This study has evaluated the diagnostic and prognostic value of hsa\_circ\_0007841 as a circRNA in MM and the results provide the basis for preclinical research. Currently, the factors of age and complications are mainly considered for individualized therapy of MM, However, to further improve treatment outcome, molecular information will be needed to guide treatment. The endogenous competitive mechanism of circRNAs provides the theoretical basis for research on the replacement of antinucleotide chemotherapy. However, studies on circRNAs are still at the preliminary stage and very little has been hitherto understood about their functions and working mechanisms. But there is a high potential for clinical translational research with the advancement of research and development of RNA-targeted drugs based on circRNAs.

#### DATA AVAILABILITY STATEMENT

This manuscript contains previously unpublished data. The name of the repository and accession number(s) are not available.

#### REFERENCES

#### ETHICS STATEMENT

The studies involving human participants were reviewed and approved by 2016121. The patients/participants provided their written informed consent to participate in this study.

#### AUTHOR CONTRIBUTIONS

MG and CL designed and performed the study. HD and LG wrote the manuscript with inputs from all authors. YF and SJ performed the analytic calculations and statistical analysis. All authors provided critical feedback and helped to shape the research, analysis, and manuscript. We thank HX for her contribution to the collection of data.

#### FUNDING

This study was supported by Hunan Provincial Natural Science Foundation of China (Grant No. 2017JJ3463).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.01261/full#supplementary-material

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Gao, Li, Xiao, Dong, Jiang, Fu and Gong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Canmei Formula Reduces Colitis-Associated Colorectal Carcinogenesis in Mice by Modulating the Composition of Gut Microbiota

#### Huayue Zhang1†, Dengcheng Hui 2†, Yuan Li 1 , Guangsu Xiong3 \* and Xiaoling Fu1 \*

*1 Department of Medical Oncology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China, 2 Department of Cirrhosis, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China, 3 Endoscopic Center, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China*

#### Edited by:

*Dong-Hua Yang, St. John's University, United States*

#### Reviewed by:

*Jianfeng Cai, University of South Florida, United States Guoxiang Cai, Fudan University Shanghai Cancer Center, China*

#### \*Correspondence:

*Xiaoling Fu<EMAIL_ADDRESS>Guangsu Xiong xiongguangsu@ shyueyanghospital.com*

*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology*

Received: *09 August 2019* Accepted: *16 October 2019* Published: *19 November 2019*

#### Citation:

*Zhang H, Hui D, Li Y, Xiong G and Fu X (2019) Canmei Formula Reduces Colitis-Associated Colorectal Carcinogenesis in Mice by Modulating the Composition of Gut Microbiota. Front. Oncol. 9:1149. doi: 10.3389/fonc.2019.01149* The gut microbiota, including pathogenic microorganisms and probiotics, has been involved in tumor initiation and progression by regulating the components of intestinal flora. Canmei formula (CMF), a traditional Chinese medicine, chronicled in the Chuang Yang Jing Yan Quan Shu, has been clinically used as an adjuvant therapy to treat patients with colorectal carcinoma (CRC) in China. In this study, we investigate the treatment effect of CMF in the azoxymethane (AOM) and dextran sodium sulfate (DSS) induced and high-fat diet augmented colitis-associated colorectal cancer *in vivo*, and explore its mechanism of action. We found that CMF treatment relieved the inflammation and alteration of the gut microbiota and significantly inhibited the development of intestinal adenoma. Linear discriminant analysis showed that the flora diversity in the normal mice, model mice and CMF treatment mice was different. At the family level, the relative abundance of Desulfovibrionaceae decreased in CMF groups. The relative abundance of Desulfovibrionaceae were lower in the CMF groups than in model group, whereas Rikenellaceae and Alistipes were increased. Altogether our results indicate that CMF treatment ameliorate colitis-associated colorectal carcinogenesis by modulating the composition of the gut microbiota *in vivo*.

Keywords: Canmei formula (CMF), traditional Chinese medicine, gut microbiota, AOM/DSS, colorectal carcinogenesis

# INTRODUCTION

Colorectal carcinoma (CRC) is the second leading cause of cancer-related death worldwide (1). There are about 1.4 million new CRC patients and 700,000 deaths each year (2). Colorectal adenoma (CRA) refers to any lesion that originates from the surface of colorectal mucosa and protrudes into the intestinal cavity. It is a precancerous lesion of CRC and has the characteristics of high recurrence and high incidence of cancerous change (3). Microscopic removal is currently the main treatment for CRA, which can effectively reduce the incidence of CRC and reduce mortality

**140**

by 67% (4). However, microscopic resection did not reduce the recurrence rate of CRA. Aspirin, celecoxib, and cyclooxygenase-2 inhibitors are used clinically to treat inflammatory bowel disease, but the efficacy is limited according to the large sample clinical data (5). To date, there is no therapeutic agent specific for this disease. It is critical to explore drugs that can safely and effectively prevent and treat the recurrence of CRA.

In China, traditional Chinese medicine (TCM) has a long history dating back several thousands of years ago. Results from both pre-clinical laboratory and human studies have indicated that TCM has been widely used in anticancer therapy. Previous studies have indicated that PHY906, a modified formulation derived from Huang-Qin-Tang, could ameliorate chemotherapyinduced GI tract toxicity (6, 7). Qingjie Fuzheng granules (QFGs) has been proven to be able to inhibit proliferation and induce apoptosis of CRC cells (8). These studies indicate that TCM play important roles in cancer treatment and could be expected to become a promising therapeutic candidate for tumor.

Over the past decade, CRC have emerged as one of the most studied human conditions link to gut microbiota. Studies indicate that changes in the composition of gut microbiota, one of the most important internal environmental factors, are closely associated with the progression of CRC (9, 10). At present, the study on the secretion of toxin-activated inflammation by the intestinal flora is the most in-depth study of CRA carcinogenesis (3). The changes of intestinal flora are positively correlated with colorectal cancer. The secretion of toxins by the bacteria is a key factor for activating inflammation and oxidative stress pathways (11, 12). A number of studies have confirmed that Enterococcus faecalis, Escherichia coli, Enterotoxin-producing Bacteroides fragilis (ETBF), Streptococcus bovis, Fusobacterium nucleatum, and Helicobacter pylori are the main species that can induce cancer (3, 12, 13). Among them, Fusarium nucleatum induces uncontrolled intestinal epithelial cell growth by activating βcatenin; E. coli produces colibactin with genotoxicity (3).

Canmei formula (CMF), a classical traditional Chinese herbal formulation with Titanium and Marci Hieronymi, is widely used to treat colorectal diseases. The active constituents in CMF, including Citric acid, DL-TYROSINE, L(-)-Carnitine, L-Tyrosine, Ambrosic acid and others, have been reported (14, 15). However, there is no study on the effect of CMF in modulating gut microbiota. In our previous studies, 16sDNA sequencing technique was used to compare the change of gut microbiota induced by CMF treatment in AOM/DSS and high-fat dietinduced CRC mice (16, 17). This study provides evidence of the modulations of gut microbiota through CMF treatment, which will help understanding the host-microbe interactions during the treatment of CRC.

#### MATERIALS AND METHODS

#### Preparation of the Extracts for CMF

Mume Sieb and Marci Hieronymi were purchased from Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine. Mume Sieb was the fruit of Prunus mume. The Marci Hieronymi is a dry body of the larvae of the camphor family, Bombyx mori linnaeus, 4 ∼ 5 years old, infected with Beauveria bassiana. They were formally identified by the department of pharmacy, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

CMF was formulated with Titanium and Marci Hieronymi in a ratio of 1:1. All the herbs were purchased from Yueyang hospital herbal pharmacy. Formulation was performed as described below. Briefly, 980 g mixture was extracted twice for 1.5 and 1 h for each time. The filtrates were concentrated in a vacuum at 60◦C and dried to obtain the CMF extract powder of 335.2 g, the rate of the extract was 32.1%.The extract obtained in this part is CMF-L/H. The same process was repeated twice for the CMF extract. Then the liquid was poured through the column of the macroporous resin, and then the resin column was rinsed with pure water, 20% ethanol and 90% ethanol. After alcohol precipitation, the filtrateswere concentrated in a vacuum at 60◦C and dried to obtain the CMF extract powder of 60.3 g, the rate of the purified product was 18%. The extract obtained in this part is used as CMF-A. The quality of preparations was controlled according to the guidelines from Chinese food and drug administration (CFDA).

#### Liquid Chromatograph and Mass Spectrometry

The solution obtained by the above preparation method was stored at 4◦C. For HPLC analysis, the solution was centrifuged at 12,000 rpm for 10 min, then the supernatant was filtered through a 0.22µm membrane before injection into the HPLC system. Analysis were performed by using Dionex UltiMate 3000 HPLC system (ThermoFisherScientific, USA) with a diode array detector. The C18 column (150 × 2.1 mm, 2.5µm) was used with a flow rate of 0.3 ml·min−1 . The injection volume was 5 µl, and the column temperature was maintained at 40◦C. The mobile phase was composed of (A) aqueous formic acid (0.1%, v/v) and (B) acetonitrile (0.1%, v/v) under following gradient elution: 95% A from 0 to 0.5 min, 95-2% A from 0.5 to 15 min, 2% A from 15 to 17 min, 2-95% A from 17 to 17.5 min, 95% A from 17.5 to 25 min. Mass spectrometry was performed on a Q Exactive high-resolution benchtop quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) using a heated electrospray ionization (HESI) source for ionization of the target compounds in positive and negative ion modes. The key parameters were as follows: ionization voltage, +3.5 kV/−3.2 Kv; sheath gas pressure, 40 arbitrary units; auxiliary gas, 10 arbitrary units; heat temperature, 350◦C. For the compounds of interest, a scan range of m/z 120–1,500 was chosen. Resolution for higher energy collisional dissociation cell (HCD) spectra was set to 17,500 at m/z 120 on the QExactive.

#### Animal Experiments and Grouping

The animal procedures and care in this study was approved by the IRB of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine. Seven-week-old female C57/BL6 mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. After arrival, the animals were quarantined for the first 7 days. Under controlled conditions: humidity 50 + 10%, 12/12 h light/dark cycle, and temperature ∼23◦C, all animals were housed in plastic cages (6 mice/cage)and provided with free drinking water and basic diet. AOM, the colonic carcinogen, was purchased from Sigma-Aldrich, USA. DSS with a molecular weight of 36,000–50,000 (No.216011080) was purchased from MPBIO Company, USA. For the induction of tumorigenesis, mice were injected with AOM (12 mg/kg body weight) intraperitoneally. Starting 5 day after the injection, animals received 2.5% DSS in the drinking water for 5 days. At the same time, the mice were given a high-fat diet. Then mice were given normal drinking water and normal diet for 2 weeks and subjected to two more DSS treatment cycles. Subsequently, mice were randomized into experimental and control groups. Group 1, NC, n = 8. Experimental groups included 5 groups: group 2: treatment with AOM and DSS (MC, n = 8); group 3: treatment with AOM, DSS, and Aspirin (Aspirin, n = 8); group 4: treatment with AOM, DSS, and CMF ethanol extract (CMF-A, n = 8); group 5: treatment with AOM, DSS, and CMF aqueous extract (CMF-L, n = 8); group 6: treatment with AOM, DSS, and CMF water extract high dose group(CMF-H, n = 8). Aspirin was given every day at the dose of 1.4 mg/kg/d. CMF-L and CMF-H were given at the doses of 3.65 and 7.3 g/kg/d, respectively. CMF-A was given at the doses of 0.657 g/kg/d. In the clinical practice of Chinese herbal medicine, the prescription dose of CMF is usually 60 g of herbal materials per day. When this human dose was converted into an animal dose (a person of 60 kg, and a conversion factor of 9.1 between human and mouse), it was equivalent to the low dose (3.65 g/kg/d) used in this study. All animals were sacrificed at the 18th week after the corresponding drug was administrated.

#### Colon Tissue Collection and Processing

After mice were sacrificed, the colorectal adenoma tissues were rapidly isolated, freed of adherent tissues and rinsed with phosphate buffer saline. Subsequently, tumor biopsy specimens of 1 cm-length were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin and sectioned into 4-µm-thick slices. Slices were hydrated and stained with hematoxylin and eosin (H&E) followed by microscopic examination. Then portions of adenoma tissues were homogenized (10% W/V) in (20 mM Tris-HCI containing 1 mM EDTA, PH 7.4) by a glass homogenizer. The homogenates were centrifuged at 3,000 × g for 20 min at 4◦C and supernatants were stored at −80◦C.

#### Real Time Quantitative Reverse Transcription PCR

According to the modified methods as described, the RNA extracts of the colorectal tissues were prepared (8). By normalizing target mRNA Ct values to those for GAPDH (Ct), the relative amount of target mRNA was determined by the comparative threshold (Ct) method. The primer sequences used were as follows:

MUS-NF-κB-F (bp1297): (ATGCACCGTAACAGCAGGAC); MUS-NF-κB-R (bp1405C): (TGTCATCCGTGCTTCCAGTG); MUS-IL-17C-F (bp269) (AGGAGGTGCTGGAAGCTGAC); MUS-IL-17C-R (bp391C) (TGCATCCACGACACAAGCAT);

GAPDH-F (5′ -GTGAGGCCGGTGCTGAGTAT-3′ ); GAPDH-R (5′ -GTGCAGGATGCATTGCTGAC-3′ );

# ELISA Assay

Concentration of IL-17C in serum and NF-κB in supernatants of colonic tissues were quantified by IL-17C assay kit and NFκB assay kit (Shanghai Pusheng Biological Technology Co., Ltd. China) according to manufacturer's instruction.

#### Fecal DNA Extraction and Illumina Miseq Sequencing

Genomic DNA was extracted from every stool sample using the FastDNA Spin Kit for Soil (MP Biomedical, LLC, catalog 116560- 200) following the manufacturer's instructions.V3 and V4 region of the 16s rDNA sequencing was amplified by Primer F (5′ -AAC GGGAAGACAACGTACGG-3′ ) and Primer R (5′ -CAGATG CAGGAGGACATGTC-3′ ) with barcode sequence. Library was constructed following the manufacturer's instructions of the Ion Plus Fragment Library Kit and sequenced by Ion S5 Sequencer.

#### Bioinformatics Analysis

Reads was filtered and chimera sequence was removed.

