is responsible for mediating chemoresistance. Although p63 was not expressed in primary melanocytes, up-regulation of p63 mRNA and protein was observed in melanoma cell lines and clinical samples, providing the first evidence of significant p63 expression in this lineage. Upon genotoxic stress, endogenous p63 isoforms were stabilized in both nuclear and mitochondrial subcellular compartments. Our data provide evidence of a physiological interaction between p63 with p53 whereby translocation of p63 to the mitochondria occurred through a codependent process with p53, whereas accumulation of p53 in the nucleus was prevented by p63. Using RNA interference technology, both isoforms of p63 (TA and ΔNp63) were demonstrated to confer chemoresistance, revealing a novel oncogenic role for p63 in melanoma cells. Furthermore, expression of p63 in both primary and metastatic melanoma clinical samples significantly correlated with melanoma-specific deaths in these patients. Ultimately, these observations provide a possible explanation for abrogation of the p53-mediated apoptotic pathway in melanoma, implicating novel approaches aimed at sensitizing melanoma to therapeutic agents.
PMCID: PMC3600906  PMID: 23420876
4.  Identification of two regions in the p140Cap adaptor protein that retain the ability to suppress tumor cell properties 
p140Cap is an adaptor protein that negatively controls tumor cell properties, by inhibiting in vivo tumor growth and metastasis formation. Our previous data demonstrated that p140Cap interferes with tumor growth and impairs invasive properties of cancer cells inactivating signaling pathways, such as the tyrosine kinase Src or E-cadherin/EGFR cross-talk. In breast cancer p140Cap expression inversely correlates with tumor malignancy. p140Cap is composed of several conserved domains that mediate association with specific partners. Here we focus our attention on two domains of p140Cap, the TER (Tyrosine Enriched Region) which includes several tyrosine residues, and the CT (Carboxy Terminal) which contains a proline rich sequence, involved in binding to SH2 and SH3 domains, respectively. By generating stable cell lines expressing these two proteins, we demonstrate that both TER and CT domains maintain the ability to associate the C-terminal Src kinase (Csk) and Src, to inhibit Src activation and Focal adhesion kinase (Fak) phosphorylation, and to impair in vitro and in vivo tumor cell features. In particular expression of TER and CT proteins in cancer cells inhibits in vitro and in vivo growth and directional migration at a similar extent of the full length p140Cap protein. Moreover, by selective point mutations and deletion we show that the ability of the modules to act as negative regulators of cell migration and proliferation mainly resides on the two tyrosines (Y) inserted in the EPLYA and EGLYA sequences in the TER module and in the second proline-rich stretch contained in the CT protein. Gene signature of cells expressing p140Cap, TER or CT lead to the identification of a common pattern of 105 down-regulated and 128 up-regulated genes, suggesting that the three proteins can act through shared pathways. Overall, this work highlights that the TER and CT regions of p140Cap can efficiently suppress tumor cell properties, opening the perspective that short, defined p140Cap regions can have therapeutic effects.
PMCID: PMC3696535  PMID: 23841028
p140Cap; breast cancer; lung cancer; colon cancer; cell signaling; Csk; Src
6.  Cross-Analysis of Gene and miRNA Genome-Wide Expression Profiles in Human Fibroblasts at Different Stages of Transformation 
We have developed a cellular system constituted of human telomerase immortalized fibroblasts that gradually underwent neoplastic transformation during propagation in culture. We exploited this cellular system to investigate gene and miRNA transcriptional programs in cells at different stages of propagation, representing five different phases along the road to transformation, from non-transformed cells up to tumorigenic and metastatic ones. Here we show that gene and miRNA expression profiles were both able to divide cells according to their transformation phase. We identified more than 1,700 genes whose expression was highly modulated in cells at at least one propagation stage and we found that the number of modulated genes progressively increased at successive stages of transformation. These genes identified processes significantly deregulated in tumorigenic cells, such as cell differentiation, cell movement and extracellular matrix remodeling, cell cycle and apoptosis, together with upregulation of several cancer testis antigens. Alterations in cell cycle, apoptosis, and cancer testis antigen expression were particular hallmarks of metastatic cells. A parallel deregulation of a panel of 43 miRNAs strictly connected to the p53 and c-Myc pathways and with oncogenic/oncosuppressive functions was also found. Our results indicate that cen3tel cells can be a useful model for human fibroblast neoplastic transformation, which appears characterized by complex and peculiar alterations involving both genetic and epigenetic reprogramming, whose elucidation could provide useful insights into regulatory networks underlying cancerogenesis.
