Debanjali Dasgupta

Hepatic miR-126 is a potential plasma biomarker for detection of hepatitis B virus infected hepatocellular carcinoma.

Controversies about the origin of circulating miRNAs have encouraged us to identify organ specific circulating miRNAs as disease biomarkers. To identify liver‐specific miRNAs for hepatocellular carcinoma (HCC), global expression profiling of miRNAs in liver tissue of HBV‐HCC and HBV‐control with no or mild fibrosis was evaluated. A total of 40 differentially expressed miRNAs were identified in HCC. Among ten highly altered miRNAs, six miRNAs were successfully validated in tissues, whereas only two miRNAs, miR‐126 and miR‐142‐3p showed increased expression in plasma of HBV‐HCC compared to HBV‐non‐HCC patients. Subsequently, ROC curve analysis revealed that neither miR‐126 nor miR‐142‐3p performed better than AFP in discriminating HCC from non‐HCC while combination of each with AFP showed significantly higher efficiency rather than AFP alone (AUC: 0.922, 0.908 vs. 0.88; sensitivity: 0.84, 0.86 vs. 0.82 and specificity: 0.92, 0.94 vs. 0.86 respectively). Interestingly, triple combination of markers (miR‐126 + miR‐142‐3p + AFP) showed no additive effect on efficiency (AUC: 0.925) over the dual combination. Again, the expression of only miR‐126 was noticed significantly higher in HBV‐HCC patients with low‐AFP [<250 ng/ml] compared to either non‐HCC or liver cirrhosis (AUC: 0.77, 0.64, respectively). Furthermore, no alteration in expression of mir‐126 in HCV‐HCC or non‐viral‐HCC revealed that miR‐126 + AFP might be specific to HBV‐HCC. To understand the physiological role of these two miRNAs in hepato‐carcinogenesis, target genes related to cancer pathways (APAF1, APC2, CDKN2A, IRS1, CRKL, LIFR, EGR2) were verified. Thus, combination of circulating miR‐126 + AFP is a promising noninvasive diagnostic biomarker for HBV‐HCC and may be useful in the management of HCC patients.

MiRNA199a-3p suppresses tumor growth, migration, invasion and angiogenesis in hepatocellular carcinoma by targeting VEGFA, VEGFR1, VEGFR2, HGF and MMP2

Increasing significance of tumor–stromal interaction in development and progression of cancer implies that signaling molecules in the tumor microenvironment (TME) might be the effective therapeutic targets for hepatocellular carcinoma (HCC). Here, the role of microRNA miR-199a-3p in the regulation of TME and development of HCC has been investigated by several in vitro and in vivo assays. Expression of miR-199a-3p was observed significantly low in HCC tissues and its overexpression remarkably inhibited in vivo tumor growth and metastasis to lung in NOD-SCID mice. In vitro restoration of miR-199a-3p expression either in endothelial cells (ECs) or in cancer cells (CACs) significantly diminished migration of ECs in co-culture assay. Again incubation of miR-199a-3p transfected ECs with either conditioned media (CM) of CACs or recombinant VEGF has reduced tube formation, in ECs and it was also dropped upon growth in CM of either anti-VEGF antibody-treated or miR-199a-3p-transfected CACs. In addition, bioinformatics and luciferase-reporter assays revealed that miR-199a-3p inhibited VEGF secretion from CACs and VEGFR1 and VEGFR2 expression on ECs and thus restricted cross talk between CACs and ECs. Again, restoration of miR-199a-3p in hepatic stellate cells (HSCs) reduced migration and invasion of CACs in co-culture assay, while it was enhanced by the overexpression of HGF suggesting miR-199a-3p has hindered HSC-CACs cross talk probably by inhibiting HGF and regulating matrix metalloproteinase MMP2, which were found as targets of miR-199a-3p subsequently by luciferase-reporter assay and gelatin zymography, respectively. Thus, these findings collectively highlight that miR-199a-3p restricts metastasis, invasion and angiogenesis in HCC and hence it may be considered as one of the powerful effective therapeutics for management of HCC patients.

Novel point and combo-mutations in the genome of hepatitis B virus-genotype D: characterization and impact on liver disease progression to hepatocellular carcinoma.

Background The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC. Methods The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence. Results Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively. Conclusions Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.

Distinct distribution pattern of hepatitis B virus genotype C and D in liver tissue and serum of dual genotype infected liver cirrhosis and hepatocellular carcinoma patients.

Aims The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored. Methods The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed. Results HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC. Conclusions Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy.

