CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein.

BACKGROUND: Circular RNAs (circRNAs) play important roles in many biological processes. However, the detailed mechanism underlying the critical roles of circRNAs in cancer remains largely unexplored. We aim to explore the molecular mechanisms of circRTN4 with critical roles in pancreatic ductal adenocarcinoma (PDAC). METHODS: CircRTN4 expression level was examined in PDAC primary tumors. The oncogenic roles of circRTN4 in PDAC tumor growth and metastasis were studied in mouse tumor models. Bioinformatics analysis, luciferase assay and miRNA pulldown assay were performed to study the novel circRTN4-miRNA-lncRNA pathway. To identify circRTN4-interacting proteins, we performed circRNA-pulldown and mass spectrometry in PDAC cells. Protein stability assay and 3-Dimensional structure modeling were performed to reveal the role of circRTN4 in stabilizing RAB11FIP1. RESULTS: CircRTN4 was significantly upregulated in primary tumors from PDAC patients. In vitro and in vivo functional studies revealed that circRTN4 promoted PDAC tumor growth and liver metastasis. Mechanistically, circRTN4 interacted with tumor suppressor miR-497-5p in PDAC cells. CircRTN4 knockdown upregulated miR-497-5p to inhibit the oncogenic lncRNA HOTTIP expression. Furthermore, we identified critical circRTN4-intercting proteins by circRNA-pulldown in PDAC cells. CircRTN4 interacted with important epithelial-mesenchymal transition (EMT)- driver RAB11FIP1 to block its ubiquitination site. We found that circRTN4 knockdown promoted the degradation of RAB11FIP1 by increasing its ubiquitination. Also, circRTN4 knockdown inhibited the expression of RAB11FIP1-regulating EMT-markers Slug, Snai1, Twist, Zeb1 and N-cadherin in PDAC. CONCLUSION: The upregulated circRTN4 promotes tumor growth and liver metastasis in PDAC through the novel circRTN4-miR-497-5p-HOTTIP pathway. Also, circRTN4 stabilizes RAB11FIP1 to contribute EMT.

Receptor tyrosine kinase fusions act as a significant alternative driver of the serrated pathway in colorectal cancer development.

Serrated polyps are a clinically and molecularly heterogeneous group of lesions that can contribute to the development of colorectal cancers (CRCs). However, the molecular mechanism underlying the development of serrated lesions is still not well understood. Here, we combined multiple approaches to analyze the genetic alterations in 86 colorectal adenomas (including 35 sessile serrated lesions, 15 traditional adenomas, and 36 conventional adenomatous polyps). We also investigated the in vitro and in vivo oncogenic properties of a novel variant of the NCOA4-RET fusion gene. Molecular profiling revealed that sessile serrated lesions and traditional serrated adenomas have distinct clinicopathological and molecular features. Moreover, we identified receptor tyrosine kinase translocations exclusively in sessile serrated lesions (17%), and the observation was validated in a separate cohort of 34 sessile serrated lesions (15%). The kinase fusions as well as the BRAF and KRAS mutations were mutually exclusive to each other. Ectopic expression of a novel variant of the NCOA4-RET fusion gene promoted cell proliferation in vitro and in vivo, and the proliferation was significantly suppressed by RET kinase inhibitors. All of these underscored the importance of mitogen-activated protein kinase (MAPK) pathway activation in the serrated pathway of colorectal tumorigenesis. In addition, we demonstrated that the kinase fusion may occur early in the precursor lesion and subsequent loss of TP53 may drives the transformation to carcinoma during serrated tumorigenesis. In conclusion, we identified kinase fusions as a significant alternative driver of the serrated pathway in colorectal cancer development, and detecting their presence may serve as a biomarker for the diagnosis of sessile serrated lesions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Loss of tumor suppressor IGFBP4 drives epigenetic reprogramming in hepatic carcinogenesis.

Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition.

Increased expression of GATA zinc finger domain containing 1 through gene amplification promotes liver cancer by directly inducing phosphatase of regenerating liver 3.

