Rajani Rai

PSCA gene variants (rs2294008 and rs2978974) confer increased susceptibility of gallbladder carcinoma in females.

BACKGROUND AND AIM PSCA is a tissue specific tumor suppressor or oncogene which has been found to be associated with several human tumors including gallbladder cancer. It is considered to be involved in the cell-proliferation inhibition and/or cell-death induction activity. Therefore, we aimed to investigate the role of PSCA gene polymorphisms in gallbladder cancer risk in North Indian population. METHODOLOGY A total of 405 gallbladder cancer patients and 247 healthy controls were included in the case-control study for risk prediction. We examined the association of two functional SNPs, rs2294008 and rs2978974 in PSCA gene by genotyping using Taqman allelic discrimination assays. Statistical analysis was done using SPSS software, version 17. Linkage disequilibrium and haplotype analysis was done with the help of SNPstats software. FDR test was used to correct for multiple comparisons. RESULTS No significant associations of rs2294008 and rs2978974 genetic variants of the PSCA gene were found with GBC risk at allele, genotype or haplotype levels. Stratifying the subjects on the basis of gallstone also did not show any significant result. However, on gender stratification, we found a significant association of Trs2294008-Grs2978974 haplotype with higher risk of GBC in females (FDR Pcorr=0.021, OR=1.6). In contrary, Trs2294008-A rs2978974 haplotype conferred significant lower risk in males (FDR Pcorr=0.013; OR=0.25). CONCLUSIONS These findings suggest that PSCA genetic variants may have a significant effect on GBC susceptibility in a gender specific manner.

DCC (deleted in colorectal carcinoma) gene variants confer increased susceptibility to gallbladder cancer (Ref. No.: Gene-D-12-01446)

BACKGROUND AND AIM GBC is a lethal and multifaceted disease. Deleted in colorectal carcinoma (DCC) is a well known tumor suppressor gene. Recently a small genome-wide association study (GWAS) identified DCC to be significantly associated with gallbladder cancer (GBC) susceptibility in a Japanese population sample. However, the study sample size was small and lacked independent replication. Therefore, the present study was carried out to replicate the association of two GWAS identified DCC SNPs (A>Grs4078288, C>Trs7504990) and two other SNPs (C>Grs2229080 and A>Grs714) previously associated with various cancers. METHODOLOGY The study was accomplished in 406 GBC cases and 260 healthy control samples from North India. Genotyping was carried out by PCR-RFLP and Taqman genotyping assays. Statistical analysis was performed by using SPSS ver16 and functional prediction of these variants was carried out using Bioinformatics tools (FAST-SNP, F-SNP). RESULT We did not observe association with GWAS-identified SNPs of DCC but other SNPs showed significant association. In addition, a DCC haplotype Grs2229080-Ars4078288-Crs7504990-Ars714 conferred high risk of GBC in India. The haplotype associated risk was independent of gallstone, sex or tobacco usages which are well-known modifiers of GBC risk. Further analysis suggested DCC A>Grs714 as a major risk conferring SNP in the Indian population. CONCLUSION This study re-affirms the role of plausible tumor suppressor DCC variants, in gallbladder carcinogenesis and the risk haplotype may be explored as a useful marker for GBC susceptibility.

MicroRNA aberrations: An emerging field for gallbladder cancer management.

Gallbladder cancer (GBC) is infrequent but most lethal biliary tract malignancy characterized by an advanced stage diagnosis and poor survival rates attributed to absence of specific symptoms and effective treatment options. These necessitate development of early prognostic/predictive markers and novel therapeutic interventions. MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a key role in tumor biology by functioning like tumor suppressor- or onco- genes and their aberrant expression are associated with the pathogenesis of several neoplasms with overwhelming clinical implications. Since miRNA signature is tissue specific, here, we focused on current data concerning the miRNAs aberrations in GBC pathogenesis. In GBC, miRNAs with tumor suppressor activity (miR-135-5p, miR-335, miR-34a, miR-26a, miR-146b-5p, Mir-218-5p, miR-1, miR-145, mir-130a) were found downregulated, while those with oncogenic property (miR-20a, miR-182, mir-155) were upregulated. The expression profile of miRNAs was significantly associated with GBC prognosis and prediction, and forced over-expression/ inhibition of these miRNAs was shown to affect tumor growth and development. Further, differential expression of miRNAs in the blood samples of GBC patients suggest miRNAs as promising noninvasive biomarker. Thus, miRNAs represent potential candidate for GBC management, though many hurdles need to be overcome before miRNAs therapy can be clinically applied to GBC prevention and treatment.