Genetic Association and Gene-Gene Interaction Reveal Genetic Variations in ADH1B, GSTM1 and MnSOD Independently Confer Risk to Alcoholic Liver Diseases in India

Genetic susceptibility is an important modifier of clinical outcome and natural history of progression in Alcoholic liver disease (ALD). While the significance of ethnicity in this evolution is very clear, subtle inter-individual genetic variant(s) might be important and thus we investigated those in an Indian population. Fourteen markers were genotyped within two alcohol metabolism genes [Alcohol dehydrogenase (ADH) gene clusters (ADH1B and ADH1C) and Aldehyde dehydrogenase (ALDH2)], one microsomal ethanol oxidizing enzyme cytochrome p450 (CYP2E1) and three oxidative stress response (OSR) genes (MnSOD, GSTT1 and GSTM1) among 490 Bengali individuals (322 ALD and 168 control) from Eastern and North-Eastern India and validation was performed in a new cohort of 150 Bengali patients including 100 ALD and 50 advanced non-alcoholic steatohepatitis (NASH). Out of 14 genetic variants, carriage of 5 genotypes (rs2066701CC in ADH1B, rs1693425TT in ADH1C, rs4880TT in MnSOD and GSTT1/GSTM1 null, p-value <0.05) were noted significantly higher among ALD patients while inter or intra group gene-gene interaction analysis revealed that addition of risk genotype of any OSR gene enhanced the possibility of ALD synergistically. Multiple logistic regression analysis showed independent association of rs2066701CC, rs4880TT and GSTM1 null genotype with ALD while lower frequencies of those genotypes in advanced NASH patients further confirmed their causal relation to ALD. Thus these findings suggest that the three variants of ADH1C, MnSOD and GSTM1 can be used to identify individuals who are at high risk to develop ALD and may be helpful in proper management of Indian alcoholics.

Oncogenic potential of hepatitis B virus subgenotype D1 surpasses D3: significance in the development of hepatocellular carcinoma

Despite widespread distribution of hepatitis B virus (HBV)-genotype D, the clinical implications of its ten subgenotypes (D1-D10) have not been well documented. Here, we have investigated the impact of two major circulating HBV/D subgenotypes, D1 and D3 in Eastern India towards pathogenesis of liver disease progression to hepatocellular carcinoma (HCC). HBV subgenotypes were determined using full-length genome sequences of HBV isolates from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) and HCC. Impact of D1 and D3 on viral lifecycle and disease progression was assessed by several in vitro assays. Phylogenetic tree analysis revealed that HBV/D1 and HBV/D3 were the two predominating HBV subgenotypes circulating in Eastern India. Interestingly, the frequency of patients infected with HBV/D1 was noticed progressively rising from CHB to HCC through LC while the increasing frequency of HBV/D3 declined suddenly in HCC implicating HBV/D1 might have greater oncogenic potential than HBV/D3. Similar to higher viral load noted in HCC patients infected with HBV/D1 than HBV/D3, the larger amount of intracellular/extracellular viral DNA and secreted HBsAg levels in transfected cell lines also implicated that HBV/D1 might replicate faster than HBV/D3. Again, higher expression of marker genes related to endoplasmic reticulum stress, epithelial-mesenchymal transition, DNA double strand breaks, angiogenesis etc. and faster rate of cellular migration and anchorage independent growth cumulatively suggested that compared to HBV/D3, HBV/D1 generates more liver injuries which eventually culminates into HCC. Therefore, our results highlight the importance of determination of subgenotypes of HBV in CHB patients, so that high-risk individual can be monitor periodically that may help to detect HCC at early stages.

Abstract 3969: Hepatic microRNA as biomarker for detection of hepatocellular carcinoma in high-risk chronic Hepatitis B patients

a) Absence of pathognomonic symptoms in patients with early phase of hepatocellular carcinoma (HCC) often leads to untreatable disease when diagnosed. Alpha-fetoprotein (AFP) with radiological images is the only potential HCC diagnosis option while disease prognosis remains dismal mandating necessity of biomarker identification with diagnostic, prognostic potentials as well as ability to assist in therapy. Thus this study aimed to identify non-invasive biomarker correlated with HCC pathogenesis by analyzing deregulated miRNAs in premalignant liver cirrhosis (LC) and in HCC. b) Among 148 study subjects, 117 were chronic Hepatitis B virus (HBV) (NAsymptomatic- control = 28, NLC = 30, NHCC = 59), 14 chronic Hepatitis C virus (HCV) infected (NLC = 7, NHCC = 7) and 17 were uninfected control. Differential expression profiling of miRNAs in each of 4 HBV infected LC and HCC tissues were performed by comparing with 8 asymptomatic controls using microarray and validated by qRT-PCR. R packages were used to determine area under receiver operating characteristics (AUROC) curve and other statistical analysis. Different softwares were used for prediction of target pathways associated with HCC development. c) Microarray analysis leads to identification of significantly altered [> or d) In conclusion, combination of miR-126 with AFP may be a better predictor of HCC in high-risk chronic hepatitis patients and miR-126 alone can show better performance in detection of HCC with low AFP. Citation Format: Amit Ghosh, Alip Ghosh, Somenath Datta, Debanjali Dasgupta, Soumyajit Das, Sukanta Ray, Subash Gupta, Simanti Datta, Abhijit Chowdhury, Saroj Kant Mohapatra, Soma Banerjee. Hepatic microRNA as biomarker for detection of hepatocellular carcinoma in high-risk chronic Hepatitis B patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3969. doi:10.1158/1538-7445.AM2015-3969