We identified that GATA zinc finger domain containing 1 (GATAD1), a transcriptional factor, was significantly up-regulated in hepatocellular carcinoma (HCC) through gene amplification. We demonstrated the critical role, molecular mechanisms, and clinical implications of GATAD1 as a novel oncogenic factor in HCC. We found that GATAD1 protein was expressed in 76.6% of primary HCCs (85/111) but silenced in normal liver tissues. Gene amplification of GATAD1 was positively correlated with its overexpression in primary HCCs (R = 0.629, P < 0.0001). GATAD1 significantly increased cell proliferation, G1 -S cell cycle transition, and migration/invasion but suppressed apoptosis in liver cell lines and promoted tumor growth and lung metastasis in both xenograft and orthotopic mouse models. Mechanistically, GATAD1 induced the transcriptional expression of phosphatase of regenerating liver 3 (PRL3) by binding to its promoter identified by RNA sequencing and chromatin immunoprecipitation-PCR analyses. PRL3 played an oncogenic role in HCC. Knockdown of PRL3 blunted the tumorigenic effect of GATAD1. In addition, GATAD1 activated Akt signaling, evidenced by increased phosphorylation levels of total Akt, Akt1, Akt2, and Akt target glycogen synthase kinase 3ß, while knockdown of PRL3 abolished this effect of GATAD1. We further unveiled that PRL3 activated Akt signaling by dephosphorylating phosphatase and tensin homolog at tyrosine residue, thus reducing phosphatase and tensin homolog protein. The PRL3 inhibitor 5-[[5-bromo-2-[(2-bromophenyl)methoxy]phenyl]methylene]-2-thioxo-4-thiazolidinone significantly suppressed HCC growth by inhibiting Akt activation. Moreover, high GATAD1 nuclear protein expression was associated with poor survival of HCC patients as an independent prognostic factor. CONCLUSION: GATAD1 plays a pivotal oncogenic role in HCC by directly inducing PRL3 transcription to activate the Akt signaling pathway. GATAD1 may serve as an independent poor prognostic factor for HCC patients. (Hepatology 2018;67:2302-2319).

Eukaryotic elongation factor-2 kinase expression is an independent prognostic factor in colorectal cancer.

BACKGROUND: Prognostication of patients with colorectal cancer (CRC) currently relies on tumor-node-metastasis (TNM) staging but clinical outcomes of patients of the same histoclinical stage are heterogeneous. It is therefore imperative to devise novel molecular tests to stratify CRC patients. Our previous work demonstrated that eukaryotic elongation factor-2 kinase (EEF2K) is a tumor suppressor in CRC. Herein, we investigated EEF2K expression in CRC and determined its relationship with clinicopathological parameters. METHODS: Quantitative RT-PCR and Westerns blots were used to examine EEF2K expression in primary tumor and the adjacent non-tumor tissues of CRC patients (n = 20). Kaplan-Meier curves and Cox regression analysis were used to assess the association between clinical outcomes of CRC patients and EEF2K protein expression determined by immunohistochemistry on tissue microarray (n = 151). RESULTS: EEF2K was significantly downregulated at both mRNA and protein levels in tumors of CRC patients. Univariate Cox regression analysis revealed that CRC patients with high tumor grade, advanced TNM staging and low EEF2K expression were associated with worse overall survival. Multivariate analysis further demonstrated that low EEF2K expression was an independent factor for predicting poorer overall survival in CRC patients (p = 0.014; Hazard ratio = 2.951; 95% confidence interval: 1.240-7.024). The 5-year survival rate was 82.8% in the EEF2K-high-expression group versus 63.9% in the EEF2K-low-expression group (p = 0.0118). The association of overall survival with EEF2K expression in CRC patients was verified in The Cancer Genome Atlas (TCGA) cohort. CONCLUSIONS: EEF2K is downregulated in CRC and its expression can be employed as a prognostic marker for CRC patients independent of TNM staging.

EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

EXOSC4 functions as a potential oncogene in development and progression of colorectal cancer.