The read pairs were de-multiplexed based on their unique molecular barcodes and overlapping reads were merged using USEARCH v7.0.1001 software. Operational taxonomic unit (OTU) picking was conducted using the QIIME (Quantitative Insights Into Microbial Ecology) software package. By using UCLUST, 16S rRNA gene sequences were clustered at a similarity cutoff value of 97%. By the SILVA reference database (https://www.arb-silva.de/download/), matching of OTUs to bacteria was conducted. By mapping the demultiplexed reads to the identified OTUs, abundances were recovered. Alpha andbeta diversity plots were also generated using QIIME. The non-weighted UniFrac approach was used to measure the beta diversity between five bacterial cave communities. The relative abundance of transporter genes was predicted and performed by picrust analysis. Independent of the taxonomic analysis, aclosed-reference OTU picking protocol (QIIME) and the Greengenes database (http://greengenes.lbl.gov) were used to pre-cluster at 97% identity, and 97% of the OTUs were picked. The obtained OTU table was normalized by 16S rRNA copy number, and the functional genes were predicted according to the Kyoto Encyclopedia ofGenes and Genomes (KEGG) catalog. By using SPSS version 15.0 statistical software (IBM, Armonk, NY, USA), Fisher's test and the Mann-Whitney U testwere used to analyze, respectively, the gender distribution of subjects and intergroup differences at the genus level in each subcluster. The linear discriminant analysis (LDA) effectsize (LEfSe) method with default settings was used to analyze the differences among groups in phyla, class, order, family, and genera levels.

#### Statistical Analysis

SPSS software version 15.0 was used for statistical analysis. The data were presented as mean ± SD. Unpaired students' t-test was used to compare the means of the two groups. One-way analysis of variance and Adonis were used to compare the means of more than two groups. A level of P < 0.05 was considered as statistically significant.

#### RESULTS

#### Identification of the Main Components in the Extract of CMF

To identify the components of Mume Sieb and Marci Hieronymi extract, Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS was performed. The main components in the extract of CMF were identified according to the elemental composition data determined from accurate mass measurements and comparison with the literature data. Fortyone compounds were identified in the CMF extract and demonstrated in **Table 1**. In addition, to exclude the possibility of manufacturing problems relating to procession, extraction, handling, and storage, two batches of CMF were evaluated by HPLC. We found that the magnitude, number, and retention time of the peaks were similar between the two batches (**Figure 1**). These results confirmed the stability of the CMF extraction process.

TABLE 1 | Major chemical constituents identified in CMF.

# CMF Attenuated AOM/DSS-Induced Colitis-Associated Tumorigenesis

To evaluate the protective effect of CMF on the development of colitis-associated cancer, the well-established AOM/DSS and high-fat diet-induced colitis-associated colorectal tumor model in C57BL/6 mice were used (**Figure 2A**). Initially, the toxicity of CMF was tested in vivo. We did not find weight loss or behavioral changes in all mice. These results indicate that each of those extracts of CMF were well-tolerated (**Figure 2B**). AOM/DSS colorectal cancer mice were induced as described. All mice were treated with CMF once a day. After CMF treatment, tumor development and growth was inhibited in AOM/DSS colorectal cancer mice (**Figures 2C–F**). As a control, aspirin treatment effectively inhibited the occurrence of intestinal adenomas of 3 mm and above, but had no inhibitory effect on the occurrence of adenomas smaller than 3 mm (**Figure 2C**). Different extracts of CMF can significantly inhibit the growth of intestinal glands. **Figures 2D–F** shows that the ethanol extract of CMF significantly inhibited the occurrence of intestinal adenoma, and there is no difference in the occurrence of adenoma in the colon and small intestine. Histological examination revealed that mucosal inflammation, aberrant crypt foci adenoma, ulcer, or dysplasia in the colon tissues of the model group. Histological studies indicated that there was a large adenocarcinoma inside mucosa with several abnormal cells exhibiting cylindrical shape, large nuclei, increasing nuclear/cytoplasmic (N/C) ratio and cellular cleavagein AOM/DSS group (**Figure 2G**). CMF remarkably relieved the condition. These results suggest that CMF treatment inhibits inflammationassociated carcinogenesis in the AOM/DSS and high-fat diet mice.

#### CMF Treatment Repressed NF-κB and IL-17C Signaling in AOM/DSS Induced Mice

As NF-κB and IL-17C play important roles in the development of CRC, the mRNA expression of IL-17C and NF-κB collected from CRC mice were detected by Quantitative real-time PCR (**Figures 3A,B**). IL-17C was upregulated in the colorectal tissues of AOM/DSS mice. However, treatment with different extracts of CMF, including water extract and alcohol extract, effectively suppressed the mRNA expression of IL-17C. Drugs treatment, including aspirin, induced the mRNA expression of NF-κB. ELISA assay was performed to detect the protein expressions of inflammatory factors. As it is illustrated in **Figure 3C**, the protein levels of IL-17C in the serum of AOM/DSS-induced mice were significantly highest among mice with other drug intervention. The level of NF-κB significantly reduced in the tumor tissues of CMF-A, CMF-H, CMF-L, and Aspirin groups when compared to MC group (**Figure 3D**). All these results indicate that CMF treatment represses the expression of NF-κB and IL-17C.

# CMF Treatment Regulated Gut Microbiota

To examine the effect of CMF on the regulation of components of gut microbiota, high-throughput pyrosequencing was performed with an Illumine MiSeq platform to generate 39584 high quality and valid sequences from 16 samples from different groups. We conducted a bar-coded pyrosequencing run to analyze the changes of gut microbiota in the five studied groups. In total, 39,584 available reads and 1461 OUTs were obtained. Shannon diversity and rarefaction curves are shown in **Figures 4A,B**. The rarefaction curves were consistent with the current sequencing, demonstrating that most of the flora diversity has been captured in all samples. What's more, the overlap of OUTs between groups revealed that 482 OUTs coexisted in both the MC group and CMF-A group, 496 OUTs coexisted in both the NC group and CMF-A group, 514 OUTs coexisted in both the NC group and MC group and 467 OUTs coexisted in both the MC group and Aspirin group (**Figures 4C,D**). In addition, we examined the flora community richness and diversity in mice. No statistical difference was observed for the ACE, Chao, Shannon, and Simpson indices among these groups (**Figures 4E–H**), although Simpson index were lower, but other indices are higher in NC group and the different CMF extract groups compared with MC group. Nonmetric multidimensional scaling (NMDS) analysis indicated the changes of overall structure of gut microbiota among different groups (**Figure 4I**). These results showed that the

mean ± SD of mice in each group.

diversity of bacterial population of the NC group was higher than that of the MC group. There was a statistical difference between the CMF-H group and the MC group. These results suggested that CMF treatment increases the diversity of the intestinal flora.

# Analysis of the Relative Abundance of Microbiota at the Family Level and Genus Level

The family muribaculaceae, bacteroidales, and prevotellaceae were the most prevalent taxa in these groups (**Figure 5A**).

It was found that these bacteria are dominant bacteria at the genus level (**Figure 5B**). Intraindividual changes were detected in these groups. Compared with the model group, the abundance of a small number of bacteria increased or decreased to varying degrees, and most of the bacteria remain unchanged. At the family level, abundance of Desulfovibrionaceae decreased in groups that were treated with CMF. Conversely, a transient CMF-treatment increase several family microbiota, including Rikenellaceae, Erysipelotrichaceae, Lactobacillaceae, Streptococcaceae, and Tannerellaceae. At the genus level, the abundance of genus Alistipes, Faecalibaculum, Lactobacillus, and Parabacterioides, were up-regulated after the treatment of CMF. The genus Parasutterella was found down-regulated in CMF treatment groups. It is worth mentioning that genus Desulfovibrionaceae\_uncultured acted to AOM/DSS-mediated gut microbiota dysbiosis had statistical difference (**Figure 5F**). There arechanges in the

gut microbiota but there is no statistical difference in these changes (**Figures 5C–E**).

To systematically examine the alteration of taxa abundance according to CMF treatment, we conducted LEfSe (LDA Effect Size). The microbial cladogram indicates that the gut microbiome significantly altered in Bacteroidaceae and Staphylococcaceae, ranging from family to genus level (**Figures 6A,C**). At the genus level, the relative abundance of Turicibacter, Bacteroides, Bacteroidaceae, Faecalibaculum, Erysipelatoclostridium, and Staphylococcus were enriched in mice treated with the CMF-A diet (**Figures 6B,D**).

#### DISCUSSION

Traditional Chinese medicine (TCM) have been used for the treatment of diseases and health conditions for several thousands of years. In the treatment of CRC, the application of TCM has

Frontiers in Oncology | www.frontiersin.org

received worldwide attention. Many studies have demonstrated the anti-tumor effect of TCM in CRC therapy. For example, Wei et al. showed that JianPi JieDu decoction (JPJD) is able to inhibit tumorigenesis, metastasis, as well as angiogenesis of tumors through the mTOR/HIF-1α/VEGF pathway (18). Wu et al. demonstrated that Gegen Qinlian decoction (GGT) can ameliorate gut-toxicity through anti-inflammatory pathways, inhibition of neutrophil migration, anti-oxidative stress via the Nrf2/Keap-1 pathway in the colon (19). Wing lam et al. reported that PHY906 reduced the CPT-11-induced gastrointestinal toxicity by inhibiting multiple steps of inflammation and the promotion of intestinal progenitor cell repopulation (20). Extensive studies have revealed that TCM antitumor drugs work by inhibiting tumor proliferation, promoting tumor apoptosis, and affecting diverse molecular targets (21–23).

The alterations of cancer genetic and molecular targets in CRC treatment have been extensively reported. Recent studies have reported that the regulation of gut microbiota may affect carcinogenesis (24–28). Intestinal flora is a direct factor of intestinal cancer. In this study, we found that CMF can ameliorate colitis-associated colorectal carcinogenesis in mice by modulating the fecal microbiota. The gut microbiota, including Fusobacterium (F.) nucleatum (29) and Firmicutes, Bacteroidetes Ley et al. (30), plays an important role in human health. In addition, many factors will affect and destroy the balance of intestinal microbiota, including the invasion of antigens, activation of immune cells, and production of cytokines. Therefore, investigating the relationship between CMF and the gut microbiota in AOM/DSS-induced colitis in miceis critical.

In our study, the anti-inflammatory role of CMF in the in vivo model of colitis-associated colorectal carcinogenesis induced by AOM/DSS and high-fat diet in C57BL/6 micewere investigated. Two different solvent extraction components of CMF were used to treat these mice to evaluate its therapeutic effect. The results showed that each of these extracts of CMF were welltolerated. Histopathology findings were consistent with previous experimental results (31). The CRC mice treated with AOM/DSS and high-fat diet presented more severe condition than CMFtreated mice. In contrast, administration of CMF decreased tumor number and tumor size. Especially the ethanol extract of CMF can effectively inhibit the occurrence of colitis-associated colorectal carcinogenesis in the colon and small intestine.

Previous studies showed that tumor-prone mice colonized with E. coli and B. fragilis and increased IL-17 expression that promotes colon tumorigenesis in AOM mice (3, 32). It was reported that spontaneous activation of NF-κB was detected in tissues of human colorectal cancer (31, 33).We found that CMF could decrease the expression level of IL-17C and inhibit the activity of NF-κB.

High-throughput sequencing determined microbiological composition in mice. Most of the diversity of microorganisms was captured in all samples. The ACE, Chao, Simpson, and Shannon indices reveal that the microbial diversity in control mice and CMF treated mice were greater than in the model mice (16). The microflora analysis show that there are significant differences between the control mice and the model mice. There was a statistical difference between the ethanol extract of CMF treated mice and the model mice. This result indicated that the ethanol extract of CMF could increase the diversity of the intestinal flora. We evaluated phylum, class, order, family, and genus, the gut microbiota community in all samples. From the experiment, Muribaculaceae, Bacteroidales, and Prevotellaceae were the dominant family found in the gut. In accordance with family criteria, these flora occupied a main tier in the genus level. Focus on certain bacteria, statistical results reveal that CMF reversed the gut microbiota dysbiosis in CRC mice, including inhibiting the growth of Desulfovibrionaceae and promoting the Rikenellaceae. Desulfovibrionaceae, a kind of sulfate-reducing and endotoxin-producing bacteria, were mostly enriched in mice with long term high-fat feeding. Previous studies found that the family Desulfovibrionacea is positively associated with obesity (9, 34–36). This phenomenon was also found in our study. Wu et al. found that relative abundances of Rikenellaceae increased in the AOM/DSS mice (32). This result was in consistent with our study. We speculate that Rikenellaceae might be a bacterium that could cause cancer.

Because the results of our study showed that the alcohol extract of CMF is more effective than that of the water extract, we analyzed the difference of flora of the mice treated with the alcohol extract of CMF and the model mice. The results showed that the gut microbiome significantly altered in Bacteroidaceae and Staphylococcaceae, ranging from family to genus level. As shown in our results, at genus level, the relative abundance of Turicibacter, Bacteroides, Faecalibaculum, Erysipelatoclostridium,

# REFERENCES

and Staphylococcus were enriched in mice treated with the CMF-A diet. In a previous study, the abundance of Bacteroidaceae had an important influence in the active progression of colitis in DSS-treated mice (16), which was in contrast to our study. Liu et al. found that the abundance of Turicibacter decreased in obesity mice (37, 38). Similarly, the Turicibacter was also reduced in AOM/DSS and high-fat diet mice in this study. Therefore, we suspect that Turicibacter, Bacteroides, Faecalibaculum, Erysipelatoclostridium, and Staphylococcus were the beneficial bacteria in the gut. But the specific role of these gut microbiota needs to be further investigated.

In summary, we report that CMF, a two-herb formulation, has reduced the incidence of CRC by inhibiting inflammatory factors and regulating gut microbiota. In addition, the mechanism of action of CMF is through the direct action on inflammatory factors or the indirect effect on regulating gut flora. Understanding the role of gut microbiota would be helpful for the prevention of CRC development.

# DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/supplementary material.

#### ETHICS STATEMENT

The animal study was reviewed and approved by IRB of Yueyang Hospital Affiliated with Shanghai University of Traditional Chinese Medicine. Written informed consent was obtained from the owners for the participation of their animals in this study.

