Joanna H.M. Tong

Viral-Human Chimeric Transcript Predisposes Risk to Liver Cancer Development and Progression

The mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects β-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.

Promoter hypermethylation of tumor-related genes in gastric intestinal metaplasia of patients with and without gastric cancer

Promoter hypermethylation is an alternative mechanism of gene silencing in human cancers including gastric cancer. While intestinal metaplasia (IM) is generally regarded as a precancerous lesion of the stomach, our study examines the presence of gene promoter hypermethylation in IM of patients with and without gastric cancer. We examined 31 samples of gastric cancer, 36 gastric IM (21 associated with gastric cancer and 15 from noncancer patients) and 10 normal gastric biopsies. Tissues containing foci of IM were carefully microdissected from paraffin‐embedded section. Bisulfite‐modifiedDNA was examined for gene promoter hypermethylation in DAP‐kinase, E‐cadherin, GSTP1, p14, p15, p16, RASSF1A and hMLH1 by methylation‐specific‐PCR. None of the control gastric tissues had hypermethylation detected, but gene promoter hypermethylation was frequently detected in gastric cancer and IM. The mean number of methylated genes in cancer and IM was 3.0 and 1.4, respectively (p < 0.0001). Methylation in IM from cancer patients was all associated with concurrent methylation in the corresponding tumor samples. The numbers of methylated genes were similar in IM obtained from cancer and noncancer patients. By examining the methylation patterns of these genes, 3 differential methylation patterns were recognized: hypermethylation was more frequent in cancer than in IM (DAP‐kinase, p14, p15 and p16); comparable frequencies of methylation in cancer and IM (E‐cadherin and hMLH1); and no methylation (GSTP1). Aberrant methylation in tumor‐related genes is frequently detected in gastric IM of both cancer and noncancer patients, suggesting their early involvement in the multistep progression of gastric carcinogenesis.

Epigenetic Therapy Using Belinostat for Patients With Unresectable Hepatocellular Carcinoma: A Multicenter Phase I/II Study With Biomarker and Pharmacokinetic Analysis of Tumors From Patients in the Mayo Phase II Consortium and the Cancer Therapeutics Research Group

PURPOSE Epigenetic aberrations have been reported in hepatocellular carcinoma (HCC). In this study of patients with unresectable HCC and chronic liver disease, epigenetic therapy with the histone deacetylase inhibitor belinostat was assessed. The objectives were to determine dose-limiting toxicity and maximum-tolerated dose (MTD), to assess pharmacokinetics in phase I, and to assess activity of and explore potential biomarkers for response in phase II. PATIENTS AND METHODS Major eligibility criteria included histologically confirmed unresectable HCC, European Cooperative Oncology Group performance score ≤ 2, and adequate organ function. Phase I consisted of 18 patients; belinostat was given intravenously once per day on days 1 to 5 every 3 weeks; dose levels were 600 mg/m(2) per day (level 1), 900 mg/m(2) per day (level 2), 1,200 mg/m(2) per day (level 3), and 1,400 mg/m(2) per day (level 4). Phase II consisted of 42 patients. The primary end point was progression-free survival (PFS), and the main secondary end points were response according to Response Evaluation Criteria in Solid Tumors (RECIST) and overall survival (OS). Exploratory analysis was conducted on pretreatment tumor tissues to determine whether HR23B expression is a potential biomarker for response. RESULTS Belinostat pharmacokinetics were linear from 600 to 1,400 mg/m(2) without significant accumulation. The MTD was not reached at the maximum dose administered. Dose level 4 was used in phase II. The median number of cycles was two (range, one to 12). The partial response (PR) and stable disease (SD) rates were 2.4% and 45.2%, respectively. The median PFS and OS were 2.64 and 6.60 months, respectively. Exploratory analysis revealed that disease stabilization rate (complete response plus PR plus SD) in tumors having high and low HR23B histoscores were 58% and 14%, respectively (P = .036). CONCLUSION Epigenetic therapy with belinostat demonstrates tumor stabilization and is generally well-tolerated. HR23B expression was associated with disease stabilization.