CYP17 polymorphism (rs743572) is associated with increased risk of gallbladder cancer in tobacco users

Gallbladder cancer (GBC) involves interplay of sex steroids, including estrogen and progesterone. Since CYP17 is a key enzyme involved in estrogen and testosterone hormone biosynthesis as well as in xenobiotic metabolism, it may be a potential candidate gene in the carcinogenesis of the gallbladder. Hence, the present study aimed to investigate the association of CYP17 (rs2486758, and rs743572) polymorphisms with GBC susceptibility. The present study included a total of 414 histologically confirmed GBC and 230 healthy controls. The CYP17 (rs2486758 and rs743572) polymorphisms were genotyped by TaqMan-Allele discrimination assays. Statistical analysis was performed by using SPSS ver. 16. Overall, both the CYP17 SNPs did not indicate any association with GBC risk at genotype, haplotype, or at the genotypic interaction levels. However, in the case-only analysis, CYP rs743572 showed association with increased risk of GBC in tobacco users at hetero genotype and dominant models, as compared to non-user GBC patients. The TCrs2486758-AGrs743572 genotypic combination was also associated with increased GBC susceptibility in tobacco users. CYP17 rs743572 is associated with increased risk of GBC in tobacco users in the North Indian population. However, the study requires confirmation in other populations.

Impact of DCC (rs714) and PSCA (rs2294008 and rs2976392) Gene Polymorphism in Modulating Cancer Risk in Asian Population

Multiple studies have investigated the association of gene variant of Deleted in colorectal carcinoma (DCC) and Prostate Stem cell antigen (PSCA) with various cancer susceptibility; however, the results are discrepant. Since SNPs are emerging as promising biomarker of cancer susceptibility, here, we aimed to execute a meta-analysis of DCC (rs714 A > G) and PSCA (rs2294008 C > T, rs2976392 G > A) polymorphism to demonstrate the more accurate strength of these associations. We followed a rigorous inclusion/exclusion criteria and calculated the pooled odds ratios (ORs) and 95% confidence intervals (CIs). Overall, the pooled analysis showed that the DCC rs714 conferred increased risk of cancer only in Asians (AA vs. GG: OR = 1.86, p ≤ 0.0001; AG vs. GG: OR = 1.43, p = 0.005; GA + AA vs. GG: OR = 1.66, p ≤ 0.0001; AA vs. GG + GA; OR = 1.52, p ≤ 0.004, A vs. G allele: OR = 1.41, p ≤ 0.0001). PSCA rs2294008 was associated with increased overall cancer risk (TT vs. CC: OR = 1.28, p = 0.002; CT vs. CC: OR = 1.21, p ≤ 0.0001; CT + TT vs. CC: OR = 1.24, p ≤ 0.0001; TT vs. CC + CT; OR = 1.17, p ≤ 0.005, T vs. C allele: OR = 1.16, p ≤ 0.0001); however, in stratified analysis this association was limited only to gastric and bladder cancer and the strength was more prominent in Asians. In contrast, the PSCA rs2976392 SNP did not modulate the cancer risk. Therefore, we concluded that rs714 and rs2294008 polymorphism may represent a potential genetic biomarker for cancer risk in Asians and gastric as well as bladder cancer, respectively. However, since our study is limited to Asians and cancer types, further larger studies involving other cancers and/or population, gene-environment interactions and the mechanism of DCC and PSCA gene deregulation are desired to define the role of genotype with overall cancer risk.