PMCID: PMC3275799  PMID: 22321013
7.  Regulation of aromatase expression in breast cancer treated with anastrozole neoadjuvant therapy 
PMCID: PMC3570232  PMID: 23408108
breast neoplasms; aromatase inhibitors; genetic polymorphism; aromatase targeting microRNAs; intrinsic resistance
8.  Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues 
PLoS ONE  2012;7(9):e45105.
Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples.
Methodology/Principal Findings
We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport.
Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach.
PMCID: PMC3441572  PMID: 23028787
9.  Overexpression of CD157 Contributes to Epithelial Ovarian Cancer Progression by Promoting Mesenchymal Differentiation 
PLoS ONE  2012;7(8):e43649.
Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for improving patients’ survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to anoikis, increased anchorage-independent growth, cell motility and mesothelial invasion. Conversely, knockdown of CD157 in OV-90 and OC314 cells reverts the mesenchymal phenotype and reduces the cells’ migratory potential. Transcriptome profiling analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and response to stress). Together, these findings implicate CD157 in the progression of EOC to metastatic disease and suggest that CD157 may represent a valuable therapeutic target.
PMCID: PMC3423388  PMID: 22916288
PLoS ONE  2012;7(4):e35423.
PMCID: PMC3327679  PMID: 22523595
11.  Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer 
BMC Cancer  2011;11:31.
Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease.
Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples.
EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression.
Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.
PMCID: PMC3040720  PMID: 21266046
BMC Cancer  2010;10:631.
Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC.
Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs.
EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis.
These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.
PMCID: PMC3000850  PMID: 21087480
13.  Absence of the K303R estrogen receptor α mutation in breast cancer patients exhibiting different responses to aromatase inhibitor anastrozole neoadjuvant treatment 
Aromatase inhibitors, such as anastrozole, are established in the treatment of hormone-dependent breast cancer. However, approximately 20% of patients treated with anastrozole do not respond, and it remains impossible to accurately predict sensitivity. Thus, novel markers to predict response are required. The K303R estrogen receptor (ER)α mutation confers resistance to tamoxifen treatment. Moreover, K303R-expressing MCF-7 cells, transfected with an aromatase expression vector and stimulated with androstenedione (an aromatase substrate), were found to be resistant to the inhibitory effect of anastrozole. The aim of this study was to verify whether the presence of the K303R ERα mutation is associated with response to 3-month neoadjuvant treatment with anastrozole (Arimidex) in a cohort of post-menopausal breast cancer patients. Of 37 patients with ER+ tumors, 19 showed a clinical response to anastrozole and 18 were resistant. Biopsies were obtained from tumors responding to the therapy or from non-responding tumors. None carried the K303R ERα mutation. To our knowledge, this is the first study to search for K303R ERα mutations in tumors clinically responsive or resistant to an aromatase inhibitor. Lack of the mutation leads us to believe that this mutation has in vivo biological significance in only a subset of breast cancers.
PMCID: PMC3446720  PMID: 22993622
breast cancer; aromatase inhibitors; estrogen receptor mutation
14.  ETS Transcription Factors Control Transcription of EZH2 and Epigenetic Silencing of the Tumor Suppressor Gene Nkx3.1 in Prostate Cancer 
PLoS ONE  2010;5(5):e10547.
ETS transcription factors regulate important signaling pathways involved in cell differentiation and development in many tissues and have emerged as important players in prostate cancer. However, the biological impact of ETS factors in prostate tumorigenesis is still debated.
