Dana Ziliak

Population differences in microRNA expression and biological implications.

Population differences observed for complex traits may be attributed to the combined effect of socioeconomic, environmental, genetic and epigenetic factors. To better understand population differences in complex traits, genome-wide genetic and gene expression differences among ethnic populations have been studied. Here we set out to evaluate population differences in small non-coding RNAs through an evaluation of microRNA (miRNA) baseline expression in HapMap lymphoblastoid cell lines (LCLs) derived from 53 CEU (Utah residents with northern and western European ancestry) and 54 YRI (African from Ibadan, Nigeria). Using the Exiqon miRCURYTM LNA arrays, we found that 16% of all miRNAs evaluated in our study differ significantly between these 2 ethnic groups (pBonferroni corrected<0.05). Furthermore, we explored the potential biological function of these observed differentially expressed miRNAs by comprehensively examining their effect on the transcriptome and their relationship with cellular sensitivity drug phenotypes. After multiple testing adjustment (false discovery rate (FDR)<0.1), we found that 55% and 88% of the differentially expressed miRNAs were significantly and inversely correlated with an mRNA expression phenotype in the CEU and YRI samples, respectively. Interestingly, a substantial proportion (64%) of these miRNAs correlated with cellular sensitivity to chemotherapeutic agents (FDR<0.05). Lastly, upon performing a genome-wide association study between SNPs and miRNA expression, we identified a large number of SNPs exhibiting different allele frequencies that affect the expression of these differentially expressed miRNAs, suggesting the role of genetic variants in mediating the observed population differences.

Germline Polymorphisms Discovered via a Cell-based Genome-wide Approach Predict Platinum Response in Head and Neck Cancers

Identifying patients prior to treatment who are more likely to benefit from chemotherapeutic agents or more likely to experience adverse events is an aim of personalized medicine. Pharmacogenomics offers a potential means of achieving this goal through the discovery of predictive germline genetic biomarkers. When applied particularly to the treatment of head and neck cancers, such information could offer significant benefit to patients as a means of potentially reducing morbidity associated with platinum-based chemotherapy. We developed a genome-wide, cell-based approach to identify single nucleotide polymorphisms (SNPs) associated with platinum susceptibility and then evaluated these SNPs as predictors for response and toxicity in head and neck cancer patients treated with platinum-based therapy as part of a phase II clinical trial. Sixty head and neck cancer patients were evaluated. Of 45 genome-wide SNPs examined, we found that 2 SNPs, rs6870861 (P=0.004; false discovery rate [FDR] <0.05) and rs2551038 (P=0.005; FDR <0.05), were associated significantly with overall response to carboplatin-based induction chemotherapy when incorporated into a model along with total carboplatin exposure. Interestingly, these 2 SNPs are associated strongly with the baseline expression of >20 genes (all P ≤10(-4)), and that 2 genes (SLC22A5 and SLCO4C1) are important organic cation/anion transporters known to affect platinum uptake and clearance. Several other SNPs were associated nominally with carboplatin-related hematologic toxicities. These findings demonstrate importantly that a genome-wide, cell-based model can identify novel germline genetic biomarkers of platinum susceptibility, which are replicable in a clinical setting with treated cancer patients and seem clinically meaningful for potentially enabling future personalization of care in such patients.

Genetic Variation That Predicts Platinum Sensitivity Reveals the Role of miR-193b* in Chemotherapeutic Susceptibility

Platinum agents are the backbone of cancer chemotherapy. Recently, we identified and replicated the role of a single nucleotide polymorphism (SNP, rs1649942) in predicting platinum sensitivity both in vitro and in vivo. Using the CEU samples from the International HapMap Project, we found the same SNP to be a master regulator of multiple gene expression phenotypes, prompting us to investigate whether rs1649942-mediated regulation of miRNAs may in part contribute to variation in platinum sensitivity. To these ends, 60 unrelated HapMap CEU I/II samples were used for our discovery-phase study using high-throughput genome-wide miRNA and gene expression profiling. Examining the relationships among rs1649942, its gene expression targets, genome-wide miRNA expression, and cellular sensitivity to carboplatin and cisplatin, we identified 2 platinum-associated miRNAs (miR-193b* and miR-320) that inhibit the expression of 5 platinum-associated genes (CRIM1, IFIT2, OAS1, KCNMA1, and GRAMD1B). We further replicated the relationship between the expression of miR-193b*, CRIM1, IFIT2, KCNMA1, and GRAMD1B, and platinum sensitivity in a separate HapMap CEU III dataset. We then showed that overexpression of miR-193b* in a randomly selected HapMap cell line results in resistance to both carboplatin and cisplatin. This relationship was also found in 7 ovarian cancer cell lines from NCI60 dataset and confirmed in an OVCAR-3 that overexpression of miR-193b* leads to increased resistance to carboplatin. Our findings highlight a potential mechanism of action for a previously observed genotype-survival outcome association. Further examination of miR-193b* in platinum sensitivity in ovarian cancer is warranted. Mol Cancer Ther; 11(9); 2054–61. ©2012 AACR.