Multi-Analytic Approach Elucidates Significant Role of Hormonal and Hepatocanalicular Transporter Genetic Variants in Gallstone Disease in North Indian Population

Objective Cholesterol gallstone disease (CGD) is a multifactorial and multistep disease. Apart from female gender and increasing age being the documented non-modifiable risk factor for gallstones the pathobiological mechanisms underlying the phenotypic expression of CGD appear to be rather complex, and one or more variations in genes could play critical roles in the diverse pathways further progressing to cholesterol crystal formation. In the present study we performed genotyping score, Multifactor dimensionality reduction (MDR) and Classification and Regression Tree analysis (CART) to identify combinations of alleles among the hormonal, hepatocanalicular transporter and adipogenesis differentiation pathway genes in modifying the risk for CGD. Design The present case-control study recruited total of 450 subjects, including 230 CGD patients and 220 controls. We analyzed common ESR1, ESR2, PGR, ADRB3, ADRA2A, ABCG8, SLCO1B1, PPARγ2, and SREBP2 gene polymorphisms to find out combinations of genetic variants contributing to CGD risk, using multi-analytical approaches (G-score, MDR, and CART). Results Single locus analysis by logistic regression showed association of ESR1 IVS1-397C>T (rs2234693), IVS1-351A>G (rs9340799) PGR ins/del (rs1042838) ADRB3-190 T>C (rs4994) ABCG8 D19H (rs11887534), SLCO1B1 Exon4 C>A (rs11045819) and SREBP2 1784G>C (rs2228314) with CGD risk. However, the MDR and CART analysis revealed ESR1 IVS1-397C>T (rs2234693) ADRB3-190 T>C (rs4994) and ABCG8 D19H (rs11887534) polymorphisms as the best polymorphic signature for discriminating between cases and controls. The overall odds ratio for the applied multi-analytical approaches ranged from 4.33 to 10.05 showing an incremental risk for cholesterol crystal formation. In conclusion, our muti-analytical approach suggests that, ESR1, ADRB3, in addition to ABCG8 genetic variants confer significant risk for cholesterol gallstone disease.

A Multiple Interaction Analysis Reveals ADRB3 as a Potential Candidate for Gallbladder Cancer Predisposition via a Complex Interaction with Other Candidate Gene Variations

Gallbladder cancer is the most common and a highly aggressive biliary tract malignancy with a dismal outcome. The pathogenesis of the disease is multifactorial, comprising the combined effect of multiple genetic variations of mild consequence along with numerous dietary and environmental risk factors. Previously, we demonstrated the association of several candidate gene variations with GBC risk. In this study, we aimed to identify the combination of gene variants and their possible interactions contributing towards genetic susceptibility of GBC. Here, we performed Multifactor-Dimensionality Reduction (MDR) and Classification and Regression Tree Analysis (CRT) to investigate the gene–gene interactions and the combined effect of 14 SNPs in nine genes (DR4 (rs20576, rs6557634); FAS (rs2234767); FASL (rs763110); DCC (rs2229080, rs4078288, rs7504990, rs714); PSCA (rs2294008, rs2978974); ADRA2A (rs1801253); ADRB1 (rs1800544); ADRB3 (rs4994); CYP17 (rs2486758)) involved in various signaling pathways. Genotyping was accomplished by PCR-RFLP or Taqman allelic discrimination assays. SPSS software version 16.0 and MDR software version 2.0 were used for all the statistical analysis. Single locus investigation demonstrated significant association of DR4 (rs20576, rs6557634), DCC (rs714, rs2229080, rs4078288) and ADRB3 (rs4994) polymorphisms with GBC risk. MDR analysis revealed ADRB3 (rs4994) to be crucial candidate in GBC susceptibility that may act either alone (p < 0.0001, CVC = 10/10) or in combination with DCC (rs714 and rs2229080, p < 0.0001, CVC = 9/10). Our CRT results are in agreement with the above findings. Further, in-silico results of studied SNPs advocated their role in splicing, transcriptional and/or protein coding regulation. Overall, our result suggested complex interactions amongst the studied SNPs and ADRB3 rs4994 as candidate influencing GBC susceptibility.

Quantitative assessment of CD44 genetic variants and cancer susceptibility in Asians: a meta-analysis.