Colorectal cancer (CRC) is a heterogeneous disease with complex mechanisms of pathogenesis. Classification systems have been proposed based on molecular features of tumors in clinical practice. Thus, more molecular markers associated with development and progression of CRC might serve as useful tools for early diagnosis even for providing more accurate molecular classification. Frequent gain of chromosome 8q was detected in CRC by array-CGH and overexpression of exosome component 4 (EXOSC4) in this region was revealed by expression microarray analysis. Through qRT-PCR and immunohistochemistry (IHC) analysis, EXOSC4 showed increased expression in CRC cell lines and clinical specimens. Higher expression of EXOSC4 was more often detected in left side, and correlated with BRAF wild type, MSI-low or MSS, CIMP-low, and MLH1-no-silence CRC patients. Functionally, EXOSC4 overexpression increased early tumorigenic capacity by promoting cell proliferation and monolayer colony formation, enhancing cell invasion and migration study and accelerating xenograft formation in nude mice. While EXOSC4 knockdown exhibited anti-oncogenic role such as inhibiting cell proliferation and invasion. EXOSC4 inhibition also resulted in G1 phase cell cycle arrest. For the downstream signaling analysis, EXOSC4 was found to be involved in multiple signaling pathways such as cell cycle, p53 pathway and Wnt pathway. In summary, our findings demonstrated the oncogenic role of EXOSC4 in development and progression of CRC. Deep understanding of EXOSC4 as a potential diagnostic molecular biomarker will provide clinical translational potential for intervention therapy.

Assessment of programmed cell death ligand-1 expression by 4 diagnostic assays and its clinicopathological correlation in a large cohort of surgical resected non-small cell lung carcinoma.

Immune checkpoint blockade targeting the PD-1/PD-L1 axis has recently demonstrated efficacy and promise in cancer treatment. Appropriate biomarker selection is therefore essential for improving treatment efficacy. However, the establishment of PD-L1 assay in pathology laboratories is complicated by the presence of multiple testing platforms using different scoring systems. Here we assessed the PD-L1 expression in 713 consecutive non-small cell lung carcinomas by four commercially available PD-L1 immunohistochemical assays, namely, 22C3, 28-8, SP142 and SP263. The analytical performances of the four assays and diagnostic performances across clinically relevant cutoffs were evaluated. The prevalence of PD-L1 (22C3) expression was 21% with a ≥50% cutoff and 56% with a ≥1% cutoff. High PD-L1 expression (using a ≥50% cutoff) was significantly associated with male sex (P = 0.001), ever smoking history (P < 0.001), squamous cell carcinoma (P = 0.001), large cell carcinoma (P < 0.001), lymphoepithelioma-like carcinoma (P = 0.006), sarcomatoid carcinoma (P < 0.001), mutant KRAS (P = 0.005) and wild-type EGFR (P = 0.003). Elevated PD-L1 expression was also significantly associated with shorter survival in patients with adenocarcinoma (log-rank P = 0.026) and remained an independent prognostic factor by multivariable analysis. Among the four assays, 22C3, 28-8 and SP263 were highly concordant for tumor cell scoring. With a cutoff of ≥50% (i.e., the threshold for first-line patient selection), inter-rater agreement was high among the three assays with percentage agreement >97%. In conclusion, three PD-L1 assays showed good analytical performance and a high agreement with each other, but not all cases were correctly classified using the same clinical cutoff. Further studies comparing the predictive value of these assays are required to address the interchangeability of these assays for clinical use.

The role of human papillomavirus in laryngeal cancer in Southern China.