Chemotherapeutic-induced apoptosis: a phenotype for pharmacogenomics studies.

Aim To determine whether cellular apoptosis is a suitable phenotypic trait for pharmacogenomics studies by evaluating caspase 3/7-mediated activity in lymphoblastoid cell lines after treatment with six chemotherapeutic agents: 5′-deoxyfluorouridine, pemetrexed, cytarabine, paclitaxel, carboplatin, and cisplatin. Materials and methods Using monozygotic twin pair and sibling pair lymphoblastoid cell lines, we identified conditions for measurement of caspase 3/7 activity in lymphoblastoid cell lines. Genome-wide association studies were performed with over 2 million single nucleotide polymorphisms (SNPs) and cisplatin-induced apoptosis in HapMap CEU cell lines (n=77). Results Although treatment with 5′-deoxyfluorouridine and pemetrexed for up to 24 h resulted in low levels of apoptosis or interindividual variation in caspase-dependent cell death; paclitaxel, cisplatin, carboplatin, and cytarabine treatment for 24 h resulted in 9.4-fold, 9.1-fold, 7.0-fold, and 6.0-fold increases in apoptosis relative to control, respectively. There was a weak correlation between caspase activity and cytotoxicity (r2=0.03–0.29) demonstrating that cytotoxicity and apoptosis are two distinct phenotypes that may produce independent genetic associations. Estimated heritability (h2) for apoptosis was 0.57 and 0.29 for cytarabine (5 and 40 &mgr;mol/l, respectively), 0.22 for paclitaxel (12.5 nmol/l), and 0.34 for cisplatin (5 &mgr;mol/l). In the genome-wide association study using the HapMap CEU panel, we identified a significant enrichment of cisplatin-induced cytotoxicity SNPs within the significant cisplatin-induced apoptosis SNPs and an enrichment of expression quantitative trait loci (eQTL). Among these eQTLs, we identified several eQTLs with known function related to apoptosis and/or cytotoxicity. Conclusion Our study identifies apoptosis as a phenotype for pharmacogenomic studies in lymphoblastoid cell lines after treatment with paclitaxel, cisplatin, carboplatin, and cytarabine that may have utility for discovering biomarkers to predict response to certain chemotherapeutics.

Integrative analyses of genetic variation, epigenetic regulation, and the transcriptome to elucidate the biology of platinum sensitivity

BackgroundUsing genome-wide genetic, gene expression, and microRNA expression (miRNA) data, we developed an integrative approach to investigate the genetic and epigenetic basis of chemotherapeutic sensitivity.ResultsThrough a sequential multi-stage framework, we identified genes and miRNAs whose expression correlated with platinum sensitivity, mapped these to genomic loci as quantitative trait loci (QTLs), and evaluated the associations between these QTLs and platinum sensitivity. A permutation analysis showed that top findings from our approach have a much lower false discovery rate compared to those from a traditional GWAS of drug sensitivity. Our approach identified five SNPs associated with 10 miRNAs and the expression level of 15 genes, all of which were associated with carboplatin sensitivity. Of particular interest was one SNP (rs11138019), which was associated with the expression of both miR-30d and the gene ABCD2, which were themselves correlated with both carboplatin and cisplatin drug-specific phenotype in the HapMap samples. Functional study found that knocking down ABCD2 in vitro led to increased apoptosis in ovarian cancer cell line SKOV3 after cisplatin treatment. Over-expression of miR-30d in vitro caused a decrease in ABCD2 expression, suggesting a functional relationship between the two.ConclusionsWe developed an integrative approach to the investigation of the genetic and epigenetic basis of human complex traits. Our approach outperformed standard GWAS and provided hints at potential biological function. The relationships between ABCD2 and miR-30d, and ABCD2 and platin sensitivity were experimentally validated, suggesting a functional role of ABCD2 and miR-30d in sensitivity to platinating agents.