#### AUTHOR CONTRIBUTIONS

XF, HZ, and GX contributed to study design, data interpretation, and manuscript preparation. YL contributed to animal rearing. DH contributed to data acquisition and analysis.

#### FUNDING

This work was supported by the National Natural Science Foundation of China (81403360). The funders play no role in data collection and analysis, design, decision to publish, or preparation of the manuscript.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zhang, Hui, Li, Xiong and Fu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Survivin Promotes Piperlongumine Resistance in Ovarian Cancer

Xing-Wei Nan1†, Li-Hua Gong1†, Xu Chen1†, Hai-Hong Zhou1 , Piao-Piao Ye1 , Yang Yang2 , Zi-Hao Xing2 , Meng-Ning Wei 2 , Yao Li 2 , Sheng-Te Wang2 , Kun Liu2 , Zhi Shi 2 \* and Xiao-Jian Yan1,3 \*

*1 Department of Gynecology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, 2 Guangdong Provincial Key Laboratory of Bioengineering Medicine, Department of Cell Biology and Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, College of Life Science and Technology, Jinan University, Guangzhou, China, 3 Center for Uterine Cancer Diagnosis & Therapy Research of Zhejiang Province, Women's Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China*

#### Edited by:

*Chang Zou, Shenzhen People's Hospital, China*

#### Reviewed by:

*Qing Wang, Sun Yat-sen University, China Shuangping Liu, Dalian University, China*

#### \*Correspondence:

*Zhi Shi<EMAIL_ADDRESS>Xiao-Jian Yan<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology*

Received: *27 September 2019* Accepted: *15 November 2019* Published: *29 November 2019*

#### Citation:

*Nan X-W, Gong L-H, Chen X, Zhou H-H, Ye P-P, Yang Y, Xing Z-H, Wei M-N, Li Y, Wang S-T, Liu K, Shi Z and Yan X-J (2019) Survivin Promotes Piperlongumine Resistance in Ovarian Cancer. Front. Oncol. 9:1345. doi: 10.3389/fonc.2019.01345* Ovarian cancer is one of the most fatal female malignancies while targeting apoptosis is critical for improving ovarian cancer patients' lives. Survivin is regarded as the most robust anti-apoptosis protein, and its overexpression in ovarian cancer is related to poor survival and apoptosis resistance. Piperlongumine (PL) extracted from peppers is defined as an active alkaloid/amide and exhibits a broad spectrum of antitumor effects. Here, we demonstrate that PL induces the rapid depletion of survivin protein levels via reactive oxygen species (ROS)-mediated proteasome-dependent pathway *in vitro*, while exerting a remarkable inhibitory influence on the proliferation of ovarian cancer cells. Overexpression of survivin raises the survival rate of ovarian cancer cells to PL. Moreover, PL inhibits ovarian cancer cells xenograft tumor growth and downregulates survivin *in vivo*. Our findings reveal a previously unrecognized mechanism of PL in suppressing survivin expression as well as survivin promotes piperlongumine resistance in ovarian cancer and suggest that ROS-mediated proteasome-dependent pathway can be exploited to overcome apoptosis resistance triggered by aberrant expression of survivin.

 It was evaluated that 22,240 ovarian cancer cases would be diagnosed while concurrently 14,070 women would succumb to the disease in 2018 (2). Between 1976 and 2015, owing to effective approaches to the clinical treatment, including aggressive cytoreductive surgery followed by system chemotherapy, the mortality rate of ovarian cancer declined by 33% (2). However, despite the markable achievement of ovarian cancer during the last decade, the prognosis for women with advanced disease remains poor and chemoresistance appears to be a significant obstacle constraining the clinical application (3). Progress in curbing the incidence and mortality of human ovarian cancer can be propelled by the development of novel agents.

Survivin, one of the inhibitor of apoptosis proteins family (IAP) firstly found in 1997, may accelerate the progress of cancer via promoting the insurgence of mutations as well as inducing resistance to chemotherapy (4). While undetectable in terminally differentiated adult tissues, the survivin gene has been proved to be overexpressed in lots of cancers including ovarian cancer (5) in which survivin overexpression is detected in 74% of case and is related to the advanced clinical stage (6). Furthermore, our previous studies showed that the cytoplasmic protein expression level of survivin was an independent prognosis marker for ovarian cancer, downregulating survivin expression could enhance apoptosis (7, 8). Thus, survivin could be a promising target for apoptosis-based treatment in ovarian cancer therapy (9).

Piperlongumine (PL) extracted from the long pepper (Piperlongum L.) belongs to a biological alkaloid, embodying potential selective cytotoxic and antitumor properties over cancer cells of several histo-types including ovarian cancer (10). Our previous studies firstly demonstrated that PL facilitated G2/M cell cycle arrest, impeded the proliferation of human ovarian cancer cells, and induced reactive oxygen species (ROS) mediated apoptosis (11). Our previous cell-based experiments (11) and data published (12) indicated that the effect of PL to suppress the growth of tumors without general toxicity and to perturb redox and ROS homeostasis verify the PL treatment as a potential therapeutic opportunity against ovarian cancer.

However, the detailed molecular mechanism of the PL-mediated antitumor effect is not well-understood and still requires further investigation. Here, we firstly demonstrate that PL depleted the expression of survivin protein via a ROSmediated proteasome-dependent pathway in ovarian cancer cells. We further evaluate the efficacy of PL to inhibit tumor growth in vivo as a potentially effective therapy for ovarian cancer treatment.

#### MATERIALS AND METHODS

#### Cell Culture

The cancer cell lines A2780, OVCAR-3, and HEK293T were cultured in DMEM (Gibco, NY, USA). Ten percent fetal bovine serum (Gibco, NY, USA) and antibiotics (10 mg/mL streptomycin and 10,000 U/mL penicillin) were additionally supplemented. The aforementioned cell lines were cultivated in a 37◦C incubator at 5% CO2.

#### Chemicals and Antibodies

PL, N-Acetyl-cysteine (NAC) and Chloroquine (CQ) were obtained from Sigma Chemical Co (St. Louis, MO, USA). MG132 was from ApexBio. The HRP secondary antibodies, anti-survivin (#2808), and anti-PARP (#9542) were obtained from Cell Signaling Technologies (Danvers, MA, USA). Anti-Vinculin (BM1611) antibody was obtained from Boster, China. Anti-β-actin (KM9001), and anti-GAPDH (KM9002) antibodies were obtained from Tianjin Sungene Biotech Co., China. MG132, CQ, and NAC were added 1 h before PL treatment in all cotreated experimented.

#### Western Blot

Cells were lysed in RIRA buffer at 4◦C for around 30 min followed by centrifugation at 13,200 × rpm for 10 min to remove nuclei and other cell debris. Total protein concentration was detected by the Micro BCA Protein Assay Kit (Sangon Biotech, C503061) and the lysates were either used immediately or stored at −80◦C. 10–12% SDS-PAGE gels were used to separate the proteins extracted before and thereby separated proteins were completely transferred to polyvinylidene difluoride (PVDF) membranes. Five percent BSA was used to block the membranes for 1 h and the indicated primary antibodies were added and incubated overnight. Membranes were probed with the chemiluminescent detection reagents, and reactive bands were visualized using UVP ChemStudio PLUS (Analytikjena) (13, 14).

#### RT-PCR

Total RNA was extracted from 5 × 106 to 5 × 107 cells using the RaPure Total RNA Mini Kit (Magen, #R4011-02), treated with DNAseI to eliminate genomic DNA, and quantitated using the Epoch spectrophotometer. Reverse transcription (RT) followed instructions provided by StarScript II First-strand cDNA Synthesis Kit-II (GenStar, #A214-05). The resulting cDNA was used as a template for the amplification of target gene transcripts by RT-PCR, using HieffTM qPCR SYBR R Green Master Mix (YEASEN, #11201ES08) on the Hema9600 PCR machine. After 35 amplification cycles, reaction products were analyzed and β-actin RNA was used as a loading control. The primer sequences were as follows: survivin forward: CCGAC GTTGCCCCCTGC; survivin reverse: TCGATGGCACGGC GCAC; β-actin forward: AAATCGTGCGTGACATTAAGC; β-actin reverse: CCGATCCACACGGAGTACTT (15, 16).

#### Lentivirus Production and Infection

pDONR201-survivin was inserted into pCDH-Neo-Venus/DEST via LR clonase (Invitrogen, #11791) to form pCDH-Neo-Venussurvivin plasmid. At a 4:3:1 ratio, the lentiviral transfer vector, packaging plasmids psPAX2 and pMD.2G were transferred to the HEK293T cells to produced lentivirus. The PEI reagent was performed for transfection. Then, the viral supernatant was, respectively, collected 24, 48, 72, 96 h following transfection, filtered through a 0.20µm filter, and concentrated. Using the polybrene (Solarbio, H8761), A2780, OVCAR-3 cells were transfected with pCDH-Neo-Venus-survivin or pCDH-Neo-Venus/DEST, followed by incubation with 48 h for the subsequent trials (17, 18).

### Cell Viability Assay

It was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Briefly, ovarian cancer cells were seeded in 96-well plates, 4,500 cells per well, supplementing with 100 µL of medium and exposed to PL at different concentrations for 72 h, followed by the addition of 10 µL of MTT solution (5 mg/mL) per well to the medium and incubation 4 h at 37◦C. After we discarded the medium, 50 µL of DMSO per well was used to elute the blue MTTformazan product, and absorbance of the solution was read at 570 nm (19, 20).

#### Nude Mice Xenograft Assay

Balb/c nude female mice with 4–5 weeks of age and 20–22 g weight were purchased from the Guangdong Medical Laboratory Animal Center. For in vivo experiments, 4 × 106 A2780 cells in 100 µL of medium were injected subcutaneously into the left and right shoulders of each mouse. After the subcutaneous tumors reached a size of 0.3 × 0.3 cm2 , mice were randomized to treatment with PL (20 mg/kg) intraperitoneally or 0.5% methylcellulose alone every day. In the end, the tumors were got rid of mice and weighed after they were sacrificed. The bodyweight and the tumor volume (V) of mice were recorded every day. Formula: V = π/6 (1/2 (A + B))3 , the rate of inhibition (IR) = 1 – Mean tumor weight of experiment group/Mean tumor weight of control group × 100% (21, 22).

#### Statistical Analysis

Statistical significance of differences was determined by student's t-test. Differences were defined as significant at P < 0.05.

# RESULTS

#### PL Downregulates the Expression of Survivin in Ovarian Cancer Cells

Our previous study firstly demonstrated that PL facilitated G2/M cell cycle arrest, impeded the proliferation of human ovarian cancer cells, and induced ROS dependent apoptosis in ovarian cancer (11). Due to the ability to inhibit the cell apoptosis, survivin may take part in various processes associated with the tumor development and progression, facilitated metastasis and angiogenesis, as well as favor cell cycle progression (9). Because

of the apoptotic effect of PL induced in ovarian cancer cells, we explored the negative regulation of PL on survivin expression. As shown in **Figures 2A,C**, A2780 and OVCAR-3 cells were exposed to increasing concentrations of PL (up to 20µM) for 6 h, and the survivin protein was detected by western blot assay. PL effectively depleted the survivin expression in both A2780 and OVCAR-3 cells at the low micromolar concentration (10 or 20µM) in contrast to the control cells. Furthermore, as demonstrated in **Figures 1B,D**, downregulation of survivin was significant as soon as 6 h following PL treatment. Together, these evidences manifested that PL induces the reduction of survivin ovarian cancer cells in vitro, which may contribute to its apoptotic effect.

# PL Does Not Alter mRNA Expression of Survivin

To deliberate the discipline of PL-mediated survivin reduction, our group detected whether PL regulates the survivin mRNA expression in ovarian cancer cells. A2780 and OVCAR-3 cells were exposed to increasing concentrations of PL for 6 h or treated for different time scales at the same concentration of PL (10 or 20µM), and the expression levels of survivin mRNA were examined by RT-PCR. In **Figures 2A–D**, the survivin mRNA expression levels were not altered both on concentration and time scales, which were not similar to the depletion of survivin protein levels. These findings strongly supported the notion

that the PL regulates survivin gene expression at the nontranscriptional level.

# PL Induces Proteasome-Dependently Degradation of Survivin

The aforementioned findings indicated that PL might induce depletion of survivin at the post-transcription level. The possible discipline expounding PL-induced decrease of survivin protein expression may associate with its rapidly increased ubiquitinproteasome degradation. In order to verify this supposition, our group preincubated A2780 and OVCAR-3 cells with proteasome inhibitors MG132 (1µM). The results presented in **Figures 3A,C** demonstrated that MG132 reversed PL-mediated survivin depletion compared with control cells, hinting that PL-mediated degradation of survivin occurs via ubiquitinproteasome mechanism.

Otherwise, the autophagy-lysosome system and the ubiquitinproteasome system are the essential intracellular proteolytic pathways in eukaryotes (23). To clarify whether the degradation of survivin protein was related to the induction of autophagy, CQ, a potent inhibitor of autophagy, was used to verify this hypothesis. However, in contrast to the result of the MG132, the expression levels of survivin stayed unchanged in response to autophagy inhibition **(Figures 3B,D)**, indicating that the cellular autophagic flux is not involved in the PL-mediated degradation of survivin expression. Collectively, these results showed that PL induces the depletion of survivin via the proteasomedependent pathway.

# PL Induces ROS-Dependently Apoptosis and Survivin Depletion

Lots of anticancer agents perform the antitumor property by the activation of intracellular ROS. Indeed, it has been elucidated that PL-induced apoptotic cell death in mammary tumors and sarcoma are closely related to the increased elevation of the intracellular ROS level (24). Of note, our previous study found that PL caused a prominent rise in ROS levels and induced significant apoptosis in ovarian cancer cells (11). Meanwhile, co-administration of PL and NAC fully restored the PL-mediated increase in ROS and inhibited the depletion of survivin **(Figures 4A,B)**. Taken together, these data offered additional support to our hypothesis that ROS generation is significant for PL-mediated ovarian cancer cells apoptosis and survivin degradation.

taking advantage of pCDH-Neo-Venus-survivin lentivirus, while pCDH-Neo-Venus/DEST was adopted as the control. The Western blotting analysis was used to confirm the result of infection. As shown in **Figures 5A,C**, A2780 and OVCAR-3 cells infected with pCDH-Neo-Venus-survivin exhibited a much higher survivin expression. To detect the role of survivin in the apoptosis effect of PL, stable cells overexpressing survivin were treated with the increasing concentrations of PL and assayed to examine the survival rate **(Figures 5B,D)**. In the presence of PL, the IC50 value of OVCAR-3 cells was 4.59µM. However, in survivin overexpression cells, the IC50 value was 9.65µM, which was 2.10-fold higher than the control group. Consistently, similar results were also demonstrated in A2780 cells. Collectively, these results suggested that survivin overexpression raises the survival rate of ovarian cancer cells.