To delineate the role of human papillomavirus (HPV) in laryngeal cancer in Southern Chinese, a retrospective cross-sectional study was conducted in a major otorhinolaryngology referral center in Hong Kong. Eighty-five Chinese patients with histology-confirmed laryngeal squamous cell carcinoma (LSCC) diagnosed between 2005 and 2010 were examined for the status of HPV by PCR, and the expression of p16 and p53 by immunohistochemistry. The HPV, p16 and p53 findings were correlated with clinicopathological features, recurrence and 5-year survival. HPV DNA was detected in one patient (1.2%, 95%CI: 0.2-6.4%) who had glottic cancer and harbored HPV-6. Overexpression of p16 and p53 were detected in 11 (12.9%) and 47 (55.3%) cases, respectively. Recurrence occurred in 22.4% of patients at a median of 13 months. The 5-year overall survival and disease-specific survival were 55.7% and 72.4%, respectively. Overexpression of p16 or p53 was not associated with clinicopathological features, recurrence or overall survival. HPV plays a limited role in laryngeal cancer in Hong Kong Southern Chinese. In contrast to oropharyngeal cancer, p16 cannot be used as a surrogate marker for oncogenic involvement of HPV and cannot predict survival in laryngeal cancer.

Concurrent fatty liver increases risk of hepatocellular carcinoma among patients with chronic hepatitis B.

BACKGROUND AND AIMS: Concurrent fatty liver in hepatitis B virus (HBV)-infected patients without significant alcohol intake is a frequent and increasingly alarming problem because of the non-alcoholic fatty liver disease pandemic. The risk of HBV-related hepatocellular carcinoma (HCC) development was increased by concomitant obesity and diabetes. Direct evidence of the hepatocarcinogenic effect of fatty liver in chronic HBV remains elusive. We aimed to evaluate the risk of concurrent histologically proven fatty liver in HBV hepatocarcinogenesis. METHODS: We conducted a retrospective cohort study on a liver biopsy cohort of HBV-infected patients without significant alcohol intake to evaluate the prevalence of concurrent histologically proven fatty liver and its association with subsequent HCC development. We also examined nine polymorphisms on six non-alcoholic fatty liver disease-related candidate genes (ADIPOQ, APOC3, GCKR, LEPR, PNPLA3, and PPARG). RESULTS: Among 270 HBV-infected patients, concurrent fatty liver was found in 107 patients (39.6%) and was associated with metabolic risks, cirrhosis (P = 0.016) and PNPLA3 rs738409 CG/GG genotype (P = 0.002). At a median follow-up of 79.9 months, 11 patients (4.1%) developed HCC, and nine of them had concurrent fatty liver. By multivariable Cox analysis, concurrent fatty liver (HR 7.27, 95% confidence interval: 1.52-34.76; P = 0.013), age, cirrhosis, and APOC3 rs2854116 TC/CC genotype (HR 3.93, 95% confidence interval: 1.30-11.84; P = 0.013) were independent factors predicting HCC development. CONCLUSIONS: Concurrent fatty liver is common in HBV-infected patients and an independent risk factor potentiating HBV-associated HCC development by 7.3-fold. The risk of HBV-related HCC is increased by APOC3 gene polymorphism, and further characterization is required by its role.

miR-508-3p concordantly silences NFKB1 and RELA to inactivate canonical NF-κB signaling in gastric carcinogenesis.

BACKGROUND: NF-κB signaling pathway plays an important role in gastric carcinogenesis. The basic expression and functional role of NFKB1 and RELA (components of canonical NF-κB pathway) in gastric cancer (GC) have not been well elucidated. In this study, the role of NFKB1 and RELA in gastric tumorigenesis will be investigated and their regulation by microRNAs (miRNAs) will be deeply explored. METHODS: The mRNA and protein expression of NFKB1 and RELA were investigated by qRT-PCR and Western blot in GC cell lines and primary tumors. The functional roles of NFKB1 and RELA in GC were demonstrated by MTT proliferation assay, monolayer colony formation, cell invasion and migration, cell cycle analysis and in vivo study through siRNA mediated knockdown. Identification of NFKB1 as a direct target of tumor suppressor miRNA miR-508-3p was achieved by expression regulation assays together with dual luciferase activity experiments. RESULTS: NFKB1 and RELA were up-regulated in GC cell lines and primary tumors compared with normal gastric epithelium cells and their upregulation correlation with poor survival in GC. siRNA mediated knockdown of NFKB1 or RELA exhibited anti-oncogenic effect both in vitro and in vivo. NFKB1 was further revealed to be a direct target of miR-508-3p in gastric tumorigenesis and their expression showed negative correlation in primary GC samples. miR-508-3p was down-regulated in GC cells compared with normal gastric epithelium samples and its ectopic expression in GC cell lines also exerts tumor suppressor function. NFKB1 re-expression was found to partly abolish the tumor-suppressive effect of miR-508-3p in GC. CONCLUSION: All these findings supports that canonical NF-κB signaling pathway is activated in GC at least by the inactivation of miR-508-3p and this might have therapeutic potential in GC treatment.