Frequent hypermethylation of promoter region of RASSF1A in tumor tissues and voided urine of urinary bladder cancer patients

High frequency loss of 3p21.3 region where RASSF1A located was demonstrated in several tumors. We aimed to investigate the methylation status of RASSF1A and the frequency of LOH in 3p21.3 region in bladder cancer. Three bladder cancer cell lines, 40 cases of bladder TCC and 14 cases of paired voided urine samples were subjected to methylation analysis. By methylation specific PCR, complete methylation of promoter region of RASSF1A gene were detected in cell lines T24 and UMUC3. Demethylation treatment re‐expressed RASSF1A in these 2 cell lines. Methylation of RASSF1A was also detected in 47.5% (19/40) of the TCC cases but not in 6 carcinoma in situ (CIS) or 6 normal urothelium samples. For LOH study, loss of 3p21.3 region was detected in 57.9% (11/19) of our cases. Interestingly, methylation of RASSF1A was found in 72.7% (8/11) of the cases with LOH but only in 12.5% (1/8) of the cases without LOH. Methylation of RASSF1A was detected in 50% (7/14) of voided urine samples, but not in normal control. It showed a higher sensitivity than conventional urine cytology in detecting cancer cells, especially for low grade cases. In conclusion, our results demonstrated a high frequency of RASSF1A methylation with frequent LOH in 3p21.3 region in bladder cancer. It suggested that it may be a potential tumor suppressor gene in this chromosomal region and can be silenced by promoter hypermethylation. Detection of aberrant gene methylation in routine voided urine was feasible and may provide a non‐invasive and sensitive approach for cancer detection.

Yes-Associated Protein 1 Exhibits Oncogenic Property in Gastric Cancer and Its Nuclear Accumulation Associates with Poor Prognosis

Purpose: Yes-associated protein 1 (YAP1) is a multifunctional protein that can interact with different transcription factors to activate gene expression. The role of YAP1 in tumorigenesis is unclear. We aimed to investigate the functional role of YAP1 in tumorigenesis of gastric cancer. Experimental Design: YAP1 expresson in gastric adenocarcinoma was evaluated. The biological function was determined by proliferation assay, colony formation, cell invasion, and flow cytometric analysis through knocking down or ectopic expressing YAP1 in gastric cancer cell lines coupled with in vivo study. The possible downstream effectors of YAP1 were investigated by expression microarray. Results: YAP1 protein expression was upregulated in gastric cancer. Nuclear accumulation of YAP1 was associated with poor disease-specific survival (P = 0.021), especially in patients with early-stage diseases (P < 0.001). Knockdown YAP1 resulted in a significant reduction in proliferation, anchorage-dependent colony formation, cell invasion, and cell motility. Ectopic YAP1 expression promoted anchorage-independent colony formation, induced a more invasive phenotype, and accelerated cell growth both in vitro and in vivo. Microarray analysis highlighted the alteration of MAPK (mitogen-activated protein kinase) pathway by YAP1. We confirmed a constitutive activation of RAF/MEK/ERK (extracellular signal-regulated kinase) in YAP1-expressing MKN45 cells and further showed that YAP1 enhanced serum/epidermal growth factor–induced c-Fos expression in gastric cancer cells. Conclusions: Our findings supported that YAP1 exhibits oncogenic property in gastric cancer. We provided the first evidence that YAP1 exerted the oncogenic function by enhancing the capacity to activate the early-response gene pathway. YAP1 could be a prognostic biomarker and potential therapeutic target for gastric cancer. Clin Cancer Res; 17(8); 2130–9. ©2011 AACR.

Hypermethylation of the tumor suppressor gene RASSFIA and frequent concomitant loss of heterozygosity at 3p21 in cervical cancers

Loss of heterozygosity (LOH) at chromosome 3p21 is frequent in cervical cancers. The candidate tumor suppressor gene, RASSF1A located at 3p21.3, is found to be inactivated in several major human cancers, implicating its significance in carcinogenesis. We aimed to investigate the status of RASSF1A in cervical cancers. The mutation and methylation status of RASSF1A were analysed in 4 cervical cancer cell lines, 50 primary cervical cancers including 33 squamous cell carcinoma (SCC), 17 adenocarcinoma (AC) and 11 normal controls. The primary cancer samples were also detected for LOH at 3p21 and human papillomavirus (HPV). Hypermethylation of RASSF1A was detected in 30% of SCC, 12% of AC and in 1 of the 4 cancer cell lines but was absent in all normal cases. Methylation of the cancer cell line was associated with loss of gene expression, which was restored by demethylation. About 67% (8 of 12) of hypermethylated primary cancers showed concomitant LOH at 3p21. No somatic mutation was found in all primary cancer samples or cell lines but 2 cases showed germline polymorphism at codon 133. Oncogenic HPV DNAs were found in most cancer samples. No correlation was detected between RASSF1A‐hypermethylation or LOH at 3p21 and age of patient, HPV genotype, tumor grade and stage. Hypermethylation of RASSF1A occurs in a subset of cervical cancers, among which concomitant LOH at 3p21 is common. The results supported that RASSF1A may be one of the cervical cancer‐related tumor suppressor genes located at 3p21 regions.

Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line

The interleukin-mediated Janus kinase (JAK)/STAT pathway plays a crucial role in carcinogenesis. Recently, increased STAT3 activity was found in hepatocellular carcinoma and multiple myeloma in which there was silencing of SOCS-1 (suppressor of cytokine signalling-1) by gene promoter hypermethylation. We investigated the expression level of interleukin-6 (IL-6) and SOCS-1 in gastric cancer cell lines. Expression of SOCS-1 correlated with IL-6 level in most of the cell lines, except for AGS cells in which SOCS-1 was absent despite a high level of IL-6 production. Methylation analysis by methylation-specific polymerase chain reaction and bisulphite sequencing revealed that CpG island of SOCS-1 was densely methylated in AGS cells. Demethylation treatment by 5′aza-deoxycytidine restored SOCS-1 expression and also suppressed constitutive STAT3 phosphorylation in AGS cells. Moreover, methylation of SOCS-1 was detected in 27.5% (11 of 40) of primary gastric tumours samples, 10% (one of 10) of adjacent noncancer tissues but not in any (zero of nine) normal gastric mucosa. Methylation of SOCS-1 also correlated with the loss of mRNA expression in some primary gastric cancers. In conclusion, this is the first report to demonstrate that hypermethylation of SOCS-1 led to gene silencing in gastric cancer cell line and primary tumour samples. Downregulation of SOCS-1 cooperates with IL-6 in the activation of JAK/STAT pathway in gastric cancer.

Promoter hypermethylation of tumor‐related genes in the progression of colorectal neoplasia

Gene promoter hypermethylation is increasingly recognized to play an important role in cancer development through silencing of gene transcription. This study determined the methylation profiles of primary colorectal cancers and adenomas to elucidate the role of epigenetic changes in different stages of colorectal carcinogenesis. We examined the methylation profiles of 47 sporadic colorectal cancers, 36 colonic adenomas from patients without cancer and 34 colonic biopsies from patients without colonic lesions. Paired adjacent dysplasia tissues obtained from 17 cancer patients were also examined. Promoter hypermethylation in 10 tumor‐related genes (APC, ATM, GSTP1, HLTF, MGMT, hMLH1, p14, p15, SOCS‐1 and TIMP‐3) were studied by methylation‐specific PCR. Promoter hypermethylation was frequently detected in more than 40% of colonic cancers and adenomas in APC, ATM, HLTF, MGMT and hMLH1 genes (p < 0.0001 vs. normal). While low level of methylation was detected in p14, p15 and TIMP‐3, there was no methylation detected in GSTP1 and SOCS‐1. The frequencies of methylation were comparable between tumors and adenomas, and advanced and nonadvanced adenoma. In contrast, K‐ras mutation was only detected in advanced adenomas and cancers. Concurrent methylation in ≥ 3 genes was found in 66.7% adenomas and 68.1% cancers but not in normal colonic tissues. Methylation was associated with reduced protein expressions in colorectal adenomas and cancers. Moreover, methylation in ATM was more common in older cancer patients (p = 0.002), but there was no significant association between promoter hypermethylation and other clinicopathologic characteristics of cancer. Our study demonstrated the early and specific involvement of promoter hypermethylation in the colorectal adenoma‐carcinoma sequence.

TOP2A overexpression in hepatocellular carcinoma correlates with early age onset, shorter patients survival and chemoresistance.

Genomic gain represents an important mechanism in the activation of proto‐oncogenes. In many instances, induced oncogenes hold clinical implications both as prognostic markers and targets for therapeutic design. In hepatocellular carcinoma (HCC), although chromosomal gains are common, information on underlying oncogenes induced remains minimal. Here, we examined 7 causal sites of HCC for overexpressed genes by array‐based transcriptional mapping. In 22 HCC cell lines and early passages of cultures studied, clusters of up‐regulated genes were indicated, where TOP2A expression ranked the highest. Distinct TOP2A transcriptions were confirmed in an independent series of HCC tumors relative to adjacent non‐tumoral liver (p = 0.0018). By tissue microarray analysis of 172 HCC, we found TOP2A expressions correlated with advance histological grading (p < 0.001), microvascular invasion (p = 0.004) and an early age onset of the malignancy (≤40 years; p = 0.007). In conjunction with P‐gp and MRP1, TOP2A were further assessed for its association with chemotherapy responsiveness and survival in 148 patients who entered our recently reported Phase III prospective randomized study. In 73 chemoresistant and 75 nonresistant patients, only TOP2A positivity correlated with chemoresistance (p = 0.029) and shorter patients survival (p < 0.0001). The potential therapeutic value in targeting TOP2A by Etoposide, as a single agent, and in combination with Doxorubicin was also explored. In vitro cytotoxic studies suggested Etoposide at IC20 readily reduced IC50 values of Doxorubicin by a magnitude of ∼3.5 to 10‐fold compared to Doxorubicin alone (p < 0.028). Our study highlighted for the first time the prognostic value of TOP2A in HCC and the potential use of TOP2A reactive agents in therapy.

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

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.