# Overexpression of Survivin Raises the Survival Rate of Ovarian Cancer Cells to PL

To explore the role of survivin involved in PL-induced ovarian cancer apoptosis, we performed survivin infection

# PL Inhibits A2780 Xenograft Tumor Growth and Downregulates Survivin in vivo

To evaluate the antitumor effects of PL in vivo, a xenografts model of A2780 cells in BALB/c mice was conducted.

Intraperitoneal injection of PL at doses of 20 mg/kg for 15 days decreased A2780 tumors weight as well as the tumor volume vs. vehicle control **(Figures 6A,B,E)**. Notably, PL administration was endurable without severe weight loss **(Figures 6C,D)**. Additionally, immunoblotting analyses of the tumor tissues showed that survivin was significantly downregulated after PL exposure for 15 days **(Figure 6F)**, which was in accordance with the results in vitro. In short, these data indicated that PL suppresses the xenograft tumor growth in vivo accompanied by a decreased survivin level.

# DISCUSSION

Because of the advanced stage at the time of diagnosis (80%) and a high recurrence rate (70–80%), human ovarian cancer surpassing other gynecological cancers lies in the fifth-leading cause of cancer-related deaths. The first-line treatment for it is the tumor cytoreductive surgery combined with platinum-taxane chemotherapy (25, 26) or neoadjuvant chemotherapy followed by debulking surgery (27). Although ∼80% of patients will have a response to the frontline therapies, 25% of women within 6 months will experience resistant cancer recurrence, and the overall 5-year survival rate ranges between 30 and 40% on a global scale (1). Thus, the explore of applicable treatment drugs remains a crucial goal for carrying out a better outcome.

Owing to the abnormal expression of anti-apoptotic proteins resulting in antitumor treatments less effective or ineffective, the reactivation of cell apoptosis becomes a potential therapy to conquer antitumor drug resistance. Survivin, the smallest member of IAP proteins, participates in the inhibition of apoptosis, affects the proper process of mitosis and promotes angiogenesis or even DNA repair (28). Generally, abnormal expression of survivin is associated with decreased apoptosis, increased tumor recurrence, poor prognosis, and high chemoresistance in human ovarian cancer (29, 30). Downregulating the expression of survivin elevated the sensitivity of ovarian cancer to chemotherapy and promoted apoptosis (7, 31). Thus, elucidating the regulatory mechanisms of survivin protein expression in cancers may propose mechanistic clues to future therapeutic strategies (32, 33). In general, survivin

FIGURE 6 | PL inhibits A2780 xenograft tumor growth and downregulates survivin *in vivo*. Each mouse was injected subcutaneously with A2780 cells (4 × 106 in 100 µL of medium) under the left and right shoulders. When the subcutaneous tumors were ∼0.3 cm × 0.3 cm2 in size, mice were randomized into two groups and received an intraperitoneal injection of the vehicle alone (0.5% methylcellulose) or PL (20 mg/kg) every day. The body weight and tumor volume were recorded. After the experiment, the mice were anesthetized, and tumor tissue was excised from the mice and weighed. The tumor volume (A), original tumors (B), body weight (C), tumor weight (D), summary data (E), and Western blot analysis of survivin (F) were shown. β-actin served as a loading control. The band quantification was performed using ImageJ software. \**P* < 0.05 vs. corresponding control.

combines with XIAP to indirectly suppress the function of caspases. XAF1 binds to XIAP resulting in the activation of ubiquitin-protein isopeptide ligase (E3) activity of XIAP, then promotes survivin ubiquitination and degradation (34). Conversely, the activation of ERK leads to the combination obstruction between survivin and the XIAP-XAF1 E3 ligase complex, which exactly stabilizes survivin proteins (35). The proapoptotic F-box protein FBXL7 acting as a ubiquitin E3 ligase interacts with Glu-126 to modulate the polyubiquitylation of survivin and thereby enhance its proteasomal degradation (36). Nonetheless, aurora kinase A (AURKA) restricts survivin to combine with FBXL7 E3 ubiquitin ligase through the negative regulation on FOXP1-FBXL7 axis, which upregulates the survivin expression (37). lncRNA LINC00473 (LNC473) remarkably suppresses the ubiquitination of survivin through recruiting deubiquitinase USP9X and then enhanced the stability of survivin (38). The Culling 9 (CUL9) is a putative E3 ligase and deplete the protein abundance of survivin via enhancing its ubiquitylation (39). Additionally, it has been reported that the ubiquitin-like-modifier proteins FAT10 (40), the heat shock protein (HSP90) (41) and CSN5/JAB1 (42) directly bind to survivin and therefore are resistant to ubiquitin-proteasome degradation. Therefore, the therapeutic regiment targeting the survivin axis is pushing the door of ovarian cancer. Such treatments include regiments that target transcription or posttranslational level, vaccines based on cytotoxic activities of immune-cells and gene therapy methods that suppress survivin's function (43).

Piperlongumine, a biologically active alkaloid/amide isolated from peppers, has selectively excellent ability to inhibit tumor cells via the induction of oxidative stress and genotoxicity, as well as the significant oral bioavailability in mice burden tumor without unbearable systemic toxicity (44). We have previously reported that exposure of ovarian cancer cells to PL selectively suppressed cell viability in a concentration- and time-dependent manner (11). We deeply take an insight into the underlying mechanism of PL-induced apoptosis in ovarian cancer and find that survivin plays a vital role in the regulation of PL-mediated apoptosis in this study. Here, we firstly elucidated that PL induced apoptosis as well as the rapid depletion of survivin in ovarian cancer in vitro and vivo. Furthermore, overexpression of survivin raised the survival rate of ovarian cancer cells, implying that PL induced apoptosis of ovarian cancer via inhibiting survivin expression. In addition, our data suggested that the levels of survivin mRNA stayed unchanged under PL administration, indicating that PL induced depletion of survivin at the nontranscriptional level. This notion may lead to a rational concern about the strategy modulating either ubiquitin-proteasome or autophagy because both of them are the two primary proteolytic

#### REFERENCES

mechanisms. Then, a blockage of proteasome activity reversed the reduction of survivin by PL, as compared with keeping stable under autophagy inhibition. Otherwise, the previous studies also observed that the elevated levels of intracellular ROS induced by PL involved in the process of apoptosis in ovarian cancer. Considering that ROS-mediated signaling in cancer cells has long been thought to participate in malignant transformation, carcinogenesis initiation, and the survival of cancer cells, it is reasonable that the ROS-dependent pathway may take part in the regulation of survivin (45). In agreement with this notion, we observed that the apoptosis and the depletion of survivin under PL were reversed by NAC.

As all the data we assembled suggest, it is reasonable to regard the ROS-mediated proteasome-dependent mechanism as the major system responsible for the depletion of survivin under PL treatment. PL is on the way to transform ovarian cancer patients' outcomes and looking for finding its place in ovarian cancer therapeutic strategies.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/supplementary material.

#### ETHICS STATEMENT

The animal study was reviewed and approved by the ethics committee of Jinan University.

#### AUTHOR CONTRIBUTIONS

X-WN, L-HG, XC, ZS, and X-JY designed the experiments, performed the experiments, analyzed the data, and wrote the paper. H-HZ, P-PY, YY, Z-HX, M-NW, YL, S-TW, and KL conducted the experiments. All authors read and approved the final manuscript.

#### FUNDING

This study was supported by funds from the National Key Research and Development Program of China No. 2017YFA0505104 (ZS), the National Natural Science Foundation of China Nos. 81772540 (ZS), 81503293 (X-JY), the Science and Technology Foundation of Wenzhou City, Zhejiang Province, China No. 2015Y0304 (L-HG), the Technology Development Funds of Wenzhou City No. Y20190014 (X-JY), and the Traditional Chinese Medicine Science and Technology Foundation of Zhejiang Province, China No. 2020ZB144 (X-JY).

CA Cancer J Clin. (2018) 68:284–96. doi: 10.3322/caac. 21456

3. Herzog TJ, Monk BJ. Bringing new medicines to women with epithelial ovarian cancer: what is the unmet medical need? Gynecol Oncol Res Pract. (2017) 4:13. doi: 10.1186/s40661-017- 0050-0

of specificity protein transcription factors. Cancer Prev Res. (2017) 10:467–77. doi: 10.1158/1940-6207.CAPR-17-0053

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Nan, Gong, Chen, Zhou, Ye, Yang, Xing, Wei, Li, Wang, Liu, Shi and Yan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# MiR-204-3p Inhibited the Proliferation of Bladder Cancer Cells via Modulating Lactate Dehydrogenase-Mediated Glycolysis

Jinan Guo1,2, Pan Zhao2,3, Zengqin Liu2,3, Zaishang Li 1,2, Yeqing Yuan1,2, Xueqi Zhang1,2 , Zhou Yu1,2, Jiequn Fang1,2 \* and Kefeng Xiao1,2 \*

*1 Department of Urology, Shenzhen Urology Minimally Invasive Engineering Center, The Second Clinical Medical College of Jinan University, Shenzhen People's Hospital, The First Affiliated Hospital of South University of Science and Technology of China, Shenzhen, China, 2 Shenzhen Public Service Platform on Tumor Precision Medicine and Molecular Diagnosis, Shenzhen Urology Minimally Invasive Engineering Center, The Second Clinical Medical College of Jinan University, Shenzhen People's Hospital, The First Affiliated Hospital of South University of Science and Technology of China, Shenzhen, China, 3 Clinical Medical Research Center, Shenzhen Urology Minimally Invasive Engineering Center, The Second Clinical Medical School of Jinan University, Shenzhen People's Hospital, The First Affiliated Hospital of South University of Science and Technology of China, Shenzhen, China*

#### Edited by:

*Chang Zou, Shenzhen People's Hospital, China*

#### Reviewed by:

*Jianqing Ruan, Soochow University Medical College, China Yan Deng, The Chinese University of Hong Kong, China Jigang Wang, China Academy of Chinese Medical Sciences, China*

#### \*Correspondence:

*Jiequn Fang<EMAIL_ADDRESS>Kefeng Xiao<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology*

Received: *16 August 2019* Accepted: *29 October 2019* Published: *29 November 2019*

#### Citation:

*Guo J, Zhao P, Liu Z, Li Z, Yuan Y, Zhang X, Yu Z, Fang J and Xiao K (2019) MiR-204-3p Inhibited the Proliferation of Bladder Cancer Cells via Modulating Lactate Dehydrogenase-Mediated Glycolysis. Front. Oncol. 9:1242. doi: 10.3389/fonc.2019.01242* MicroRNAs (miRNAs) are endogenous non-coding RNAs that negatively regulate the expression of downstream targeted mRNAs. Increasing evidence has suggested that miRNAs act as tumor suppressors or oncogenes to interfere the progression of cancers. Here, we showed that miR-204-3p was decreased in bladder cancer tissues and cell lines. Down-regulation of miR-204-3p was significantly associated with a poor prognosis in bladder cancer patients. Overexpression of miR-204-3p inhibited proliferation and induced apoptosis in bladder cancer cells. Furthermore, miR-204-3p was found to bind to the 3′ -untranslated region (UTR) of the lactate dehydrogenase (LDHA), which consequently reduced the expression of both mRNA and protein of LDHA. Interestingly, overexpression of miR-204-3p decreased glucose consumption and lactate production of bladder cancer cells. Overexpression of LDHA relieved the growth inhibition and cell apoptosis enhancement by miR-204-3p in bladder cancer cells. These results demonstrated that miR-204-3p negatively modulated the proliferation of bladder cancer cells via targeting LDHA-mediated glycolysis. MiR-204-3p might be a promising candidate for designing anticancer medication.

Keywords: bladder cancer, miR-204-3p, lactate dehydrogenase, glycolysis, proliferation

# INTRODUCTION

Bladder cancer (BC) is one of the most common malignancies in the urological system (1). There are approximately 356,000 new cases and 145,000 deaths annually around the world (1–3). The high occurrence and mortality of BC make it a big threat to the health of patients. Although remarkable progress has been made in the treatment of BC including surgical resection and chemotherapy, the 5-year overall survival of BC remains poor (4, 5). Therefore, it is urgent to identify novel biomarkers and targets of BC.

**163**

MicroRNAs (miRNAs) are a class of single-stranded noncoding RNAs with the length of 22–24 nucleotides (6, 7). MiRNAs negatively regulate the gene expression via binding with the 3 ′ -untranslated region (UTR) of targets, which leads to the degradation or translation inhibition of mRNAs (8). MiRNAs are reported to be involved in both physiological and pathological conditions including cell proliferation, differentiation, and apoptosis (9, 10). Recent research found that aberrant expression of miRNAs was associated with the tumorigenesis of BCs (9– 12). Therefore, detecting the abundance of miRNA might provide novel evidence for the diagnosis of BC and benefit the outcome of cancer patients. To get a whole picture about the expression of miRNAs in BC, we reviewed the previous publications relevant to the altered miRNAs in BC and performed a meta-analysis. The data showed that miR-204-3p was significantly down-regulated in the urine supernatant of BC patients (13). Interestingly, miR-204-3p was frequently found to be down-regulated in cancers, which acted as a tumor suppressor in tumorigenesis (14). Abnormal expression of miR-204-3p was observed in gastrointestinal stromal tumors, breast cancer, and hepatocellular carcinoma (HCC) (15, 16). However, the expression and function of miR-204-3p in BC remain largely unknown.