Steatotic hepatocellular carcinoma: a variant associated with metabolic factors and late tumour relapse.

AIMS: Steatosis in hepatocellular carcinoma (HCC) has been recognized for decades and found most commonly in small, well-differentiated HCC. However, the clinicopathological features and pathogenesis of HCC with steatosis is not well characterized. There are few data concerning whether HCC with steatosis should be regarded a distinct histological variant, steatotic HCC. METHODS AND RESULTS: A retrospective cohort of 516 patients undergoing curative surgery for primary HCC was recruited. The median follow-up was 45.5 (range: 0.2-166.0) months. Steatotic HCC was defined as HCC with significant steatosis (≥5% of tumour cells). Associations with immunohistochemical expression of fatty acid binding protein-1 (FABP1), sonic hedgehog (SHH) and gene polymorphism of patatin-like phospholipase 3 (PNPLA3) were analysed. Steatotic HCC was found in 21.1% of patients and was associated with higher metabolic risks [diabetes mellitus (36.7% versus 18.2%) and hypertension (44.0% versus 28.7%)], underlying fatty liver (60.6% versus 37.8%), steatohepatitis (30.3% versus 13.0%), smaller tumour size, lower frequency of major vessel (1.8% versus 11.3%) and microvascular invasion (20.2% versus 32.4%), earlier tumour stages and lower serum alpha-fetoprotein. It was associated with developing late tumour relapse (hazard ratio 2.15, P = 0.002) independently of underlying cirrhosis and non-anatomical excision. Steatotic HCC did not differ from HCC without significant steatosis in immunohistochemical expression of FABP1 and SHH and PNPLA3 gene polymorphism. CONCLUSION: Steatotic HCC is a common histological variant of HCC with distinct association with underlying fatty liver, steatohepatitis and metabolic risks. Despite more favourable baseline tumour features, it was associated with late tumour relapse.

Targeting of YAP1 by microRNA-15a and microRNA-16-1 exerts tumor suppressor function in gastric adenocarcinoma.

BACKGROUND: MicroRNAs (miRNAs) have been reported to play an important role in tumorigenesis. In this study, the role of miR-15a and miR-16-1 in gastric adenocarcinoma (GAC) was investigated. METHODS: The expression of miR-15a and miR-16-1 in cell lines and primary tumors was examined by miRNA qRT-PCR. Proliferative assays, colony formation, cell invasion and migration, flow cytometry analysis and in vivo study were performed by ectopic expression of miR-15a and miR-16-1. The putative target genes of miR-15a and miR-16-1 were explored by TargetScan and further validated. RESULTS: We found that miR-15a and miR-16-1 were down-regulated in GAC cell lines and primary tumor samples compared with normal gastric epithelium. Functional study demonstrated that ectopic expression of miR-15a and miR-16-1 suppressed cell proliferation, monolayer colony formation, invasion and migration, and xenograft formation in vivo. In addition, miR-15a and miR-16-1 induced G0/G1 cell cycle arrest which was further confirmed by Western blot and qRT-PCR of related cell cycle regulators. YAP1 was confirmed to be a functional target of miR-15a and miR-16-1 in GAC. YAP1 re-expression partly abrogated the inhibitory effect of miR-15a and miR-16-1 in GAC cells. In clinical samples, YAP1 protein expression shows negative correlation with miR-15a and miR-16-1 expression. CONCLUSION: In conclusion, targeting YAP1 by tumor suppressor miRNA miR-15a and miR-16-1 plays inhibitory effect and this might have a therapeutic potential in GAC.

let-7b/g silencing activates AKT signaling to promote gastric carcinogenesis.

