Ajay Goel

Aberrant Methylation of Multiple Tumor Suppressor Genes in Aging Liver, Chronic Hepatitis, and Hepatocellular Carcinoma

Aberrant DNA methylation is an important epigenetic alteration in hepatocellular carcinoma (HCC). However, the molecular processes underlying the methylator phenotype and the contribution of hepatitis viruses are poorly understood. The current study is a comprehensive methylation analysis of human liver tissue specimens. A total of 176 liver tissues, including 77 pairs of HCCs and matching noncancerous liver and 22 normal livers, were analyzed for methylation. Methylation of 19 epigenetic markers was quantified, and the results were correlated with different disease states and the presence or absence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Based on methylation profiles, the 19 loci were categorized into 3 groups. Normal liver tissues showed methylation primarily in group 1 loci (HIC‐1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC), which was significantly higher than group 2 (CDH1, RUNX3, RIZ1, SFRP2, MINT31) and group 3 markers (COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, DCC) (P < 0.0001). Noncancerous livers demonstrated increased methylation in both group 1 and group 2 loci. Methylation was significantly more abundant in HCV‐positive livers compared with normal liver tissues. Conversely, HCC showed frequent methylation at each locus investigated in all 3 groups. However, the group 3 loci showed more dense and frequent methylation in HCV‐positive cancers compared with both HBV‐positive cancers and virus‐negative cancers (P < 0.0001). Conclusion: Methylation in HCC is frequent but occurs in a gene‐specific and disease‐specific manner. Methylation profiling allowed us to determine that aberrant methylation is commonly present in normal aging livers, and sequentially progresses with advancing stages of chronic viral infection. Finally, our data provide evidence that HCV infection may accelerate the methylation process and suggests a continuum of increasing methylation with persistent viral infection and carcinogenesis in the liver. (HEPATOLOGY 2008.)

Characteristic patterns of altered DNA methylation predict emergence of human hepatocellular carcinoma

We aimed to identify the specific subset of tumor suppressor genes (TSGs) that are methylation‐silenced during the earliest steps of hepatocarcinogenesis, and to further evaluate whether these genes can serve as predictive biomarkers of hepatocellular carcinoma (HCC) emergence. A total of 482 liver tissues including 177 pairs of HCCs and matched nontumor livers and 128 liver biopsies from chronic hepatitis C (CHC) patients were analyzed for quantitative methylation analysis in 24 TSG promoters and three MINT loci. The tumors were classified as early, less‐progressed, and highly progressed HCCs using histology and radiological approaches. A subset of TSGs that harbored distinctly high levels of methylation in early HCCs were selected. Based on the methylation profiles of these genes, Kaplan‐Meier analyses were performed to determine time‐to‐HCC occurrence in CHC patients. Subsequently, multivariate analysis was performed using age, gender, fibrosis stage, and number of methylated TSGs as covariates. Among TSGs analyzed, a subset of eight TSGs (HIC1, GSTP1, SOCS1, RASSF1, CDKN2A, APC, RUNX3, and PRDM2) demonstrated a distinct cluster by hierarchical clustering and receiver operating characteristic analyses. This subset of TSGs showed significantly higher methylation levels in the early HCCs (P < 0.0001). In the CHC patients, methylation frequencies in these TSGs were associated with shorter time‐to‐HCC occurrence (P < 0.0001), and number of methylated genes was an independent risk factor for HCC (hazard ratio = 5.21, 95% confidence interval = 2.25‐11.76, P = 0.0002). Conclusion: Epigenetic inactivation of a subset of TSGs plays a critical role in the earliest steps of hepatocarcinogenesis. Furthermore, epigenetic inactivation of these genes in CHC provides a prognostic value for determining the risk for developing HCC later in life. (HEPATOLOGY 2012;56:994–1003)

JC Virus Infection in Colorectal Neoplasia That Develops after Liver Transplantation