Aerobic glycolysis has been considered as a distinct hallmark of cancer cells, which represents that cancer cells addictively depend on glycolysis to metabolize glucose even in oxygen-rich condition (17–19). Therefore, understanding the mechanisms that contribute to the glycolysis process will provide novel cues for therapeutic strategies for cancer treatment. The lactate dehydrogenase (LDHA) is a critical enzyme of the glycolysis, which catalyzes the production of lactate (20). Up-regulation of LDHA has been found in a variety of human cancers, which is associated with cell growth, metastasis, and poor prognosis of cancer patients (21–24). Notably, miRNAs are reported to target LDHA and negatively regulate the expression of LDHA. For example, repression of LDHA by miR-34a inhibited the glycolysis and suppressed the growth of breast cancer cells (20). Recent study showed that miR-142-3p targeted LDHA, which reduced the glycolysis and growth of HCC cells (25). These reports suggest that the negative modulation of LDHA by miRNA is an important strategy to regulate the progression of cancers.

In this study, we investigated the function of miR-204-3p in BC. The data showed that miR-204-3p was down-regulated in BC tissues and cell lines. Overexpression of miR-204-3p decreased the proliferation of BC cells via modulating LDHAmediated glycolysis.

#### MATERIALS AND METHODS

#### Meta-Analysis

The studies that reported the alternation of miRNAs in BCs was searched using the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) and EBI ArrayExpress databases with the keywords "human" and "bladder" and "miRNA." For a meta-analysis, priority was given to the peer-reviewed publications that investigated the expression of miRNAs in both the BC and corresponding normal controls. For the studies without data of control group, only cell-culture-based results were excluded. Data extraction from the selected publications contained the first author's name, publication time, sample size, patient's age, type of case, origin of the studied population, tumor stage, detection method, and cutoff values for down-regulation or up-regulation. The quality of the publications was evaluated by the Newcastle-Ottawa scale, which was generally used for assessing the quality of nonrandomized studies in meta-analyses. Each article was subjected to assessment with eight methodology items with the score ranging from 0 to 9. The higher score indicated better quality of the publication. The articles with score of 7 or more were recruited for the meta-analysis.

#### Clinical Samples and Cell Lines

A total of 60 paired BC and adjacent normal tissues were obtained from the BC patients who underwent surgery in the Shenzhen People's Hospital. The tissues were confirmed by three pathologists independently. None of the patients received radiotherapy or chemotherapy before the surgical resection. All the tissues were maintained in liquid nitrogen until use. All the experimental procedures were approved by the Research Ethics Committee of Shenzhen People's Hospital, and each patient signed the written informed consent.

The normal human urothelial SV-HUC-1 cells were purchased from the Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China). BC cell lines including SW780, J82, UMUC3, 5637, and T24 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml of penicillin and 100 mg/ml of streptomycin (Invitrogen, Carlsbad, CA, USA) at 37◦C with 5% CO2.

#### Reverse Transcriptase Quantitative PCR Analysis

MiRNAs were extracted from the cell lines or bladder tissues using the miRNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instruction; 0.5 µg of RNA was reversely transcripted into cDNA with the PrimeScript RT Master Mix (Takara, Dalian, China). Real-time PCR analysis was performed with the SYBR Select Master Mix (Takara, Dalian, China) on the ABI7500 platform (Applied Biosystems, Foster City, CA, USA). The expression of U6 RNA and GAPDH was detected as the respective endogenous control. The expression level of miR-204-3p and LDHA was normalized to U6 and GAPDH and calculated using the 2−11CT method. The primers were as follows: miR-204-3p, F, 5′ -ACACTCCAGCTG GGGCTGGGAAGGCAAAGGG-3′ and R, 5′ -CTCAACTGG TGTCGTGGA-3′ ; LDHA, F, 5′ -AGCCCGATTCCGTTACCT-3′ and R, 5′ -CACCAGCAACATTCATTCCA-3′ ; U6, F, 5′ -CTC GCTTCGGCAGCACA-3′ and R, 5′ -AACGCTTCACGAATT TGCGT-3′ ; and GAPDH, F, 5′ -TGACGCTGGGGCTGGCAT TG-3′ and R, 5′ -GCTCTTGCTGGGGCTGGTGG-3′ .

#### Cell Viability

The proliferation of BC cells with the transfection of control miRNA or miR-204-3p mimics was evaluated by the Cell Counting Kit-8 assay (CCK-8, Beyotime, Shanghai, China). Briefly, 10 µl of CCK-8 reagent was added into the medium at the indicated time points of 1, 2, 3, 4, and 5 days and incubated for 4 h at 37◦C. The absorbance value (optical density [OD]) of each well at 450 nm was detected with a microplate reader (Bio-Rad, CA, USA). The results were obtained from three independent experiments.

# Cell Colony Formation

The BC cells expressing the indicated miRNA were seeded in a 6-well plate with the density of 1,000 cells per well. Cells were cultured with the RPMI-1640 medium for 2 weeks. And then the medium was discarded, and the cells were washed twice with phosphate-buffered saline (PBS). Cells were fixed with methanol for 15 min at room temperature (RT). After being washed with PBS, the colonies were stained with 1% crystal violet, and the number of colonies was counted with light microscopy.

#### Western Blot

Protein was extracted from the BC cells with the NP-40 lysis buffer (150 mM of NaCl, 1% NP-40, 50 mM of Tris–HCl (pH 8.0), and 1 mM of EDTA) in the presence of proteinase inhibitor (Millipore, Braunschweig, Germany). The protein concentration was evaluated using the bicinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China); 15 µg of proteins was loaded and separated by the 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore, Braunschweig, Germany). The membrane was firstly blocked with 5% non-fat milk at RT for 1 h, followed by incubation with primary antibodies at RT for 2 h and by incubation of the membrane with goat anti-mouse IgG secondary antibody (1:3,000, Bio-Rad, CA, USA), and then analyzed using the chemiluminescent horseradish peroxidase (HRP) conjugated substrate (Millipore, Braunschweig, Germany). GAPDH was used as the loading control. Antibodies including anti-LDHA (#2012, 1:3,000, Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-GAPDH (#5174, 1:3,000, Cell Signaling Technology, Inc., Danvers, MA, USA) were commercially obtained.

#### Luciferase Reporter Assay

The wild-type (WT) or mutant (MUT) 3′ -UTR of LDHA containing the putative binding sites of miR-204-3p was inserted into the pmiR-GLO vector. BC cells were transfected with the control miRNA or miR-204-3p mimics in the presence of luciferase reporter vector. After transfection for 48 h, cells were harvested, and the luciferase activity was determined with the dual-luciferase reporter system (Promega, Madison, WI, USA). The experiment was performed in triplicate.

### Measurement of the Glucose Consumption and Lactate Production

BC cells transfected with the corresponding miRNAs were cultured in RPMI-1640 medium without phenol red (Gibco, New York, NY, USA) for 24 h, and then the medium was collected. The glucose consumption and lactate production were determined using the Glucose Assay Kit (GAHK20-1KT; Sigma-Aldrich, USA) and Lactate Assay Kit (BioVision, CA, USA) according to manufacturers' instructions, respectively. The protein level in each group was measured using the BCA assay kit (Beyotime, Shanghai, China) for the normalization.

#### Statistical Analysis

Results were presented as mean ± standard deviation from three independent experiments. The data were analyzed with the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Significant differences between/among treatment groups were analyzed using unpaired Student's t-test or one-way ANOVA followed by Dunnett's post hoc test. P < 0.05 was considered to be statistically significant.

# RESULTS

# MiR-204-3p Was Down-Regulated in Bladder Cancer Tissues and Cell Lines

To obtain a whole picture of miRNA expression in BC, a miRNA meta-analysis was performed using previous publications. The data found that miR-204-3p was significantly aberrantly expressed in the urine supernatant of the BC patients (**Figure 1A**). To confirm this result, the expression of miR-204- 3p was detected by reverse transcriptase quantitative PCR (RTqPCR) with paired BC tissues and corresponding normal tissues. The result showed that the level of miR-204-3p was significantly decreased in BC tissues in comparison with that of the normal counterparts (**Figure 1B**). To further confirm the aberrant expression of miR-204-3p in BC, the abundance of miR-204-3p in several BC cell lines was examined. As indicated in **Figure 1C**, miR-204-3p was remarkably decreased in BC cells, compared with the normal cells, including SW780, J82, UMUC3, 5637, and T24. These results suggested the down-regulation of miR-204-3p in BC. To further characterize the association between miR-204-3p expression and the clinicopathological features, the abundance of miR-204-3p in BC patients with or without lymph node metastasis was compared. The data revealed that lower expression of miR-204-3p was correlated with positive lymph node metastasis (**Figure 1D**). Consistently, decreased expression of miR-204-3p was also observed in BC patients with larger tumor size (**Figure 1E**). These results demonstrated that miR-204-3p was down-regulated in BC and associated with a poor prognosis of cancer patients.

#### Overexpression of MiR-204-3p Inhibited the Proliferation and Induced Apoptosis of Bladder Cancer Cells

As miR-204-3p was decreased in BC, to investigate the effect of aberrant expression of miR-204-3p on the growth

miR-204-3p in 60 paired bladder cancer tissues and the corresponding adjacent normal tissues was determined by RT-qPCR. (C) The expression of miR-204-3p in bladder cancer cells and normal bladder epithelial cell SV-HUC-1 was determined by the RT-qPCR analysis. (D) The expression of miR-204-3p in the bladder cancer patients with or without lymph node metastasis was determined by RT-qPCR. (E) The expression of miR-204-3p in bladder cancer tissues stratified by tumor size was determined by RT-qPCR. \*\**P* < 0.01 and \*\*\**P* < 0.001. RT-qPCR, reverse transcriptase quantitative PCR.

of BC cells, SW780 and 5637, which harbored the lowest expression of miR-204-3p among all the cell lines we tested (**Figure 1C**), were transfected with miR-204-3p mimics to upregulate the expression of miR-204-3p. The overexpression of miR-204-3p in both SW780 and 5637 cells was detected by RT-qPCR (**Figure 2A**). The influence of miR-204-3p on the proliferation of BC cells was determined by CCK-8 assay. As showed in **Figures 2B,C**, the growth of both SW780 and 5637 cells was significantly inhibited with overexpressed miR-204-3p. Consistently, the colony formation assay indicated that overexpression of miR-204-3p dramatically decreased the number of colonies compared with those of the control cells (**Figure 2D**). In addition, to further confirm the negative regulation of overexpressed miR-204-3p on the growth of BC cells, the apoptosis rate of both SW780 and 5637 cells was examined with the fluorescence-activated cell sorting (FACS) analysis. The result suggested that ectopic expression of miR-204-3p significantly enhanced the apoptosis of BC cells (**Figure 2E**). These data demonstrated that overexpression of miR-204-3p inhibited the growth of BC cells, which

suggested the potential tumor suppressive function of miR-204- 3p in BC.

#### Lactate Dehydrogenase Was a Target of MiR-204-3p in Bladder Cancer Cells

To further explore the underlying molecular mechanism by which miR-204-3p modulated the growth of BC cells, the targets of miR-204-3p were predicted using the miRDB database (http://mirdb.org). Among the candidates, LDHA was found as a possible target of miR-204-3p (**Figure 3A**). To confirm the potential binding between miR-204-3p with the LDHA 3′ -UTR (position 515-521), the WT or MUT 3′ -UTR of LDHA containing the putative binding sites of miR-204-3p was inserted into the pmiR-GLO vector. Luciferase reporter assay was performed by co-transfecting negative control miRNA or miR-204-3p mimics with WT or MUT 3′ -UTR of LDHA. The results showed that overexpression of miR-204-3p significantly reduced the luciferase activity of the WT 3′ -UTR of LDHA; however, no remarkable decrease was observed when cells were transfected with MUT 3 ′ -UTR of LDHA (**Figures 3B,C**). This result indicated that miR-204-3p bound the 3′ -UTR of LDHA.

To detect whether the binding of miR-204-3p at the 3′ -UTR affected the mRNA stability of LDHA, both SW780 and 5637 cells were transfected with control miRNA or miR-204-3p mimics, and the mRNA level of LDHA was examined with the RT-qPCR assay. As shown in **Figure 3D**, overexpression of miR-204-3p significantly reduced the mRNA level of LDHA in BC cells. Additionally, the protein level of LDHA in SW780 and 5637 cells expressing miR-204-3p was also investigated by Western blot using the anti-LDHA antibody. The data showed that the level of LDHA was down-regulated with the transfection of miR-204-3p in SW780 and 5637 cells (**Figure 3E**). These results demonstrated that LDHA was a target of miR-204-3p and negatively regulated by miR-204-3p in BC cells.

#### MiR-204-3p Suppressed the Glycolysis of Bladder Cancer Cells by Modulating Lactate Dehydrogenase

LDHA was critical for the glucose metabolism of cancer cells. Considering the negative regulation of miR-204-3p on the expression of LDHA, the influence of miR-204-3p on the glycolysis of BC cells was determined by measuring the glucose consumption and lactate production. The results showed that overexpression of miR-204-3p significantly decreased the glucose uptake of SW780 and 5637 cells (**Figure 4A**). Consistently, the generation of lactate of BC cells was also dramatically reduced

\*\**P* < 0.01 and \*\*\**P* < 0.001. LDHA, lactate dehydrogenase; UTR, untranslated region; RT-qPCR, reverse transcriptase quantitative PCR.

in the presence of high level of miR-204-3p (**Figure 4B**). To further confirm these results, miR-204-3p was knocked down in SW780 and 5637 cells by transfecting miR-204-3p antagomir. The reduction of miR-204-3p was verified by RTqPCR assay (**Figure 4C**). The glycolysis of BC cells harboring depleted miR-204-3p was explored. As shown in **Figure 4D**, down-regulation of miR-204-3p remarkably elevated the glucose consumption of both SW780 and 5637 cells. Increased lactate production was also obtained with the decreased expression of miR-204-3p (**Figure 4E**). These results indicated that miR-204-3p was a negative regulator of the glycolysis of BC cells.

To further investigate whether the inhibitory effect of miR-204-3p on the proliferation of BC cells was achieved via LDHA, CCK-8 assay was performed by transfecting LDHA in miR-204-3p overexpressed cells. The results showed that highly expressed miR-204-3p suppressed the proliferation and growth and induced cell apoptosis of both SW780 and 5637 cells, while rescue the expression of LDHA attenuated the inhibitory effect of miR-204-3p on the proliferation growth of BC cells and the enhanced effect of miR-204-3p on the cell apoptosis of BC cells (**Figures 4F–I**). These data demonstrated that miR-204-3p decreased the expression of LDHA, inhibited the glycolysis, and suppressed the growth of BC cells.