BACKGROUND: Aberrant AKT activation contributes to gastric cancer cell survival and chemotherapy resistance, however its regulation is poorly understood. microRNAs have been established to be important regulators in gastric carcinogenesis. Here, we showed the functional role and putative target of let-7b and let-7g (let-7b/g) in gastric carcinogenesis. METHODS: The expression of let-7b/g in gastric cancer cell lines and primary tumors were evaluated by miRNA qRT-PCR. The putative target gene of let-7b/g was explored by TargetScan followed by further validation. Functional analyses including MTT proliferation, monolayer colony formation, cell invasion assays and in vivo study were performed in both ectopic expression and knockdown approaches. RESULTS: let-7b/g was found down-regulated in gastric cancer and its downregulation was associated with poor survival and correlated with lymph node metastasis. let-7b/g inhibited AKT2 expression by directly binding to its 3'UTR, reduced p-AKT (S473) activation and suppressed expression of the downstream effector pS6. AKT2 mRNA expression showed negative correlation with the expression of let-7b/g in primary tumors. Short interfering RNA (siRNA) mediated knockdown of AKT2 phenocopied the tumor-suppressive effects of let-7b/g. Moreover, AKT2 re-expression partly abrogated the growth-inhibitory effect of let-7b/g. CONCLUSION: In conclusion, our findings reveal decreased let-7b/g contributes to aberrant AKT activation in gastric tumorigenesis and provide a potential therapeutic strategy for gastric cancer.

Yin Yang 1 contributes to gastric carcinogenesis and its nuclear expression correlates with shorter survival in patients with early stage gastric adenocarcinoma.

BACKGROUND: Yin Yang 1 (YY1) is a transcription factor that regulates diverse biological processes and increasing recognized to have important roles in carcinogenesis. The function and clinical significance of YY1 in gastric adenocarcinoma (GAC) have not been elucidated. METHODS: In this study, the functional role of YY1 in gastric cancer was investigated by MTT proliferation assays, monolayer colony formation, cell cycle analysis, signaling pathway analysis, Western blot analysis and in vivo study through YY1 knockdown or overexpression. Immunohistochemical study with YY1 antibody was performed on tissue microarray consisting of 247 clinical GAC samples. The clinical correlation and prognosis significance were evaluated. RESULTS: YY1 expression was up-regulated in gastric cancer cell lines and primary gastric cancers. Knocking down YY1 by siYY1 inhibited cell growth, inducing G1 phase accumulation and apoptosis. Ectopic YY1 expression enhanced cell proliferation in vitro and in vivo. Knocking down YY1 in gastric cancer cells suppressed proliferation by inhibiting Wnt/ß-catenin pathway, whereas its overexpression exerted oncogenic property by activating Wnt/ß-catenin pathway. In primary GAC samples, YY1 nuclear expression correlated with shorter survival and predicted poor prognosis in early stage GACs. CONCLUSION: Our data demonstrated that YY1 contributes to gastric carcinogenesis in gastric cancer. In early stage GACs YY1 might serve as a poor prognostic marker and possibly as a potential therapeutic target.

Epstein-Barr virus-associated lymphoepithelioma-like cholangiocarcinoma: a rare variant of intrahepatic cholangiocarcinoma with favourable outcome.

AIMS: Lymphoepithelioma-like cholangiocarcinoma (LELCC) is a rare variant of intrahepatic cholangiocarcinoma (IHCC). Only 16 cases have reported previously in the literature, and about 62.5% of these cases were associated with Epstein-Barr virus (EBV). METHODS AND RESULTS: We present the largest series (seven cases) of LELCC with descriptions of clinical and pathological characteristics, investigations of aberrant DNA methylation and mutation analyses of EGFR and KRAS. The evaluation also included 11 cases of stage-matched conventional IHCC for comparison. RESULTS: All seven patients of LELCC were female and had stage I disease. LELCC had significantly better 2- and 5-year overall survival than IHCC (100% versus 52.8%, and 100% versus 13.2%, respectively, P = 0.003). All seven LELCCs were EBV-associated and composed exclusively of adenocarcinoma with varied glandular differentiation, dense lymphoplasmacytic infiltrate, and variable expression of biliary-type cytokeratins (CK7 and CK19) and stemness markers (CD133 and EpCAM). Gene hypermethylation was more frequent in LELCC than IHCC. CRBPI (85.7% versus 9.1%, P < 0.003) and CRBPIV (85.7% versus 0%, P < 0.001) showed statistically higher methylation frequencies in LELCC than IHCC. No LELCC harboured any EGFR or KRAS mutation. CONCLUSION: Epstein-Barr virus-associated LELCC is a variant of IHCC, characterized by marked female predominance, favourable overall survival and distinctively frequent DNA hypermethylation.