Genome-wide discovery of genetic variants affecting tamoxifen sensitivity and their clinical and functional validation

BACKGROUND Beyond estrogen receptor (ER), there are no validated predictors for tamoxifen (TAM) efficacy and toxicity. We utilized a genome-wide cell-based model to comprehensively evaluate genetic variants for their contribution to cellular sensitivity to TAM. DESIGN Our discovery model incorporates multidimensional datasets, including genome-wide genotype, gene expression, and endoxifen-induced cellular growth inhibition in the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide findings were further evaluated in NCI60 cancer cell lines. Gene knock-down experiments were performed in four breast cancer cell lines. Genetic variants identified in the cell-based model were examined in 245 Caucasian breast cancer patients who underwent TAM treatment. RESULTS We identified seven novel single-nucleotide polymorphisms (SNPs) associated with endoxifen sensitivity through the expression of 10 genes using the genome-wide integrative analysis. All 10 genes identified in LCLs were associated with TAM sensitivity in NCI60 cancer cell lines, including USP7. USP7 knock-down resulted in increasing resistance to TAM in four breast cancer cell lines tested, which is consistent with the finding in LCLs and in the NCI60 cells. Furthermore, we identified SNPs that were associated with TAM-induced toxicities in breast cancer patients, after adjusting for other clinical factors. CONCLUSION Our work demonstrates the utility of a cell-based model in genome-wide identification of pharmacogenomic markers.BACKGROUND Beyond estrogen receptor (ER), there are no validated predictors for tamoxifen (TAM) efficacy and toxicity. We utilized a genome-wide cell-based model to comprehensively evaluate genetic variants for their contribution to cellular sensitivity to TAM. DESIGN Our discovery model incorporates multidimensional datasets, including genome-wide genotype, gene expression, and endoxifen-induced cellular growth inhibition in the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide findings were further evaluated in NCI60 cancer cell lines. Gene knock-down experiments were performed in four breast cancer cell lines. Genetic variants identified in the cell-based model were examined in 245 Caucasian breast cancer patients who underwent TAM treatment. RESULTS We identified seven novel single-nucleotide polymorphisms (SNPs) associated with endoxifen sensitivity through the expression of 10 genes using the genome-wide integrative analysis. All 10 genes identified in LCLs were associated with TAM sensitivity in NCI60 cancer cell lines, including USP7. USP7 knock-down resulted in increasing resistance to TAM in four breast cancer cell lines tested, which is consistent with the finding in LCLs and in the NCI60 cells. Furthermore, we identified SNPs that were associated with TAM-induced toxicities in breast cancer patients, after adjusting for other clinical factors. CONCLUSION Our work demonstrates the utility of a cell-based model in genome-wide identification of pharmacogenomic markers.

Integrative “Omic” Analysis for Tamoxifen Sensitivity through Cell Based Models

It has long been observed that tamoxifen sensitivity varies among breast cancer patients. Further, ethnic differences of tamoxifen therapy between Caucasian and African American have also been reported. Since most studies have been focused on Caucasian people, we sought to comprehensively evaluate genetic variants related to tamoxifen therapy in African-derived samples. An integrative “omic” approach developed by our group was used to investigate relationships among endoxifen (an active metabolite of tamoxifen) sensitivity, SNP genotype, mRNA and microRNA expressions in 58 HapMap YRI lymphoblastoid cell lines. We identified 50 SNPs that associate with cellular sensitivity to endoxifen through their effects on 34 genes and 30 microRNA expression. Some of these findings are shared in both Caucasian and African samples, while others are unique in the African samples. Among gene/microRNA that were identified in both ethnic groups, the expression of TRAF1 is also correlated with tamoxifen sensitivity in a collection of 44 breast cancer cell lines. Further, knock-down TRAF1 and over-expression of hsa-let-7i confirmed the roles of hsa-let-7i and TRAF1 in increasing tamoxifen sensitivity in the ZR-75-1 breast cancer cell line. Our integrative omic analysis facilitated the discovery of pharmacogenomic biomarkers that potentially affect tamoxifen sensitivity.

Comprehensive Evaluation of the Contribution of X Chromosome Genes to Platinum Sensitivity