*(Continued)*

FIGURE 4 | miR-204-3p antagomir transfection was determined by the RT-qPCR. (D,E) The effects of miR-204-3p knockdown on the glucose uptake and lactate generation ofthe SW780 and 5637 cells were determined by Glucose Assay Kit and Lactate Assay Kit, respectively. (F,G) The cell viability of SW780 and 5637 with co-transfection with miRNAs and plasmid vectors was determined by CCK-8 assay. (H) The cell growth of SW780 and 5637 with co-transfection with miRNAs and plasmid vectors was determined by colony formation assay. (I) The cell apoptosis of SW780 and 5637 cells with co-transfection with miRNA and plasmid vectors was evaluated by flow cytometry. *N* = 3; \*\**P* < 0.01 and \*\*\**P* < 0.001. RT-qPCR, reverse transcriptase quantitative PCR; CCK-8, Cell Counting Kit-8.

#### DISCUSSION

Although remarkable progression has been made in the treatment of BC with the application of surgery and chemotherapy, the prognosis of BC is still poor. Moreover, most patients are diagnosed at the advanced stage and showed unsatisfactory response to treatments. Notably, increasing evidence has suggested the critical roles of miRNAs in the initiation and progression of cancers (9–11). Here, we showed that miR-204-3p was decreased in both BC tissues and cell lines, which was associated with the poor prognosis of BC patients. Further gain-of-function analysis demonstrated that overexpression of miR-204-3p suppressed the growth of BC cells, which suggested the potential suppressive function of miR-204-3p in modulating the progression of BC.

The involvement of miR-204-3p in cancer has been highlighted by the studies that aberrant expression of miR-204-3p can be used to distinguish the malignant progression in gastric cancer (16) and breast cancer (26). Significant decrease of miR-204-3p was observed in HCC, which suppressed the growth of HCC via targeting fibronectin 1 (15). Recent study by Chen et al. showed that xanthohumol, a prenylated chalcone potential for anticancer therapy, up-regulated the expression of miR-204-3p in glioma cells and induced the cell apoptosis through modulating the insulin-like growth factor (IGF)-binding protein (27). In clear cell renal cell carcinoma (ccRCC), miR-204-3p was found to be down-regulated by the ERβ-suppressed circular RNA ATP2B1, which increased the expression of fibronectin 1 and promoted the invasion of ccRCC cells (28). These findings suggested the potential tumor suppressive function of miR-204-3p in cancers. In the present study, our data revealed that miR-204-3p was decreased in BC tissues compared with the normal tissue. Down-regulation of miR-204-3p was correlated with lymph node metastasis and larger tumor size. Further investigation might be of interest to explore the relationship between the expression of miR-204-3p with the 5-year overall survival of BC patients. Consistent with the decreased level of miR-204-3p in BC, overexpression of miR-204-3p significantly suppressed the proliferation and induced apoptosis of BC cells. In vivo tumorigenesis assay might be necessary to further confirm the modulating of miR-204-3p on the growth of BC cells in the future study.

The function of miRNAs was achieved via regulating the expression of downstream targets. In this study, the possible targets of miR-204-3p were predicted with the bioinformatics, and LDHA was identified as one of the binding partners of miR-204-3p. Overexpression of miR-204-3p significantly decreased both the mRNA and protein levels of LDHA in BC cells. Consistently, overexpression of miR-204-3p reduced the glucose consumption and lactate production of BC cells. LDHA deficiency results in a decrease in lactate production accompanied by inhibition of cell migration and invasion. Therefore, LDHA has been the target of different miRNAs in cancer cells to regulate the tumorigenesis. Among them, miR-142-3p was reported to target LDHA and inhibited the aerobic glycolysis of HCC (25). In breast cancer, miR-30-5p inhibited the cell growth and metastasis through suppression of LDHAmediated glucose metabolism (29). These results collectively demonstrated that reprograming the glycolysis via targeting LDHA has been a promising way to modulate the progression of cancers.

Our study still presented several limitations. Firstly, the relationship between miR-204-3p and the overall survival of the BC patients has not been determined in the study, which needs further examination. Secondly, the present study lacks the in vivo animal studies, and the effects of miR-203-3p on the in vivo BC growth should be considered in future studies. Thirdly, the targets of miR-204-3p were not limited to LDHA, and other potential targets may be explored in future studies.

In conclusions, our results demonstrated that miR-204-3p was down-regulated in BC and correlated with the progression of the BC patients. Overexpression of miR-204-3p decreased the growth of BC cells via down-regulating LDHA, which consequently suppressed the glucose metabolism of BC cells. These data indicated that targeting the miR-204-3p-LDHA pathway might interfere with the tumorigenesis of cancer cells.

#### DATA AVAILABILITY STATEMENT

All datasets for this study are included in the article/supplementary material.

#### ETHICS STATEMENT

The studies involving human participants were reviewed and approved by the Institutional Review Board of Shenzhen people's Hospital. The patients/participants provided their written informed consent to participate in this study.

#### AUTHOR CONTRIBUTIONS

JG and KX designed the whole study. JG, PZ, ZLiu, and ZLi performed the experiments. YY, XZ, and ZY performed the statistical analysis. JF and KX drafted the manuscript.

#### FUNDING

This study was supported by the grants from Sanming Project of Medicine in Shenzhen (SZSM201412014), the Science and Technology Foundation of Shenzhen (JCYJ20170307095620828 and JCYJ20160422145718224), and the Shenzhen Urology Minimally Invasive Engineering Center (GCZX2015043016165448). All sources of funding received for the research being submitted.

#### REFERENCES

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Guo, Zhao, Liu, Li, Yuan, Zhang, Yu, Fang and Xiao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Binding of PD-L1 and Akt Facilitates Glioma Cell Invasion Upon Starvation via Akt/Autophagy/F-Actin Signaling

Ruo Qiao Chen1†, Xiao Hong Xu2†, Feng Liu2 , Chun Yang Li 2 , Yuan Jun Li 2 , Xiang Rui Li 1 , Guo Yong Jiang1 , Feng Hu3 , Di Liu4 , Feng Pan4 \*, Xin Yao Qiu2 \* and Xiao Qian Chen2 \*

1 School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 2 Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 3 Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 4 Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

#### Edited by:

Pascale Cohen, Université Claude Bernard Lyon 1, France

#### Reviewed by:

Anupam Mitra, University of California, Davis, United States Jiajun Fan, Fudan University, China

#### \*Correspondence:

Feng Pan<EMAIL_ADDRESS>Xin Yao Qiu<EMAIL_ADDRESS>Xiao Qian Chen<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology

Received: 06 August 2019 Accepted: 15 November 2019 Published: 03 December 2019

#### Citation:

Chen RQ, Xu XH, Liu F, Li CY, Li YJ, Li XR, Jiang GY, Hu F, Liu D, Pan F, Qiu XY and Chen XQ (2019) The Binding of PD-L1 and Akt Facilitates Glioma Cell Invasion Upon Starvation via Akt/Autophagy/F-Actin Signaling. Front. Oncol. 9:1347. doi: 10.3389/fonc.2019.01347 Glioma, especially glioblastoma, is pathologically characterized by high aggressiveness, which largely contributed to the ineffectiveness of current therapies. It has been recently reported that intrinsic PD-L1 can regulate tumor malignancy, whereas underlying mechanisms remain mostly unclear. Here, we report a novel mechanism by which PD-L1 promotes glioma cell infiltration. In orthotopic glioma models, PD-L1 expression was up-regulated predominantly in glioma cells in the infiltrating front. For PD-L1-overexpressed glioma cells, PI3K/Akt and actin regulations were among the top six most altered signaling pathways as detected by RNA-sequencing. PD-L1 significantly activated Akt/F-actin signaling while suppressed autophagic signaling upon cell starvation. Mechanistically, PD-L1 preferentially bound to Akt among various PI3K/Akt signaling proteins. Serial truncation identified the interaction between the 128-237aa fragment of PD-L1 and the 112-480aa fragment of Akt, which facilitates the membrane translocation/activation of Akt, and was unaffected by Perifosin (specific p-Akt inhibitor targeting Akt PH-domain). Taken together, our data indicate that in glioma cells, PD-L1 is induced to prevent autophagic cytoskeleton collapse via Akt binding/activation, facilitating glioma cell invasion upon starvation stress.

#### Keywords: glioblastoma multiforme, CD274, p62, autophagic influx, ischemia

# INTRODUCTION

Glioma, the most common primary intracranial tumor, originates from various types of cells. The most malignant glioma is glioblastoma, or glioblastoma multiforme (GBM), which accounts for around 21% of glioma and can be primary or secondary from grade II-III glioma. Recently, glioma is also classified into three types based on its molecular markers: 1p/19q deletion, IDH mutation and TERT promoter mutation. Among them, grade IV glioma mainly has the TERT promoter mutation accompanied by EGFR/PTEN mutation. Current treatments for glioma remain conventional surgery, radiotherapy and chemotherapy. However, glioma is extremely easy to relapse after treatment, with an average survival time of only 14 months for GBM patients after treatment (1). It is urgent to find new and effective therapies in order to prolong the survival time of GBM patients.

**172**

The pathological features of GBM are necrosis, high invasiveness and microvascular hyperplasia (2), which are closely related to abnormal energy metabolism. It is generally considered that solid malignant tumors including glioma suffer ischemia/hypoxia throughout their growth. For example, it is estimated that glioma tissues have wide but heterogeneous hypoxia with oxygen concentrations ranging from 0.1 to 2.5% (3). Clinical data reveal that severer GBM necrosis closely correlates to faster GBM progress and shorter patient survival time (1, 2). Upon energy stress such as ischemia, autophagy is a major mechanism to maintain cellular energy homeostasis, which is highly regulated by conserved autophagic influx signaling involving PI3K/Akt/mTOR, p62, Beclin-1 and LC3 (4–6). Higher activity levels of autophagy are widely detected in glioma tissues, particularly around necrotic tissues (7). It is reported that inducing autophagy can either improve the efficacy of chemotherapy (8) or induce chemotherapy-resistance in glioma (9). Large-scale gene knockout screening shows that mitochondrial energy-metabolism genes are necessary for anoxic adaptive growth of a variety of tumors, including GBM (10). Specifically, Kim et al. (11) reported in recent research an increase of mitochondrial SHMT2 (serine hydroxymethyltransferase) in ischemic necrosis tissues of GBM patients, this enhances the anaerobic energy metabolism of GBM cells and promotes survival of GBM cells under ischemic environment. The mechanisms by which ischemia stimulates GBM development are extremely complicated and far from clear.

Programmed cell death ligand-1 (PD-L1, i.e., CD274 or B7H1) is a key negative regulator for immune inhibitory axis signaling controlling T-lymphocyte infiltration inside solid tumors. Previous studies reported that PD-L1 is widely expressed in glioma cell lines (12, 13) and most human glioma specimens (14). However, data of PD-L1 levels and subcellular distributions in human glioma tissues vary greatly (15). Most studies reported that higher PD-L1 expression was correlated with higher glioma grades (16–18) and worse prognosis (17, 19–22), while opposite results were also reported (14, 17, 23). PTEN mutation/deletion (in 36% of glioma) is closely associated with higher PD-L1 expression in glioma (24). Present evidence suggests that PI3K/Akt is a major pathway controlling PD-L1 expression in cancer cells. For mTOR, a key downstream signaling molecule in PI3K/Akt pathway, mTOR complex 1 (mTORC1) mainly mediates PI3K/Akt-induced cell autophagy (25) and mTORC2 mediates Akt-induced cell survival (26).

PD-L1 is recently considered to be oncogenic. PD-L1 knockdown significantly decreases tumor volume in murine ovarian cancer, melanoma (27), murine medulloblastoma (28), and U87 glioma in nude mice (29), while PD-L1 overexpression promotes glioma development (29). Mechanistically, PD-L1 regulates cell growth, proliferation, apoptosis, autophagy, migration and invasion in various cancers via modulating PI3K/Akt/mTOR and Ras/Erk/EMT signalings (27, 29–31). Further exploration of biological roles and mechanisms of PD-L1 in glioma will provide therapeutic cues including targeted immunotherapies for glioma.

In the present study, we first reported that PD-L1 was mostly prominent in those highly aggressive infiltrating glioma cells in vivo. Our data revealed that PD-L1 promoted glioma cell infiltration via starvation-induced Akt/autophagy/Factin signaling. Particularly, we dissected PD-L1-Akt binding fragments and elucidated how PD-L1/Akt interactions activated its downstream cascades.

# MATERIALS AND METHODS

#### Drugs and Antibodies

Cloroquine (CQ, Selleck Chemicals, Shanghai, China) was used at a final concentration of 25µM. LY294002 (PI3K inhibitor, Cell Signaling Technology, MA, USA) was used at a final concentration of 0–50µM. Perifosine (inhibitor of Akt PHdomain, Selleck Chemicals, Shanghai, China) was used at a final concentration of 0–25µM. Primary antibodies against phospho-Akt (Ser473, #D9E), total Akt (#9272), phospho-mTOR, total mTOR,pP62, phospho-P70S6K, LC3B were purchased from Cell Signaling Technology (MA, USA). Antibodies against PD-L1/CD274 (PA5-28115, Thermo Fisher, IL, USA), PD-L1 (ABM4E54, Abcam, Cambridge, UK), GST (Z-5, Santa Cruz Biotechnology, TX, USA), Beclin1 (Santa Cruz Biotechnology, TX, USA), N-terminal GFP (Sigma), β-actin (20536–1-AP, Proteintech, Wuhan, China), Na+/K+ ATPase α1 (Proteintech, Wuhan, China), β-tubulin (Proteintech, Wuhan, China) were commercially purchased.

