Citations

Abstract BACKGROUND Metformin, a widely used anti-diabetic drug, is being considered as a highly promising agent for treatment and prevention of many types of cancer, including breast cancer. Although the exact mechanism of action of metformin is unknown, it is thought that metformin is an AMPK activator. To better individualize metformin therapy and explore additional underlying molecular mechanism associated with metformin action, we conducted a metformin pharmacogenomic study using 266 genomic data-enriched lymphoblastoid cell lines (LCLs). METHODS Genome-wide microarray data were generated for all LCLs, including 1.3 million SNPs, 485K DNA methylation probes and 54K mRNA expression probe sets. Metformin cytotoxicity assay was performed using MTS assay. Genome-wide association (GWA) analyses and integrated analyses using all the genomic data and metformin cytotoxicity (IC50 value) were performed to identify top candidate genes for functional validation in breast cancer cell lines, MDA-MB 231 and Hs578T, using siRNA screening approach. Further mechanistic study was performed to investigate the role of candidates in regulation of AMPK activity. RESULTS GWA analyses in LCLs identified 198 mRNA expression probe sets and 210 DNA methylation probes associated with metformin IC50 with p-value <10-4, respectively. Analysis of 1.3 million SNPs found 12 loci (a region containing at least 1 SNP of p-value <10-5 and 1 SNP of p-value <10-3 within 50kb) that were associated with metformin IC50. Integrated SNP loci-mRNA expression-IC50 analysis indicated that the SNP loci on chromosome 16 was associated with metformin IC50 through a trans-regulation of expression of 7 genes with p-value <10-4. Integrated methylation-mRNA expression-IC50 analysis showed that 15 DNA methylation probes in 6 genes were associated with metformin IC50 through both cis- and trans-regulation of expression of 48 genes with p-value <10-4, including 4 methylation probes in 3 of those 6 genes that were cis-correlated with its own gene expression. Ingenuity pathway analysis of top 62 candidate genes identified four major pathways and 55 genes within those pathways were selected for functional validation in breast cancer cell lines. The knockdown experiment showed that down regulation of 25 genes significantly altered metformin sensitivity in MDA-MB231, and 14 of them also showed the same effect in Hs578T. Preliminary mechanistic experiment indicated that knockdown of 7 genes significantly reduced AMPK activity in MDA-MB231 and 3 of them had same effect in Hs578T. CONCLUSIONS GWAS using a genomic data-enriched LCL model system, together with functional validation in breast cancer cell lines, could help us to identify novel genetic and epigenetic biomarkers involved in metformin response and help us to better understand the mechanisms of metformin in cancer treatment. Citation Format: Nifang Niu, Xianglin Tan, Brooke L. Fridley, Daniel J. Schaid, Ryan P. Abo, Anthony Batzler, Erin E. Carlson, Gregory Jenkins, Sebastian Moran, Holger Andrea Heyn, Manel Esteller Badosa, Liewei Wang. Metformin pharmacogenomics: A genome-wide associate study to identify genetic and epigenetic biomarkers involved in metformin response. [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 2271. doi:10.1158/1538-7445.AM2013-2271

Abstract Definitive radiotherapy combined with chemotherapy is associated with enhanced locoregional control and clinical outcomes in non-small cell lung cancer (NSCLC) patients who are not candidates for surgery. However, radiation-induced pneumonitis and esophagitis are common dose-limiting inflammatory conditions that occur in these patients. These events can compromise the effect of the overall treatment and reduce quality of life in these patients. It is not clear what factors contribute to differences in the development of toxicities among patients with same clinical or dosimetric parameters. In this study, we conducted a large scale pathway-based analysis to identify inflammation-related genetic variants associated with radiation-induced pneumonitis or esophagitis. In the initial discovery phase, 11,930 inflammation-related genetic variants were genotyped in 243 stage I-III NSCLC patients who were treated with definitive radiotherapy at MD Anderson Cancer Center. A fast-track validation was performed for the top candidate SNPs in an additional 212 NSCLC cases following the same criteria. A total of 14 SNPs and 12 SNPs were validated for pneumonitis and esophagitis, respectively. Meta-analysis was used to summarize the results from two phases. The most significant SNPs for esophagitis and pneumonitis mapped to chromosome 18p (OR=9.05, 95%CI=2.68-30.59, P-meta=3.90×10−4) and 14q (OR=4.75, 95%CI=2.17-10.41, P-meta =9.61×10−5), respectively. To explore the potential underlying mechanisms, we analyzed radiation sensitivity and eQTL relationships of the identified SNPs and their host genes in 277 ethnically defined human lymphoblastoid cell lines. Among the 24 host genes for the 26 identified SNPs, three genes (DDX58, GZMB, CDC2) showed significant association with radiation responses in these cell lines (P<0.05). Moreover, a significant eQTL relationship was identified between CDC2 expression and the genotype of the identified CDC2 SNP (P=0.022). Interestingly, this CDC2 SNP is predicted to create a new miRNA binding site. Luciferase reporter assays to test the influence of this SNP on miRNA regulation is currently ongoing. Our results provide evidence that inflammation-related genetic variants contribute to the development of radiation-induced pneumonitis and esophagitis. The results also suggest that gene expression changes through altered miRNA binding may be an underlying mechanism for these events. Citation Format: Xia Pu, Liewei Wang, Michelle AT Hildebrandt, Yuanqing Ye, Joe Y. Chang, Charles Lu, Heath D. Skinner, Nifang Niu, Gregory D. Jenkins, Ritsuko Komaki, John D. Minna, Jack A. Roth, Richard M. Weinshilboum, Xifeng Wu. Inflammation-related genetic variants predict radiation-induced toxicity following definitive radiotherapy for non-small cell lung cancer. [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 3626. doi:10.1158/1538-7445.AM2013-3626