Abstract 2306: MicroRNA 199a*: a potent suppressor of tumor metastasis and angiogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The growth of a solid tumor depends on diffusion of nutrients from the tumor microenvironment through vascular system. Angiogenesis, the new blood vessels formation is the primary route by which tumor cells get nutrients as well as exit from primary site and enter into the blood circulation. The proliferation and migration of vascular endothelial cells are triggered by angiogenic growth factors secreted by tumor cells, such as VEGF, TGF-β etc. Recent evidences showed HGF also plays an important role in angiogenesis by enhancing the proliferative activity and intracellular signaling. HGF/its receptor, c-MET and VEGF/VEGFR1 are two independent pathways for angiogenesis and metastasis. Thus detection of a molecule that hits both pathways could be a potent therapeutic agent that could facilitate the inhibition of improved barrier function. Recently mir199a* has been shown to target both CD44 and cMET molecule to block the metastasis in cancer. The present study aim to show that mir199a* also target HGF, the key modulator of the HGF/c-Met signaling cascade in epithelial cells to shut off the mesenchymal transition and VEGFR1 to prevent angiogenesis more effectively. Luciferase 3’ UTR assay was performed to investigate HGF as a target of mir-199a*. HGF mRNA and protein expression was determined by real time RT-PCR, Western blot analysis in liver stellate cell line LX2 transfected with mir-199a* and HGF shRNA. The cultured media of mir-199a* transfected LX2 cells was used to verify the effect of mir-199a* on HGF induced migration and invasion ability on metastatic cell lines using Boyden chamber assay with or without matrigel coated membrane and angiogenesis by tube formation assay. The mir-199a* binding site was detected by pictar, target scan analysis of the 3’UTR of HGF and VEGFRs. The binding of mir-199a* to the 3′UTR of HGF and VEGFR1 were confirmed by luciferase assay, mutagenesis and western blotting. Down regulation of HGF protein expression in HGF producing hepatic stellate cell, LX2 was observed by mir-199a*. Addition of LX2 conditioned media transfected with mir199a* to metastatic cell reduces the migration and invasion and also block angiogenesis in endothelial cells. As Mir-199a* could target HGF/cMET, CD44-cMET and VEGF/VEGFR1 signaling cascade, it could be used as a potential therapeutic target of HCC. Citation Format: Alip Ghosh, Amit Ghosh, Debanjali Dasgupta, Shrabasti Roychowdhury, Shrabasti Roychowdhury, Simanti Datta, Abhijit Chowdhury, Soma Banerjee. MicroRNA 199a*: a potent suppressor of tumor metastasis and angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2306. doi:10.1158/1538-7445.AM2013-2306

Abstract LB-71: Carcinogenic significance of integrated hepatitis b viral factors and its genotype in hepatocarcinogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Integration of Hepatitis B Virus (HBV) is one of the major causes underlying Hepatocellular carcinoma (HCC) development. Recent studies revealed that altered expression of multiple cellular genes due to multiple random integration of viral factors lead to development of HCC. The present study aims to identify the carcinogenic significance of integrated HBV genome and its genotype in HCC development. Host-Viral Junctions were determined in HCC (T) /adjacent tissues (NT) and cirrhotic (-HCC) tissues by sequencing of Alu PCR product followed by sequence blast. Host gene expression was checked by real time PCR. Competition between two coexisting genotypes was studied by replication assay using genotype specific PCR from media and viral core particle isolated from genotype specific plasmid transfected HepG2 cell line. Similarly, each viral genotype mediated DNA damage (γH2aX foci by confocal microscopy), ROS generation (DCFDA quantitation by Facs analysis and ER stress marker GRP78 luciferase assay), homologous recombination efficiency were also compared. HBV integration was observed in 66.7% of T (8/12), 33.3% (4/12) NT and 75% (6/8) cirrhotic tissue samples in 18, 4 and 8 different locations respectively. These viral integration analysis within or close to several host genes, such as genes involved in differentiation, signaling, stress response, cell cycle, telomere regulations (8/30, 26.7%) etc and most of them showed altered expression. Interestingly, C-terminal truncated HBX which lost its growth suppressive domain were observed more in T (5/8, 62.5%) than NT (2/4, 50%) tissue samples. Most importantly, upon genotype analysis of each five clones in serum and tissue showed genotype C preferentially integrated in coexistence of both C and D genotype as “free virus” in tissue but D genotype observed in circulation. Genotype specific PCR from HBV/C & D cotransfected media showed that in presence of HBV/C, D replicates better than C but HBV/C with high recombination frequency generates more DNA damage and facilitate viral integration. Thus random integration of HBV in host chromosome causes alterations in oncogenic gene expression and these alterations presumably lead to clonal selection of hepatocyte that acquires a growth advantage and proceeds towards HCC. Genotype of HBV is an important determining factor for HBV integration and liver inury. Citation Format: Soma Banerjee, Somenath Datta, Shrabasti Roychoudhury, Debanjali Dasgupta, Amit Ghosh, Gaurav Roy, Simanti Datta, Abhijit Chowdhury. Carcinogenic significance of integrated hepatitis b viral factors and its genotype in hepatocarcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-71. doi:10.1158/1538-7445.AM2013-LB-71