# Plasmids and Transfection

GFP-LC3 plasmid was a gift from Dr. He Li (Huazhong University of Science and Technology). GSTkRas/PTEN/Akt1/Akt2/Akt3/EGFR VIII/PKA plasmids were gifts from Dr. Haian Fu (Emory University). PD-L1-EGFP expressing-plasmid was purchased from GeneChem (Shanghai, China). Full-length and truncated human PD-L1 coding cDNA was PCR amplified from PD-L1-EGFP plasmids and cloned into NV (N-terminal Venus 1-157aa) vector including NV-PD-L1 FL (full length 1-290 aa), NV-PD-L1-T1-259 (truncate 1-259aa), T128-259, T128-239, T1-178. PD-L1 cloned into pDEST-26 or p-FU-Venus vector was used to express GST-PD-L1 and Venus-PD-L1 FL1-290 or T19-290. Full-length and truncated human Akt1 cDNA was PCR amplified from GST-Akt1 plasmid and cloned into p-FU-Venus vector for Venus-Akt1, Akt1-T1-111, and Akt1-T122-480. Akt1-Y176A and Akt1-K14R mutants were constructed by using overlapping-PCR from p-FU-Venus-Akt1 plasmid. Transfection of plasmids was performed by using Lipofectamine 2000 (11668, Life Technologies, CA, USA) according to the manufacturer's instructions.

# RNA-Sequencing and Enrichment Analysis

Three 100-mm dishes of U251/PD-L1 and U251/Vec stable cell lines were subjected to total mRNA isolation, cDNA libraries construction and sequencing at Illumina HiSeq sequence platform (PE150) with 6G clean data by Novogene Bioinformatics Institute (Shanghai, China). Differential gene expression analysis between U251/PD-L1 and U251/Vec groups was performed using the DESeq2 R package (1.10.1). The resulting P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate.  KEGG pathways and GO terms with Padj < 0.05 were considered significantly enriched by DEGs. We have submitted our data to NCBI (https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107581).

#### Orthotopic Mouse Glioma Model

All animal handling and experiments were performed in accordance with NIH guidelines and approved by the Ethics Committees of Huazhong University of Science and Technology. The mice were group housed in the Animal Core Facility of Tongji Medical College under a 12 h light/dark cycle with ad libitum access to food and water. Briefly, adult Kunming male mice (18–20 g) were anesthetized with chloral hydrate (350 mg/kg) and a burr hole was drilled in the skull 0.5 mm posterior to the bregma and 2.0 mm lateral to the midline. A 10-µl Hamilton syringe (26 gauge, Reno, NV) containing 20,000 G422 cells (mouse GBM cell line) in 1 µl of PBS was advanced to a depth of 3.5 mm from the skull surface and then withdrawal 0.3 mm. Cell suspension was delivered at the rate of 1 µl/min. After cell implantation, the needle was left in place for 6 min before withdrawal. After 6–14 d of cell inoculation, the mice were perfused with 4% paraformaldehyde (PFA) and the brains were paraffin-embedded.

#### Hematoxylin-Eosin (HE) Staining and Immunohistochemistry (IHC)

IHC was performed as previously reported (29). Paraffinembedded mouse brain tissues (bearing tumor) were cut into 4 µm-thick slices for H&E staining and IHC analysis. Briefly, the slices were deparaffinized in xylene and antigenretrieved by microwave processing. After 1 h of blocking with 5% bovine serum albumin in PBS, the slices were incubated with primary antibodies (PD-L1, Abcam, UK) overnight at 4 ◦C, followed by corresponding secondary antibody incubation (Polink-1 HRP DAB Detection System, ZSGB-BIO, China). The immunoreaction was visualized with diaminobenzidine tetrachloride. The brain images were scanned with an automatic slice scanning system-SV120 (Olympus, Tokyo, Japan). The tumor parenchyma rim was delineated with black dashed ellipse circle, while the infiltration frontiers was delineated with blue or white dashed irregular circle.

#### Cell Culture and Starvation

Human glioblastoma cell lines U251, LN229, and human embryonic kidney 293T cell line were purchased from American Tissue Culture Collection (MA, USA) or China Center for Type Culture Collection (Wuhan, China). U251, LN228, U87MG with stable PD-L1 overexpression (U251/vec or PD-L1, LN229/vec or PD-L1) or knockout (U251/sgGFP or sgPD-L1) were generated as previously described (29). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, CA, USA) supplemented with 10% FBS (Gemini, CA, USA) and 1% Penicillin-Streptomycin Solution (Hyclone, Thermo, Beijing, China). Fresh Earle's balanced salt solution (EBSS, GIBCO BRL, USA) media was used to induce cell starvation at 24 h after transient transfection or initial seeding. Cells were washed with EBSS media for three times and then incubated with EBSS media for various time points.

#### Western Blotting Analysis

Western blotting analysis was performed as previously reported (29). Briefly, the cell lysates were collected and dispersed in radio-immunoprecipitation assay lysis buffer containing phenylmethane-sulfonyl fluoride.  The blots were incubated with corresponding primary and secondary IRDye 800 or IRDye 680 CW-conjugated goat anti-rabbit or anti-mouse IgG antibodies (LI-COR Biosciences, Lincoln, USA). The labeled bands were visualized and quantified by Odyssey Infrared Imaging System (LI-COR Biosciences, MA, USA).

# Palloidin Staining and Immunofluorescence

Immunofluorescence staining was performed as previously reported (29). Cells in 35-mm culture dishes were fixed, permeabilized, blocked, and then incubated with primary and corresponding Dylight 488-labeled secondary antibodies (Abbkine, CA, USA). F-actin was stained with rhodaminephalloidin (Yisheng Bioengineering Institute, China) according to the manufacturer's instructions. Hoechst 33342 was used to stain the nucleus. For paraffin-embedded tissues, rat brain slices were deparaffinized, rehydrated, antigen unmasked, blocked with 5% bovine serum albumin (BSA) and then incubated with primary antibodies and corresponding Dylight 488/594 labeled secondary antibodies. Micrographs were taken under the same conditions with a conventional fluorescent microscope (Olympus, Tokyo, Japan).

#### GFP-LC3 Punctate Quantification

U251/sgGFP or U251/sgPD-L1 cells were transiently transfected with pGFP-LC3 plasmids for 24 h. Then, the cultures were subjected to EBSS treatment for 12 h and fixed. U251 cells expressing GFP-LC3 were randomly photographed under 400× magnifications under the same conditions. Cells with five or more GFP-LC3 vacuole dots (puncta) were considered autophagypositive. An average percentage of puncta-positive cells from nine fields/culture (total cells > 500) was calculated and used for statistical analysis.

#### Glutathione S-Transferase (GST)-Pull Down Assay

GST-pull down assay was performed as previously described (29). 293 cells were transfected with indicated plasmid at 1:1 for 2 d. Cell lysates were extracted with GST-lysis buffer and 400 µg of total soluble proteins from each sample were incubated with 30 µl of glutathione sepharose bead slurry (GE Healthcare Life Sciences, Piscat-away, USA) overnight at 4◦C. After extensive washing, the immunoprecipitates were subjected to Western blotting analysis. Anti-GST, anti-N-GFP or anti-PD-L1 antibodies were used to probe corresponding proteins.

#### Membrane and Cytosol Protein Detection

U251 cells were transfected with p-FU-Venus or p-FU-Venus-PD-L1 plasmid for 24–36 h. Membrane and cytosol proteins were extracted using Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) according to manufature's instructions. Membrane and cytosol proteins were then subjected to Western blotting with Na+/K+ ATPase α1 and β-tubulin as internal control of membrane and cytosol proteins respectively.

#### Statistical Analysis

All experiments were repeated independently for at least three times. The values were expressed as means ± SEM. Unpaired Student's test was used to compare between two groups of in vitro experiments. Paired Student's test was used to compare between two groups of animal experiments. Comparisons among multiple groups were performed through one-way ANOVA with Student–Newman–Keuls post-test. P < 0.05 was considered statistically significant.

# RESULTS

#### PD-L1 Is Prominently Elevated in Invasive Frontier GBM Cells in vivo

To ascertain the relationships between intrinsic PD-L1 and glioma cell invasive behavior during GBM development, we examined PD-L1 expression in the whole brain at various stages of GBM development in a highly aggressive orthotopic

FIGURE 1 | PD-L1 is preferentially elevated in invasive frontier GBM cells in vivo. Mouse G422 glioma cells (20,000 cells) were inoculated into right striatum of Kunming mice. 6, 10, and 12 days after initial cell implantation, the mice were perfused with PFA and paraffin-embedded (3 mice/group). Serial mouse brain slices were cut and the slices with maximal tumor-load were used for PD-L1 (3 slice/brain) or H&E staining (3 slice/brain). (A) Representative PD-L1 staining of glioma on day 6. Inner black-dot ellipse delineated the boundary of tumor parenchyma while the outer irregular dot circle delineated the infiltration boundary. Left panels showed the whole brain and right panels showed enlarged square images. Arrows indicated the invasive frontier glioma cells. (B) Representative H&E staining of glioma at day 6. (C,E) Representative PD-L1 staining of glioma at day 10 and 12. (D,F) Representative H&E staining of glioma at day 10 and 12.

FIGURE 2 | the following creteria: P < 0.005, Padj < 0.05 and absolute fold change ≥1.2 (n = 3). (A) DO (Disease Ontology) enrichment analysis results showed that 42 of significantly altered-genes upon PD-L1 overexpression were associated with malignant glioma (indicated by red line). (B) BP (biological process) from GO enrichment analysis results showed that PD-L1 overexpression was closely associated with various cell migration pathways (indicated by red lines). (C) CC (cellular component) from GO enrichment analysis results showed that PD-L1 overexpression was closely associated with several actin signaling pathways (indicated by red lines). (D) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis results showed that PI3K-Akt and actin regulations are among the top altered pathways by PD-L1 overexpression (indicated by red lines). (E) Overall survival of glioma patients with PD-L1 expression from database http://gepia.cancerpku.cn. Survival significant longer for patients with low PD-L1(p = 0). (F–H) Correlation analysis of PD-L1 with PIK3CG, SQSTM1 (p62), and ACTB (β-actin) showed that mRNA levels of PD-L1 were positively associated with those of PIK3CG, p62, and β-actin (p = 0) in glioma patient tissues (http://gepia.cancer-pku.cn).

mouse G422 model (**Figure 1**). PD-L1 as well as H&E staining clearly showed that the glioma tissue grew rapidly from day 6 to 12 (D6, D10, D12) after initial G422 cell-implantation. The tumor parenchyma in the left hemisphere was delineated with black-dot ellipse, and the tumor frontiers was delineated with blue (PD-L1, **Figures 1A,C,E**) or white-dot irregular circle (H&E, **Figures 1B,D,F**). Clearly, invasive GMB cells could migrate far away from its parenchyma mass at the early stage (D6) during GBM development. Along with the growth of glioma parenchyma, infiltration frontiers were also enlarged correspondingly. The frontier (blue) and inner (black) square micrographs in brain coronal sections were enlarged in right panels. It is clear that PD-L1 was prominently elevated in invasive G422 cells (indicated by arrows) in GBM infiltration frontier (**Figures 1A,C,E**). Such evidence establishes a strong positive correlation between PD-L1 and the aggressiveness of GBM cells.

### PD-L1-Altered Gene Expression Are Highly Enriched in Migration and PI3K/Akt-Actin Signaling

To explore the underlying mechanisms of PD-L1+-GBM cells in the infiltration frontier, mRNA sequencing was conducted in PD-L1-overexpressed U251 glioma cells. Differential expressed genes (DEGs) between U251/PD-L1 and U251/Vec cells (i.e., CD274 vs. Vec) were selected by three criteria (i.e., P < 0.005, Padj < 0.05 and absolute fold change ≥1.2) and the selected DEGs were subjected to various systemic analysis. DO (Disease Ontology) enrichment showed that PD-L1-altered gene expression was significantly associated with malignant glioma (**Figure 2A**, indicated by red line). Gene ontology (GO) enrichment analysis showed that PD-L1-altered gene expression was largely associated with cell migration (BP: biological process, **Figure 2B**, indicated by red lines), actin-structure functions (CC: cellular component, **Figure 2C**, indicated by red lines), PI3K-Akt signaling pathway and Regulation of actin cytoskeleton (KEGG: Kyoto Encyclopedia of Genes and Genomes, **Figure 2D**, indicated by red lines). Further analysis of human glioma database (http://gepia.cancer-pku.cn) revealed that PD-L1 (CD274) was negatively associated with the prognosis of glioma patients (p = 0, **Figure 2E**). In addition, PD-L1 expression was significantly positively correlated with PI3K (p = 0, **Figure 2F**), SQSTM1 (p62, a negative marker of autophagic influx signaling) (32) (p = 0, **Figure 2G**) and ACTB (β-actin) (p = 0, **Figure 2H**) in human glioma tissues. The evidence together indicates that PD-L1 greatly affects glioma cell invasion and PI3K/Akt-actin signaling.

# PD-L1 Regulates Akt-p62-Autophagic Influx Signaling Upon Starvation

It is well known that GBM tissues suffer severe ischemia that causes necrosis and promotes GBM cell aggressiveness (1–3), suggesting that energy-deprivation stress is a major driving force for glioma cell invasion. Since PI3K/Akt signaling is pivotal in promoting cell survival and suppressing autophagy (6, 33), we further investigated the role of PD-L1 in regulating Akt-autophagic influx signaling in glioma cells with a EBSSinduced starvation model. Western blot results showed that PD-L1 overexpression did not evidently alter p-Akt/mTOR/p70S6K signaling under normal culture conditions (**Figure 3A**, EBSS-0 h and **Figure S1**). Upon EBSS incubation, p-Akt and p62 (a negative maker for autophagy influx) was prominently reduced in U251/Vec cells (**Figures 3A,B**), while LC3II was evidently increased (–CQ2, **Figure 3C**) as detected by Western blot. Cells with PD-L1 overexpression significantly reversed EBSS-induced p-Akt reduction, p62 reduction as well as LC3II elevation compared to their corresponding Vec controls (indicated by red boxes, **Figures 3A–C**). Consistently, fluorescent immunostaining showed that PD-L1 evidently reduced LC3B and Beclin-1 while increased p62 in starved LN229 glioma cells at EBSS-6 h (**Figure 3D**).

We further examined the effects of endogenous PD-L1 on autophagy influx signaling in glioma cells by knocking-out PD-L1 using CRISPR/Cas9 technique. Western blot results demonstrated that endogenous PD-L1 was prominently decreased in stable U251/sg-PD-L1 cell lines (single colnes #2 and #3, **Figure 4A**). Under normal culture conditions, PD-L1 knockout did not evidently affected p-Akt (**Figure 4B**) or p-mTOR (**Figure S2**) in U251 cells. Upon EBSS incubation (6 and 12 h), LC3II was significantly elevated in U251/sgPD-L1 cells as compared to its corresponding U251/sgGFP controls (**Figure 4C**). Consistently, fluorescent images of GPF-LC3-puncta clearly showed that the average number of GFP-LC3-puncta (representing autophagosome) per cell was significantly increased in U251/sgPD-L1 cells compared to its U251/sgGFP control at EBSS-12h (**Figure 4D**).