Abstract Integrative genomics has the potential to uncover relevant loci, as clinical outcome and response to chemotherapies are most likely not due to a single gene (or data type) but rather a complex relationship involving genetic variation, mRNA, DNA methylation, and copy number variation. In addition to this complexity, many complex phenotypes are thought to be controlled by the interplay of multiple genes within the same molecular pathway or gene set (GS). To address these two challenges, we propose an integrative gene set analysis approach and apply this strategy to a cisplatin (CDDP) pharmacogenomics study involving lymphoblastoid cell lines for which genome-wide SNP and mRNA expression data was collected. Application of the integrative GS analysis implicated the role of the RNA binding and cytoskeletal part GSs. The genes LMNB1 and CENPF, within the cytoskeletal part GS, were functionally validated with siRNA knockdown experiments, where the knockdown of LMNB1 and CENPF resulted in CDDP resistance in multiple cancer cell lines. This study demonstrates the utility of an integrative GS analysis strategy for detecting novel genes associated with response to cancer therapies, moving closer to tailored therapy decisions for cancer patients.

Abstract The selective estrogen receptor modulators (SERM) tamoxifen and raloxifene can reduce the occurrence of breast cancer in high-risk women by 50%, but this U.S. Food and Drug Administration-approved prevention therapy is not often used. We attempted to identify genetic factors that contribute to variation in SERM breast cancer prevention, using DNA from the NSABP P-1 and P-2 breast cancer prevention trials. An initial discovery genome-wide association study identified common single-nucleotide polymorphisms (SNP) in or near the ZNF423 and CTSO genes that were associated with breast cancer risk during SERM therapy. We then showed that both ZNF423 and CTSO participated in the estrogen-dependent induction of BRCA1 expression, in both cases with SNP-dependent variation in induction. ZNF423 appeared to be an estrogen-inducible BRCA1 transcription factor. The OR for differences in breast cancer risk during SERM therapy for subjects homozygous for both protective or both risk alleles for ZNF423 and CTSO was 5.71. Significance: This study identified novel, functionally polymorphic genes involved in the estrogen-dependent regulation of BRCA1 expression, as well as a novel mechanism for genetic variation in SERM therapeutic effect. These observations, and definition of their underlying mechanisms, represent steps toward pharmacogenomically individualized SERM breast cancer prevention. Cancer Discov; 3(7); 812–25. ©2013 AACR. See related commentary by Machiela and Chanock, p. 728 This article is highlighted in the In This Issue feature, p. 705

Genome‐wide association studies (GWAS) of complex traits have generated many association signals for single nucleotide polymorphisms (SNPs). To understand the underlying causal genetic variant(s), focused DNA resequencing of targeted genomic regions is commonly used, yet the current cost of resequencing limits sample sizes for resequencing studies. Information from the large GWAS can be used to guide choice of samples for resequencing, such as the SNP genotypes in the targeted genomic region. Viewing the GWAS tag‐SNPs as imperfect surrogates for the underlying causal variants, yet expecting that the tag‐SNPs are correlated with the causal variants, a reasonable approach is a two‐phase case‐control design, with the GWAS serving as the first‐phase and the resequencing study serving as the second‐phase. Using stratified sampling based on both tag‐SNP genotypes and case‐control status, we explore the gains in power of a two‐phase design relative to randomly sampling cases and controls for resequencing (i.e., ignoring tag‐SNP genotypes). Simulation results show that stratified sampling based on both tag‐SNP genotypes and case‐control status is not likely to have lower power than stratified sampling based only on case‐control status, and can sometimes have substantially greater power. The gain in power depends on the amount of linkage disequilibrium between the tag‐SNP and causal variant alleles, as well as the effect size of the causal variant. Hence, the two‐phase design provides an efficient approach to follow‐up GWAS signals with DNA resequencing.