Using a genome-wide gene expression data set generated from Affymetrix GeneChip Human Exon 1.0ST array, we comprehensively surveyed the role of 322 X chromosome gene expression traits on cellular sensitivity to cisplatin and carboplatin. We identified 31 and 17 X chromosome genes whose expression levels are significantly correlated (after multiple testing correction) with sensitivity to carboplatin and cisplatin, respectively, in the combined HapMap CEU (Utah residents with ancestry from northern and western Europe) and YRI (Yoruba in Ibahan, Nigeria) populations (false discovery rate, FDR < 0.05). Of those, 14 overlap for both cisplatin and carboplatin. Using an independent gene expression quantification method, the Illumina Sentrix Human-6 Expression BeadChip, measured on the same HapMap cell lines, we found that 4 and 2 of these genes are significantly associated with carboplatin and cisplatin sensitivity, respectively, in both analyses. Two genes, CTPS2 and DLG3, were identified by both genome-wide gene expression analyses as correlated with cellular sensitivity to both platinating agents. The expression of DLG3 gene was also found to correlate with cellular sensitivity to platinating agents in NCI-60 cancer cell lines. In addition, we evaluated whether the expression of X chromosome genes contributed to the observed differences in sensitivity to the platinums between CEU and YRI-derived cell lines. Of the 34 distinct genes significantly correlated with either carboplatin or cisplatin sensitivity, 14 are differentially expressed (defined as P < 0.05) between CEU and YRI. Thus, sex chromosome genes play a role in cellular sensitivity to platinating agents and differences in the expression level of these genes are an important source of variation that should be included in comprehensive pharmacogenomic studies. Mol Cancer Ther; 10(3); 472–80. ©2011 AACR.

Abstract 3636: Systems biology approach links genetic, epigenetic, and transcriptomic features to tamoxifen sensitivity.

Utilizing existing genome-wide genotype, transcriptome and microRNA (miRNA) datasets, we developed an integrative approach to evaluate the role of genetic-epigenetic regulatory networks in human complex traits. In this study, we focused on cellular sensitivity to tamoxifen, a commonly prescribed breast cancer medication as a phenotype of interest. A sequential multi-dimensional model was constructed to first identify genes/miRNAs whose expression levels are correlated with tamoxifen sensitivity. This in turn facilitated the identification of SNPs associated with potential functionally relevant genomic features. Finally, associations between these selected SNPs and cellular sensitivity to tamoxifen were evaluated. A permutation-based false discovery rate (FDR) procedure showed that association findings from our approach have much lower FDRs when compared to those obtained through a traditional single-step genome-wide association study (GWAS) between SNP and drug sensitivity. Our model identified 50 unique SNPs associated with 30 miRNAs and 34 gene expression levels, all of which associated with cellular sensitivity to tamoxifen phenotypes in HapMap YRI samples. Among them, several miRNAs and genes have been previously implicated to play a role in tamoxifen sensitivity; most are novel. Functional studies on select miRNAs/genes are currently underway in both the HapMap samples and breast cancer cell lines. Citation Format: Eric R. Gamazon, Liming Weng, Hae Kyung Im, Xiaotong Zhang, Dana M. Ziliak, R. Stephanie Huang. Systems biology approach links genetic, epigenetic, and transcriptomic features to tamoxifen sensitivity. [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 3636. doi:10.1158/1538-7445.AM2013-3636

Abstract 1875: Identifying genetic variants contributing to cellular susceptibility to tamoxifen using a genome-wide cell-based model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tamoxifen is one of the most commonly used agents in the treatment and prevention of estrogen receptor positive breast cancers. Beyond the presence of hormone receptors, there are no validated predictors of tamoxifen efficacy and toxicity. Therefore, we utilized a genome-wide cell-based model to comprehensively evaluate genetic variants for their contribution to cellular susceptibility to tamoxifen. Our model incorporates multi-dimensional datasets including genome-wide genotype, gene expression, and cellular growth inhibition following treatment in lymphoblastoid cell lines from the International HapMap project. Growth inhibition was measured using AlamarBlue Assay in 60 unrelated CEU (individuals of northern and western European decedent) and 60 unrelated YRI (individuals from Ibadan, Nigeria) samples. All cell lines were treated with increasing concentrations of endoxifen, an active metabolite of tamoxifen, for 72 hours. Log2 transformed percent survival at each concentration and IC50 were used as cellular susceptibility to drug phenotypes. A step-wise genome-wide association studies were performed among SNPs, mRNA expression, and endoxifen sensitivity phenotypes. We identified 10 and 74 SNPs associated with endoxifen sensitivity through the expression of 13 and 92 genes in the separate CEU and YRI population. Interestingly 3 genes (STS, TES and SMARCA2) identified through this method are known to play a role in hormone biosynthesis pathway. Furthermore, a large portion of identified genes was found to play a role in tamoxifen sensitivity in the NCI60 cancer cell lines including CHPT1, a known tumor progression marker. This approach has made possible the identification of genetic variants/genes that have not been previously associated with tamoxifen response. Further validation of these cell-based model findings may significantly improve our ability to predict tamoxifen treatment efficacy and toxicity in breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1875. doi:1538-7445.AM2012-1875