Expression of stemness markers (CD133 and EpCAM) in prognostication of hepatocellular carcinoma.

AIMS: The expression of stemness markers in hepatocellular carcinoma (HCC) is suggested to be associated with poor clinical outcome after surgical resection. There are few data on their independent prognostic role in addition to the existing AJCC TNM staging system. METHODS AND RESULTS: The immunohistochemical expression of CD133, EpCAM, CK19 and CD56 was studied in a cohort of 282 surgical specimens collected from patients undergoing resection of primary HCC. CD133-positive HCCs were usually smaller in size (P = 0.002) and arose more frequently in cirrhotic liver (P = 0.002). CD133 expression was an independent prognostic factor for overall survival (hazard ratio 2.30, P < 0.001), and a highly potent prognostic factor in patients with stage I disease (hazard ratio 3.91, P = 0.001). EpCAM expression was associated with younger age (P < 0.001), smaller tumour size (P = 0.018) and poorer histological differentiation (P = 0.042). EpCAM immunoreactivity was an independent factor for disease-free survival in HCCs at all stages (hazard ratio 2.05, P = 0.001), stage II (hazard ratio 3.66, P = 0.010) and stages III/IV (hazard ratio 3.22, P = 0.001). Neither CK19 nor CD56 offered any independent prognostic value. CONCLUSION: The prognostic role of CD133 was most significant in TNM stage I disease, while the prognostic role of EpCAM was more apparent in more advanced TNM stages.

Genome-wide identification of Epstein-Barr virus-driven promoter methylation profiles of human genes in gastric cancer cells.

BACKGROUND: Aberrant methylation of tumor-related genes has been reported in Epstein-Barr virus (EBV)-associated gastric cancers. This study sought to profile EBV-driven hypermethylation in EBV-infected cells. METHODS: The EBV-positive AGS gastric cancer cell line (AGS-EBV) and EBV-negative AGS cells were used in this study. DNA methyltransferase-3b (DNMT3b) activity was assessed by EpiQuick activity assay, and genome-wide DNA methylation profiles were assessed by methyl-DNA immunoprecipitation microarray assay. RESULTS: EBV infection was confirmed in AGS-EBV cells by EBV-encoded RNA in situ hybridization. Expression and activity of DNA methyltransferase-3b (DNMT3b) was significantly increased in AGS-EBV compared to AGS. Ectopic expression of LMP2A (latent membrane protein 2A) in AGS increased activity of DNMT3b. A total of 1065 genes were differentially methylated by EBV infection (fold-changes ≥ 2, P < .05) in AGS-EBV compared to AGS cells. The majority of the differentially methylated genes (83.2%, 886 of 1065 genes) had cytosine-guanine dinucleotide (CpG) hypermethylation in AGS-EBV (fold-changes 2.43∼65.2) versus that found in AGS cells. Gene ontology analysis revealed that hypermethylated genes were enriched in the important cancer pathways (≥ 10 genes each, P ≤ .05) including mitogen-activated protein kinase signaling, cell adhesion molecules, wnt signaling pathway, and so forth. Six novel hypermethylated candidates (IL15RA, REC8, SSTR1, EPHB6, MDGA2, and SCARF2) were further validated. Higher levels of DNA methylation were confirmed for all these genes in AGS-EBV cells by bisulfite genomic sequencing. Furthermore, these candidates were silenced or down-regulated in AGS-EBV cells, but can be restored by demethylation treatment. CONCLUSIONS: EBV infection in AGS cells induced aberrant CpG hypermethylation of 886 genes involving in important cancer-related pathways. Induction of promoter methylation by EBV is regulated by up-regulation of DNMT3b through LMP2A.