Purpose: Liver transplant recepients (LTRs) have an increased risk of colorectal neoplasia. The mechanism responsible for this is unknown. JCV encodes for TAg and has been implicated in colorectal carcinogenesis. We hypothesized that the use of immunosuppression in LTRs facilitates activation of JCV and is responsible for the increased risk of neoplasia. Experimental Design: JCV TAg DNA and protein expression were determined in normal colonic epithelium (n = 15) and adenomatous polyps (n = 26) from LTRs and compared with tissue samples from control patients (normal colon, n = 21; adenomas, n = 40). Apoptosis and proliferation were determined by M30 and Ki-67 immunoreactivity, respectively. Results: JCV TAg DNA was found in 10 of 15 (67%) of normal colonic mucosa from LTRs compared with 5 of 21 (24%) of control normal mucosa (P = 0.025). JCV TAg DNA was detected in 16 of 26 (62%) of the adenomas from LTRs and in 20 of 40 (50%) of control adenomas. JCV TAg protein was expressed in 13 of 26 (50%) adenomas from LTRs versus 2 of 40 (5%) of adenomas from controls (P < 0.001). In adenomas from LTRs, the mean proliferative activity was higher compared with controls (60.3 ± 3.2% versus 42.7 ± 2.8%, P < 0.001), whereas mean apoptotic indices were lower in LTRs (0.29 ± 0.08% versus 0.39 ± 0.06%, P = 0.05). Conclusions: The presence of JCV in the colorectal mucosa and adenomas from LTRs, in concert with the use of immunosuppressive agents, suggests that JCV may undergo reactivation, and the subsequent TAg protein expression might explain the increased risk of colorectal neoplasia in LTRs.

Unique Association between Global DNA Hypomethylation and Chromosomal Alterations in Human Hepatocellular Carcinoma

Global DNA hypomethylation is a characteristic feature of cancer cells that closely associates with chromosomal instability (CIN). However, the association between these characteristics during hepatocarcinogenesis remains unclear. Herein, we determined the relationship between hypomethylation and CIN in human hepatocellular carcinoma (HCC) by analyzing 179 HCCs, 178 matched non-tumor livers and 23 normal liver tissues. Hypomethylation at three different repetitive DNA (rDNA) sequences and hypermethylation of 12 CpG loci, including 11 tumor suppressor gene (TSG) promoters, were quantified using MethyLight or combined bisulfite restriction analysis. Fractional allelic loss (FAL) was used as a marker for CIN, calculated by analyzing 400 microsatellite markers. Gains and losses at each chromosome were also determined using semi-quantitative microsatellite analysis. The associations between rDNA hypomethylation and FAL, as well as between TSG hypermethylation and FAL were investigated. Significantly more hypomethylation was observed in HCC tissues than in normal liver samples. Progression of hypomethylation during carcinogenesis was more prominent in hepatitis C virus (HCV)-negative cases, which was in contrast to our previous reports of significantly increased TSG methylation levels in HCV-positive tumors. Absence of liver cirrhosis and higher FAL scores were identified as independent contributors to significant hypomethylation of rDNA in HCC. Among the chromosomal alterations frequently observed in HCC, loss of 8p, which was unique in the earliest stages of hepatocarcinogenesis, was significantly associated with hypomethylation of rDNA by multivariable analysis (pu200a=u200a0.0153). rDNA hypomethylation was also associated with a high FAL score regardless of tumor differentiation (pu200a=u200a0.0011, well-differentiated; pu200a=u200a0.0089, moderately/poorly-differentiated HCCs). We conclude that DNA hypomethylation is an important cause of CIN in the earliest step of HCC, especially in a background of non-cirrhotic liver.

Two Isoforms of the Guanine Nucleotide Exchange Factor, Daple/CCDC88C Cooperate as Tumor Suppressors

Previously Aznar et al., showed that Daple enables Wnt/Frizzled receptors to transactivate trimeric G proteins during non-canonical Wnt signaling via a novel G-protein binding and activating (GBA) motif. By doing so, Daple serves as a double-edged sword; earlier during oncogenesis it suppresses neoplastic transformation and tumor growth, but later it triggers epithelial messenchymal transition (EMT). We have identified and characterized two isoforms of the human Daple/CCDC88c gene. While both isoforms cooperatively suppress tumor growth via their GBA motif, only the full-length transcript triggers EMT and invasion. Aspirin suppresses the full-length transcript and protein but upregulates the short isoform. Both isoforms are suppressed during colon cancer progression, and their reduced expression carries additive prognostic significance. These findings provide insights into the opposing roles of Daple during cancer progression and define the G protein regulatory GBA motif as one of the minimal modules essential for Daple’s role as a tumor suppressor.