#### PD-L1 128-237aa Fragment Interacts With Akt 112-480aa Fragment

In order to dissect the mechanism of PD-L1 action, we screened the binding partners of PD-L1 with various key upstream signaling proteins in PI3K/Akt pathway by GSTpull down experiment. The results clearly showed that PD-L1 preferentially bound to AKT1 and AKT2 compared to KiRAS, PTEN-1, PTEN-2, AKT3, EGFRVIII and PKA under normal

culture conditions (**Figure 5A**) as well as EBSS-4h incubation (**Figure 5B**). Then, we further dissected the exact binding domain of PD-L1 for its AKT binding. We made a series of PD-L1 (full length 290 aa, FL) truncates by deleting 260–290 aa (PD-L1 T1-259), 179-290 aa (PD-L1 T1-178) from its C-terminal and 1-127 aa (PD-L1 T128-259), 1-127/238-290 (PD-L1 T128- 259) (**Figure 5C**), 1-18 aa (PD-L1 T19-259) (**Figure 5D**) based on PD-L1 structure (upper panel, **Figure 5E**). GST-pull down results showed that PD-L1 FL1-290, T19-290, and T128-237 were the major truncates binding to AKT1 (indicated by red rectangles, **Figures 5B,C**). These results identified that PD-L1 128-237 fragment was required for its Akt binding.

We then seeked to identify the binding domain or key amino acid (aa) of Akt based on its structure (lower panel, **Figure 5E**). GST-pull down clearly showed that AKT1-FL (1-480 aa), T112- 480 but not T1-111 bound to PD-L1 (**Figure 5F**). Single amino acid mutant of AKT Y176A (loss of TNK2 binding and membrane localization) (34) and K14R (substantial reduction of

FIGURE 5 | Identification of PD-L1-Akt binding domains. Venus (or N-terminal Venus)-tagged plasmids were co-transfected with GST-tagged plasmids in 293T cells. Equal amount of total soluble proteins were subjected to GST-pull down analysis. (A,B) Western blot analysis of the binding between Venus-PD-L1 and GST-KiRas, PTEN-1, PTEN-2, AKT1, AKT2, AKT3, EGFRVIII, and PKA under normal culture conditions (A) and EBSS-4 h (B). WCL, whole cell lysate. (C,D) Western blot analysis of the binding between GST-AKT1 and NV-PD-L1 truncates. FL, full length; T, truncate. (E) Schematic diagram of PD-L1 and AKT truncates. (F) Western blot analysis of the binding between GST-PD-L1 and Venus-AKT truncates. All experiments were repeated at least three times independently.

ubiquitination and loss of PIP3 binding) (35) did not evidently affect its interaction with PD-L1 (**Figure 5F**), suggesting that PD-L1/Akt interaction occurred in the cytosol via independent AKT K14 and Y176 sites.

#### PD-L1 Facilitates Akt Membrane-Translocation and F-Actin Formation in Starved Glioma Cells

Since PD-L1 selectively interacted with Akt 112–480 fragment but not its PH domain (1–111 aa) (**Figure 5F**), it is interesting to further figure out how PD-L1 promoted Akt activation upon starvation (**Figure 3A**). Full activation of Akt depends on Akt translocation from cytoplasm to cell membrane (via its PH domain) and PIP3/PDK action on cell membrane (36, 37), which can be pharmacologically inhibited by specific inhibitor Perifosin (37) and LY294002, respectively. Administration of LY294002 (GFP, **Figure 6A**) and Perifosine (GFP, **Figure 6B**; Vec, **Figure 6C**) evidently reduced p-Akt in U251/GFP cells. In PD-L1-overexpressed U251 cells, however, LY294002 but not Perifosin could reduce p-Akt level (PD-L1, **Figures 6A–C**). Such evidence suggests that PD-L1 protects the membranetranslocation of Akt. Western blot results demonstrated that membrane-bound Akt was evidently increased upon PD-L1 overexpression compared to Vec control (**Figure 6D**). Consistently, fluorescent immunostaining of Akt showed that membranous Akt (indicated by arrows) was more evident in PD-L1-overexpressed U251 cells, which was well co-localized with F-actin (Palloidin staining) (**Figure 6E**).

Finally, we examined the effects of PD-L1 on F-actin structure in starved glioma cells. Fluorescent images of Palloidin staining clearly showed that F-actin formation was evidently increased in PD-L1-overexpressed U251 cells under normal culture conditions as well as EBBS-12 h incubation (**Figure 7A**). Taken together, the evidence suggested that PD-L1 facilitated Akt translocation to cell membrane and Akt activation to modify cellular morphology via F-actin cytoskeleton during energydeprivation stress.

# DISCUSSION

In our present study, we discovered that PD-L1 elevation occurred predominantly in highly aggressive glioma cells. RNA sequencing revealed that high levels of PD-L1 in glioma cells mainly mediated cell migration and PI3K/Akt/actin signaling. PD-L1 reduced starvation-induced Akt inhibition, autophagic influx and F-actin collapse in glioma cells. Mechanistically, PD-L1 directly interacted with Akt, and this process involved PD-L1 128-237aa and Akt 112-480aa. The binding of PD-L1 to Akt facilitated membrane-translocation of Akt and thus elicited downstream biological effects in frontier glioma cells. These findings reveal a novel invasive mechanism in GBM.

PD-L1 is a pivotal negative regulatory molecule at the immune checkpoint axis and has complicated biological functions besides immune regulation (38). Detection of PD-L1 level in cancer cells is of primary importance for predicting its biological functions as well as prognosis of immune therapy in various cancers such as melanoma. Previous studies have reported that PD-L1 is widely expressed in most glioma cell lines including U251 (12, 29) and human glioma tissues (14), indicating important biological functions of PD-L1 in glioma. Here, we clearly demonstrated that PD-L1+-glioma cells were highly aggressive (**Figure 1**). Compared to rat/C6 and nude mouse/U87 glioma models, in which 1,000,000 or 250,000 glioma cells were implanted in the striatum and only a few glioma cells migrated outside from the tumor rim at day 14 or 24 later (29), in our orthotopic glioma model, only 20,000 G422 cells were microinjected into the striatum of mouse brain. In very short time (6 d) after initial glioma cell injection, PD-L1+-glioma cells had already migrated far from the glioma center (**Figures 1A,B**). Afterwards, invasive PD-L1+-glioma cells quickly reached the outer surface of cerebral cortex at day 10 (**Figures 1C,D**) and middle line of two hemispheres at day 12 (**Figures 1E,F**) after the initial glioma cell implantation. Obviously, the G422 glioma model mimics well the invasive pathological process of GBM. Since PD-L1 in cell membrane (39), exosome (40), and cytoplasm (29) are all pivotal for cancer development and progress, we can infer that the elevation of PD-L1 levels in those infiltrating glioma cells heavily contribute to their invasiveness.

To systematically reveal the role of PD-L1 in glioma cells, we conducted RNA sequencing in PD-L1-overexpressed human glioma U251 cells, which might mimic the status of higher PD-L1 levels in those infiltrating glioma cells. Bioinformatics analysis of DEGs clearly pointed out that increased PD-L1 in glioma was closely associated with glioma malignancy (**Figure 2A**), as well as distinct signaling pathways in invasive malignant glioma, such as cell migration, PI3K/Akt and actin organization (**Figures 2B–D** and **Figures S1, S3**). In a database of 674 glioma human samples, mRNA levels of PD-L1(i.e., CD274) were significantly positively correlated to those of PI3K (i.e., PI3KCG) and β-actin (i.e., ACTB) (**Figures 2F,H**), supporting our RNAsequencing data. Also, bioinformatics analysis of glioma patient samples in opensource database showed that higher PD-L1 expression indicates poorer patient survival (**Figure 2E**), supporting that PD-L1's increase in infiltrating glioma cells is of clinical importance.

It is well known that GBM tissues suffer severe ischemic starvation, which is an important pathological factor driving aggressiveness (1, 2). PI3K/Akt is a pivotal signaling pathway promoting cellsurvival and suppressing autophagy under normal and ischemic conditions, while ischemic starvation is a major pathological stimulus for autophagy and actin cytoskeleton collapse (41, 42). It is conceivable that boundary GBM cells encounter energy-deprivation and higher PD-L1 expression may prevent GBM cell death and promote invasion via Aktautophagy-actin signaling. Indeed, bioinformatics analysis of our RNA-sequencing data showed that PD-L1 affected autophagic signaling (**Figure S2**). By applying the glioma patient database, we found that mRNA levels of PD-L1 were significantly positively correlated to those of p62 (i.e., SQSTM1) (**Figure 2G**). Consistently, our experimental results demonstrated that PD-L1 significantly resumed EBSS starvation-induced Akt-autophagy signaling in glioma cells in vitro (**Figure 3**) but had minor effects under normal culture conditions (**Figure 4B** and **Figure S4**).

The effects of PD-L1 overexpression on Akt increase and LC3 decrease were verified in vivo in a orthotopic rat/C6 glioma model (**Figure S5**). Further, via double-fluorescent immunostaining, we verified that higher PD-L1 levels in individual GBM cells were associated with lower Beclin1 and LC3 levels in GBM patient tissues (**Figure S6**). Such evidence consistently supported that PD-L1 suppressed autophagy via Akt induction/activation. In addition, PD-L1 increased F-actin formation and Akt/Factin co-localization beneath cell membrane in glioma cells upon starvation (**Figures 6E**, **7A**). In other cancer cells such as sarcoma cells, melanoma and ovarian cancer cells, PD-L1 also regulates Akt-mediated autophagic signaling (27, 31). This data largely supported our proposed PD-L1-Akt-p62 autophagy-actin-invasion mechanism in frontier GBM cells. Further collection of evidence is helpful to elucidate the exact invasive mechanism of GBM in vivo.

Importantly, we identified a novel mechanism by which PD-L1 regulate PI3K/Akt signaling. Among various upstream signaling proteins in PI3K/Akt pathway, we found that PD-L1 preferentially bound to Akt isoforms, and this became more evident upon EBSS incubation (**Figure 5** and **Figure S7**). Moreover, we dissected the binding element of PD-L1 and Akt. Our data revealed that the 128-237aa fragment of PD-L1 and the 112-480aa fragment of Akt were responsible for PD-L1/Akt interaction (**Figure 5**). Mutation of K14 and Y176, two active sites for the membrane-translocation of Akt, did not alter PD-L1/Akt interactions (**Figure 5F**), suggesting that PD-L1/Akt interaction occurred mostly in the cytoplasm. Upon PD-L1 overexpression, membrane-bound Akt was prominently increased (**Figure 6D**), supporting that cytoplasmic PD-L1/Akt interactions facilitated Akt translocation from cytoplasm to cell membrane, which is required for full activation of Akt (36). Interestingly, LY294002 (inhibiting p-Akt via PI3K) but not Perifosin (inhibitor of membrane-translocation of Akt and p-Akt via PH domain) (37) could abolished Akt phosphorylation in the presence of PD-L1 overexpression (**Figures 6A–C**). Such evidence suggested that the binding of PD-L1 to non-PH domain of Akt may prevented Perifosin action via some unknown mechanism, thus strengthened the attachment of Akt PHdomain to cell membrane and facilitated its full phosphorylation (**Figure 7B**). Further study on the mechanisms of PD-L1/Akt interactions requires more experimental data in the future.

In summary, our findings indicate the following model for PD-L1 actions in GBM (**Figure 7B**): Along with the rapid GBM growth, inner GBM cells suffer severe starvation, which causes cytoskeleton collase; while frontier GBM cells suffer less severe starvation, which induces PD-L1 expression and its binding to Akt non-PH domain. This reinforces Akt's membrane-binding and leads to increased full activation of Akt as well as its downstream p62/LC3/F-actin signaling, preventing starvationinduced Akt inhibition and facilitating GBM cell invasion. By combining our previous findings that PD-L1 promotes EMT of glioma cells, we speculate that PD-L1/Akt/autophagy/F-actin is a key driving force for GBM aggressiveness, which serves as a potential therapeutic target for GBM.

#### REFERENCES

#### DATA AVAILABILITY STATEMENT

The datasets generated for this study can be found in the NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE107581).

#### ETHICS STATEMENT

The animal study was reviewed and approved by Experimental Animal Ethics Committee, Tongji Medical College, Huazhong University of Science and Technology.

#### AUTHOR CONTRIBUTIONS

RC, XX, XQ, FL, CL, YL, XL, and GJ performed the experiment. RC, FP, XQ, and XC data analysis, wrote the manuscript, and contributed to discussion. FH and DL contributed to discussion. RC, FP, XQ, and XC designed and wrote the manuscript.

#### FUNDING

This work was supported by grants from the National Nature Science Foundation of China (Grant Nos. 81471386, 81672504 to XC, Grant No. 81873854 to FP, Grant No. 81602202 to FH). The Fundamental Research Funds for the Central Universities, HUST (Grant No. 2017KFYXJJ048 to XC). Natural Science Foundation of Hubei Province (No. 2017 CFB639), Hubei Province Health and Family Planning Scientific Research Project (No. WJ2015Q002) and Science and Technology Planning Project of Wuhan (No.<PHONE_NUMBER>010202) to FP. We declare all sources of funding received for the research being submitted.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.01347/full#supplementary-material

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Chen, Xu, Liu, Li, Li, Li, Jiang, Hu, Liu, Pan, Qiu and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# FAP-a and GOLPH3 Are Hallmarks of DCIS Progression to Invasive Breast Cancer

Li-Na Yu1,2,3, Zhen Liu4 , Yan Tian1,2,3, Pei-Pei Zhao1,2,3 and Xing Hua5 \*

1 Department of Pathology, Nanfang Hospital, Guangzhou, China, 2 Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China, 3 Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, Guangzhou, China, 4 Department of Pathology, School of Clinical Medicine, Guizhou Medical University, Guiyang, China, 5 Department of Pathology, Guangzhou Red Cross Hospital, Medical College, Jinan University, Guangzhou, China