Zinc finger E-box binding factor 1 plays a central role in regulating Epstein-Barr virus (EBV) latent-lytic switch and acts as a therapeutic target in EBV-associated gastric cancer.

BACKGROUND: The role of Epstein-Barr virus (EBV) infection in gastric carcinogenesis remains largely unknown. The authors studied the effects of zinc finger E-box binding factor 1 (ZEB1) on latent-lytic switch of EBV infection in gastric cancer and explored the importance of EBV in gastric carcinogenesis. METHODS: Loss or gain of ZEB1 function was obtained by ZEB1 small-interfering RNA (siRNA) knock-down or forced ZEB1 re-expression. Cell growth was evaluated by cell viability and colony formation assays, and the cell cycle was assessed by flow cytometry. EBV was detected using quantitative polymerase chain reaction (PCR) and in situ hybridization analyses. RESULTS: ZEB1 knock-down in a latent EBV-infected gastric cancer cell line (YCC10) increased lytic gene BamHI W leftward reading frame 1 (BZLF1) expression and decreased the expression of latent gene EB nuclear antigen 1 (EBNA1) concomitant with the inhibition of cell viability (P < .05) and S-phase DNA synthesis (P < .01). ZEB1 depletion combined with ganciclovir revealed a further reduction in cell viability (P < .001). ZEB1 knock-down induced cell apoptosis and the up-regulation of caspase 3 and poly(adenosine diphosphate-ribose) polymerase cleavage. Conversely, ectopic overexpression of ZEB1 in a lytic EBV-infected gastric cancer cell line (AGS-EBV) inhibited BZLF1 promoter (Zp) activity, BZLF1 expression, and apoptosis and promoted cell growth. EBV infection was detected in 11.3% (80 of 711) of gastric cancers. The presence of EBV was associated with age, men, and intestinal type cancer. CONCLUSIONS: ZEB1 was confirmed as a key mediator of the latent-lytic switch of EBV-associated gastric cancer, a distinct subtype with different clinicopathologic features. The current results indicated that inhibition of ZEB1 may be a potential target for EBV-associated gastric cancer therapy.

Dapper homolog 1 is a novel tumor suppressor in gastric cancer through inhibiting the nuclear factor-κB signaling pathway.

Dapper homolog 1 (DACT1) is a disheveled partner in the planar cell polarity pathway. By using genome-wide promoter methylation screening, dapper homolog 1 (DACT1) was found to be frequently methylated in gastric cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. We demonstrated that DACT1 was silenced in 7 of 10 gastric cancer cell lines and in primary gastric cancers. Transcriptional gene silence of DACT1 was mainly regulated by promoter hypermethylation. Ectopic expression of DACT1 in silenced gastric cancer cell lines (AGS, BGC823 and MGC803) by stable transfection suppressed colony formation (P < 0.001), induced cell apoptosis (P < 0.01) and retarded tumorigenesis in nude mice (P < 0.001). The tumor suppressive effect of DACT1 was further confirmed by loss of DACT1 function experiment. The proapoptotic and antiproliferative effect by DACT1 was associated with inhibition of nuclear factor (NF)-κB activation and its downstream factors, including B-cell CLL/lymphoma-2, Bcl-X, interleukin-8 and tumor necrosis factor-α. Moreover, promoter methylation of DACT1 was detected in 29.3% (60/205) of primary gastric tumors. DACT1 methylation was significantly associated with tumor metastasis (P < 0.05), invasion (P < 0.05) and advanced tumor stage (P < 0.0005). These findings provided insight into the role of DACT1 as a novel functional tumor suppressor in gastric cancer through inhibiting NF-κB signaling pathway. Promoter methylation of DACT1 is associated with tumor aggressiveness.