Precision Medicine for CRC Patients in the Veteran Population: State-of-the-Art, Challenges and Research Directions.

Colorectal cancer (CRC) accounts for ~9% of all cancers in the Veteran population, a fact which has focused a great deal of the attention of the VA’s research and development efforts. A field-based meeting of CRC experts was convened to discuss both challenges and opportunities in precision medicine for CRC. This group, designated as the VA Colorectal Cancer Cell-genomics Consortium (VA4C), discussed advances in CRC biology, biomarkers, and imaging for early detection and prevention. There was also a discussion of precision treatment involving fluorescence-guided surgery, targeted chemotherapies and immunotherapies, and personalized cancer treatment approaches. The overarching goal was to identify modalities that might ultimately lead to personalized cancer diagnosis and treatment. This review summarizes the findings of this VA field-based meeting, in which much of the current knowledge on CRC prescreening and treatment was discussed. It was concluded that there is a need and an opportunity to identify new targets for both the prevention of CRC and the development of effective therapies for advanced disease. Also, developing methods integrating genomic testing with tumoroid-based clinical drug response might lead to more accurate diagnosis and prognostication and more effective personalized treatment of CRC.

Abstract 4720: Novel findings for the clinical significance of RNA editing status of AZIN1 and ADAR 1 and 2 expression levels in gastric cancer patients

Background. Despite recent advances in surgical techniques and treatment options, gastric cancer (GC) remains the third most common cause of cancer-related deaths worldwide. Besides DNA sequence mutations, epigenetic alterations have emerged as significantly major players in cancer development. A-to-I RNA editing is a post-transcriptional modification that converts adenosines to inosines in both coding and noncoding RNA transcripts, and has recently been recognized has a novel epigenetic mechanism in GC pathogenesis. More specifically, A-to-I editing of AZIN1 transcripts was shown to be regulated by an adenosine deaminase acting on RNA-1 (ADAR1), and edited AZIN1 resulted in an aggressive phenotype during disease progression in some human cancers. The aim of this study was to clarify the clinical consequences of RNA editing status of AZIN1 and the RNA editing enzymes (ADAR1 and 2) in GC patients. Methods. Two hundred eighty-eight gastric specimens from one hundred forty-four patients who underwent surgery for GC were evaluated. We analyzed the RNA editing status of AZIN1 by RNA editing site-specific quantitative PCR (RESSqPCR). RESSqPCR allows quantitation of RNA editing levels using wild-type or edited AZIN1-specific primers, and RNA editing levels are calculated by determining the ratios of Ct values between edited vs. wild-type transcript expression levels. Furthermore, expression levels of ADAR1 and ADAR2 were evaluated by qPCR in GC tissues. Results. We observed a higher frequency of the AZIN1 RNA editing in tumors compared with normal mucosa in GC. RNA editing status of AZIN1 was significantly increased in a stage-dependent manner, and high frequency of RNA editing in AZIN1 significantly correlated with advanced T stage and presence of lymph node metastasis in GC patients. Multivariate analysis revealed that high frequency of RNA editing in the AZIN1 gene was an independent prognostic factor for poor disease free survival and overall survival. Furthermore, significant upregulation of ADAR1 and downregulation of ADAR2 was also observed in GC tissues compared with matched normal mucosa, and these alterations also significantly correlated with disease progression factors and poor prognosis in GC patients. Interestingly, ADAR1 expression level was positively correlated with RNA editing status of AZIN1 in GC tissues. Conclusions. Our findings revealed that altered gene-specific A-to-I editing events mediated by ADAR1 promote disease progression in GC. We conclude that assessment of RNA editing status of the AZIN1 gene might offer a novel and superior biomarker for a more accurate diagnosis of disease staging and may help predict clinical outcomes in GC patients. Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Kunitoshi Shigeyasu, Takashi Ichikawa, Satoshi Oki, Koichiro Mori, Yuka Nagano, Hiromi Yasuda, Shigeyuki Yoshiyama, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Toshimitsu Araki, Yasuhiko Mohri, Motoyoshi Tanaka, Chikao Miki, Ajay Goel, Masato Kusunoki. Novel findings for the clinical significance of RNA editing status of AZIN1 and ADAR 1 and 2 expression levels in gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4720. doi:10.1158/1538-7445.AM2017-4720