Methodology/Principal Findings
We performed an analysis of the ETS gene family using microarray data and real-time PCR in normal and tumor tissues along with functional studies in normal and cancer cell lines to understand the impact in prostate tumorigenesis and identify key targets of these transcription factors. We found frequent dysregulation of ETS genes with oncogenic (i.e., ERG and ESE1) and tumor suppressor (i.e., ESE3) properties in prostate tumors compared to normal prostate. Tumor subgroups (i.e., ERGhigh, ESE1high, ESE3low and NoETS tumors) were identified on the basis of their ETS expression status and showed distinct transcriptional and biological features. ERGhigh and ESE3low tumors had the most robust gene signatures with both distinct and overlapping features. Integrating genomic data with functional studies in multiple cell lines, we demonstrated that ERG and ESE3 controlled in opposite direction transcription of the Polycomb Group protein EZH2, a key gene in development, differentiation, stem cell biology and tumorigenesis. We further demonstrated that the prostate-specific tumor suppressor gene Nkx3.1 was controlled by ERG and ESE3 both directly and through induction of EZH2.
These findings provide new insights into the role of the ETS transcriptional network in prostate tumorigenesis and uncover previously unrecognized links between aberrant expression of ETS factors, deregulation of epigenetic effectors and silencing of tumor suppressor genes. The link between aberrant ETS activity and epigenetic gene silencing may be relevant for the clinical management of prostate cancer and design of new therapeutic strategies.
PMCID: PMC2866657  PMID: 20479932
15.  CD38/CD31 Interactions Activate Genetic Pathways Leading to Proliferation and Migration in Chronic Lymphocytic Leukemia Cells 
Molecular Medicine  2009;16(3-4):87-91.
Human CD38 is a pleiotropic glycoprotein belonging to a family of enzymes/receptors involved in the catabolism of extracellular nucleotides. CD38-receptor activities are regulated through binding to the nonsubstrate ligand CD31. CD38 expression above a critical threshold is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients. Activation of CD38 by means of agonistic monoclonal antibodies or the CD31 ligand induces proliferation and immunoblast differentiation of CLL cells. Here we define the genetic signature that follows long-term in vitro interactions between CD38+ CLL lymphocytes and CD31+ cells. The emerging profile confirms that the CD31/CD38 axis activates genetic programs relevant for proliferative responses. It also indicates a contribution of this pathway to the processes mediating migration and homing. These results further support the notion that the CD31/CD38 axis is part of a network of accessory signals that modify the microenvironment, favoring localization of leukemic cells to growth-permissive sites.
PMCID: PMC2785473  PMID: 19956559
16.  Identification of novel vascular markers through gene expression profiling of tumor-derived endothelium 
BMC Genomics  2008;9:201.
Targeting tumor angiogenesis and vasculature is a promising strategy for the inhibition of tumor growth and dissemination. Evidence suggests that tumor vasculature expresses unique markers that distinguish it from normal vasculature. Our efforts focused on the molecular characterization of endothelial cells (EC) in the search for selective markers of tumor vasculature that might be helpful for the development of effective therapeutic approaches.
We investigated by microarray analysis the gene expression profiles of EC purified and cultured from tumor (ovarian carcinoma [HOC-EC]) and normal (human adrenal gland [HA-EC]) tissue specimens. We found distinct transcriptional features characterizing the EC of different origin, and identified 158 transcripts highly expressed by HOC-EC. We analyzed four of these genes, ADAM23, FAP, GPNMB and PRSS3, which were not previously known to be expressed by endothelium. In vitro experiments confirmed the higher expression of the selected genes in tumor-derived endothelium with no expression in tumor cells. In vivo investigation by in situ hybridization established that ADAM23, GPNMB and PRSS3 expression is localized on blood vessels of human cancer specimens.
These findings elucidate some of the molecular features of the tumor endothelium. Comparative transcriptomic analysis allowed us to determine molecular differences of tumor and normal tissue-derived endothelium and to identify novel markers that might be exploited to selectively target tumor vasculature.
PMCID: PMC2410137  PMID: 18447899
17.  Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect 
The Journal of Clinical Investigation  2007;117(11):3236-3247.
Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors γ-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA–binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy.
PMCID: PMC2000812  PMID: 17932567

Results 1-17 (17)