CD44 is a well-established cancer stem cell marker playing a crucial role in tumor metastasis, recurrence and chemo-resistance. Genetic variants of CD44 have been shown to be associated with susceptibility to various cancers; however, the results are confounding. Hence, we performed a meta-analysis to clarify these associations more accurately. Overall, rs13347 (T vs. C: OR=1.30, p=<0.004, pcorr=0.032; CT vs. CC: OR=1.29, p=0.015, pcorr=0.047; TT vs. CC: OR=1.77, p=<0.000, pcorr=0.018; CT+TT vs. CC: OR=1.34, p=<0.009, pcorr=0.041) and rs187115 (GG vs. AA: OR=2.34, p=<0.000, pcorr=0.025; AG vs. AA: OR=1.59, p=<0.000, pcorr=0.038; G vs. A allele OR=1.56, p=0.000, pcorr=0.05; AG+GG vs. AA: OR=1.63, p=<0.000, pcorr=0.013) polymorphisms were found to significantly increase the cancer risk in Asians. On the other hand, rs11821102 was found to confer low risk (A vs. G: OR=0.87, p=<0.027, pcorr=0.04; AG vs. GG: OR=0.85, p=<0.017, pcorr=0.01; AG+AA vs. GG: OR=0.86, p=<0.020, pcorr=0.02). Based on our analysis, we suggest significant role of CD44 variants (rs13347, rs187115 and rs11821102) in modulating individuals cancer susceptibility in Asians. Therefore, these variants may be used as predictive genetic biomarkers for cancer predisposition in Asian populations. However, more comprehensive studies involving other cancers and/or populations, haplotypes, gene-gene and gene-environment interactions are necessary to delineate the role of these variants in conferring cancer risk.

Abstract 1798: Mortalin precursor as potential marker for chemoprevention with SHetA2

Introduction: The flexible heteroarotinoid (Flex-Het), SHetA2, is a novel anticancer drug that induces both intrinsic and extrinsic apoptosis and autophagy in cancer cells, but not in normal cells. Protein isolation / mass spectrometry analysis using SHetA2-coupled polystyrene magnetic beads yielded three SHetA2-binding proteins all belonging to HSPA family namely, HSPA9/mortalin, HSPA8/Hsc70 and HSPA5/BiP. Mortalin, in addition to its vital chaperoning roles in other organelles of the cell, is essential for import and folding of nuclear-encoded mitochondrial proteins. The precursor form of mortalin has a 46-amino acid N-terminal region that functions as a mitochondrial localization sequence (MLS) and is cleaved by proteases after import into the mitochondrial matrix. Hypothesis: SHetA2 binding to mortalin causes alterations that can be measured to study mechanism and monitor drug effects in animal models and clinical trials. Methods: Western blots of whole cell or subcellular protein extracts made from cultures of normal human fallopian tube secretory or mammary epithelial cells, rat mammary tumors or human ovarian cancer cell lines treated with SHetA2 or solvent were probed with an antibody that recognizes total mortalin, a custom-made rabbit polyclonal antibody specific for the mortalin MLS (PA8238) or antibodies to loading control proteins. Immunocytochemistry using these antibodies was performed with an automated (Leica Bond III) IHC work station on cells treated with SHetA2 or solvent. Results: Using a commercial antibody against total mortalin, we observed a lower-mobility band closely moving above the specific (mature) mortalin band in Western blots. Our mortalin MLS-specific antibody recognized only the lower-mobility band confirming it as the precursor form of mortalin in the SHetA2-treated cell extracts. Subcellular fractionation of the drug-treated cells revealed that the precursor protein accumulated only in the cytoplasm and not in the mitochondria. Staining of whole cells with an antibody to total mortalin showed no effect of SHetA2 treatment on the punctate pattern of expression consistent with mitochondrial localization. In contrast, staining with the mortalin MLS-specific antibody demonstrated that SHetA2 increased the intensity of the diffuse cytoplasmic staining consistent with localization of mortalin throughout the cytoplasm. These results were consistently observed among the various cell types. Conclusions: SHetA2 is the only known drug that blocks the translocation of the precursor mortalin to mitochondria and its processing to mature protein. Such cytoplasmic accumulation of the precursor form of mortalin can potentially serve as pharmacodynamic endpoint to study SHetA2’s effect in laboratory experimental tumor models as well as in human clinical trials. Funding: NCI PREVENT Task Order HHSN26100002 Citation Format: Elangovan Thavathiru, Vishal Chandra, Rajani Rai, Doris Benbrook. Mortalin precursor as potential marker for chemoprevention with SHetA2 [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 1798. doi:10.1158/1538-7445.AM2017-1798