The Epigenetics in Intestinal Tumorigenesis

Colorectal cancer develops as a result of stepwise accumulation of genetic and epigenetic alterations, which in turn, regulate the expression of various oncogenes and tumor suppressor genes. Epigenetic alterations include DNA methylation, histone modifications, chromatin remodeling, and noncoding RNAs. Data gathered in recent years clearly highlight that in contrast to genetic alterations, epigenetic alterations occur far more frequently, participate in virtually every step of colorectal cancer development, and are potentially reversible. More importantly, considering the ubiquitous nature of epigenetic alterations in human cancers, these molecular signatures have garnered significant attention for development as potential diagnostic, prognostic and predictive biomarkers in human colorectal cancer. This article reviews the current state of literature in the field, and focuses on the clinical relevance of various epigenetic alterations as potential biomarkers for the personalized treatment of patients suffering from this common malignancy worldwide.

Abstract 867: Serum microRNAs as diagnostic biomarkers for early colorectal neoplasms

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnObjectives: Early detection of precancerous lesions and early-stage cancers are essential to reduce mortality from colorectal cancer (CRC).nnSerum/plasma microRNAs (miRs) have been reported as diagnostic and prognostic biomarkers of CRC, but their utility in detecting patients with early lesions has not been fully explored. The aim of this study is to identify serum miRs which are potentially useful as diagnostic biomarkers for early colorectal neoplasms, and potential confounders in a blood test.nnMethods: Candidate biomarker miRs were selected from previously published studies. Serum samples were collected from patients with low-grade intraepithelial neoplasias (LGINs) including tubular adenomas and tubulovillous adenomas, high-grade intraepithelial neoplasias (HGINs), early cancers, and healthy volunteers, and divided into discovery and validation sets. Total RNA was extracted from serum samples and levels of candidate miRs were measured by real-time RT-PCR. To evaluate the impact of hemolysis on serum miR levels, serially diluted hemolysed controls were prepared from stock solution made by hemolysing the red blood cells in distilled water. Levels of candidate miRs in hemolysed controls were also examined by real-time RT-PCR. Absorbance of serum samples at 560 nm, 576 nm, and 592 nm was measured by spectrophotometry and hemoglobin levels were estimated from a standard curve.nnResults: Fourteen miRs were selected as candidate diagnostic biomarkers of early colorectal neoplasms from a review of the literature. Serum levels of five miRs were too low to be accurately quantified by real-time RT-PCR, and excluded from further evaluation. Sera from 12 patients with LGINs, 8 patients with HGINs, 4 patients with cancers, and 25 healthy volunteers were included in the discovery phase. Of 9 miRs examined, miR21 (p = 0.0007), miR29a (p < 0.0001) and miR125b (p = 0.0198) showed significantly higher levels in sera from early colorectal neoplasms compared to those from healthy volunteers. Higher levels of miR29a and miR125b in sera from early colorectal neoplasms were confirmed in the validation phase using independent set of samples. Levels of miR21, 29a, and miR125b in sera significantly correlated with the degree of hemolysis.nnConclusions: Serum miRs may be useful biomarkers to detect patients with early colorectal neoplasia, including adenomas. Hemolysis affects serum miR levels, and will confound measurement. Since hemolysis occurs frequently during blood collection, its impact on serum miRs should be further investigated.nnCitation Format: Atsushi Yamada, Takahiro Horimatsu, Yoshinaga Okugawa, Naoshi Nishida, Tadayuki Kou, Toshihiro Kusaka, Hajime Honjo, Yusuke Amanuma, Osamu Kikuchi, Manabu Muto, Ajay Goel, C. Richard Boland. Serum microRNAs as diagnostic biomarkers for early colorectal neoplasms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 867. doi:10.1158/1538-7445.AM2014-867

Abstract 4196: MSH3 mediates sensitization of colorectal cancer cells to cisplatin, oxaliplatin and a poly(ADP-ribose) polymerase inhibitor

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnIntroduction:The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2 forms the MutSβ heteroduplex, which interacts with interstrand crosslinks (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. MethodsIn this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Results: Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of pH2AX after oxaliplatin treatment in comparison to MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSB). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a Poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited more than 10-fold increase in toxicity compared to either treatment individually. Conclusion:Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4196. doi:10.1158/1538-7445.AM2011-4196