Abstract Epithelial ovarian cancer (EOC) is the 6th most commonly diagnosed cancer among women in the world, accounting for 4% of all female cancers. On a worldwide basis, an estimated 204,000 new cases are diagnosed and 125,000 women die of EOC annually, with 5-year survival rate at 45%. Metastasis and its complications are the usual cause of EOC-related deaths. Thus, factors related to tumor aggressiveness, response to therapy, and the underlying health state of the patient often predict survival. Candidate gene and genome-wide association studies (GWAS) for survival from EOC have discovered only a few the genetic variants, accounting for only a small proportion of the heritability. One explanation for the missing heritability is that the common analysis approach _ assessing the effect of each single nucleotide polymorphism (SNP) individually _ is not well suited to the detection of small effects of multiple SNPs. Gene set analysis (GSA) overcomes this limitation by assessing the overall evidence of association of a phenotype with all measured variation in a set of genes or pathway. Therefore, we conducted a GSA using data from an EOC GWAS, using the Illumina 610-quad SNP array, to determine novel gene sets (GSs) associated with overall survival (OS) from EOC (N cases = 1057, N deaths = 495). All subjects were non-Hispanic, non-Jewish Caucasians, with sample and SNP call rates >95%. Subjects with ambiguous gender, unresolved identical genotypes, and less than 80% European ancestry were also excluded. SNPs were mapped to genes within 20 kb, with genes then mapped to GSs defined by the following: KEGG, Gene Ontology (biological, cellular, molecular) and PharmGKB. GSA was completed in which the association of each gene with OS was assessed using a principal component gene-level test (adjusting for study site, age and population sub-structure based on the first eigenvector from EigenStrat), followed by the aggregation of the gene-level p-values using the Gamma method with a soft truncation threshold of 0.15. Empirical p-values were determined using permutation methods. Of the 2,573 GSs assessed, 30 GSs had a p-value < 0.01. The top GSs were related to: mismatch repair (p = 3.0E-4), platelet formation (p=0.001), leukocyte adhesive activation (p=0.002), antigen processing and presentation of endogenous antigens (p = 0.002) or peptide antigens (p = 0.002), platinum pathway (p = 0.003), aging and lifespan (p = 0.004, p = 0.007), immune system development (p = 0.004) and somatic diversification (p=0.007). These results illustrate the usefulness of GSA for the development of novel hypotheses that can be followed-up with additional clinical or functional studies. Moreover, application of GSA to survival from EOC allowed the detection of significant GSs (and the corresponding genes/SNPs within these GSs) that impact OS that were not detected by assessing each SNP individually, where the smallest p-value from single SNP analysis of the GWAS for OS was 1.3E-05. Citation 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 2738. doi:10.1158/1538-7445.AM2011-2738

Abstract Introduction: Androstenedione is a precursor for estrogen biosynthesis catalyzed by aromatase. Plasma androstenedione concentrations in postmenopausal women with early stage breast cancer display large interindividual variation (Ingle et al., Cancer Res. 70:3278–86, 2010). We performed a GWAS to identify single nucleotide polymorphisms (SNPs) associated with plasma levels of androstenedione in 776 postmenopausal women with resected early stage breast cancer. Methods: Plasma androstenedione concentrations were measured by GC-MS/MS. GWAS was performed using Illumina Human610-Quad BeadChips. The GWAS results were controlled for population stratification (PS) by Eigenstrat analyses. Plasma androstenedione concentrations were associated with genotype by using a quasi-likelihood model with adjustment for PS, BMI, age and other clinical variables associated with androstenedione concentrations (p<0.01). Imputation using HapMap2 data was performed within 200 kb on either side of regions containing SNPs with p<E-05. Results: 563,945 SNPs were analyzed, followed by imputation. The genotyped SNP with the lowest p value, rs4736317 (p=4.61E-06), was on chromosome 8 near the CYP11B1 and CYP11B2 genes. This SNP had a multiplicative effect of 1.128 (95% Cl: 1.07, 1.19) and a MAF of 0.40. After imputation, 20 SNPs with apparent p<E-06 were observed in this same region, with the lowest p value for rs9773022 (p=8.1E-08). We then performed functional genomic studies, and observed that two of the SNPs with low p values (rs4736312, p=1.49E-05 and rs4736350, p=6.34E-06) created apparent Oct-1 transcription factor binding sites (TFBS). Differential nuclear protein binding of Oct-1 to probes for these SNPs was verified with EMS and supershift assays using nuclear extract from A549 and NCI-H295R cells. Treatment of these two cell lines with 10 nM androstenedione led to the disruption of Oct-1 binding with the A (wild type) allele on rs4736312. rs4736312 is located in the 3'-UTR of CYP11B1 15 bp distant from response elements for the progesterone receptor and the glucocorticoid receptor. We also demonstrated that CYP11B1 and CYP11B2 were induced by estradiol in U2OS cell lines that express ER receptor and by androstenedione in H295R cells that express the androgen receptor. Transfection of a reporter gene construct with the rs4736312 SNP sequences into COS-1 and H295R cells suppressed reporter gene activity for the variant SNP sequence as compared to wild type, an observation that may correlate with the higher plasma levels of androstenedione in women (549× 328 vs 410×208 mean×SD, M/ml) with the variant allele. Conclusion: We have identified a series of SNPs in a region of chromosome 8 in and around CYP11B1 and CYP11B2 that are associated with elevated plasma androstenedione concentrations in postmenopausal women with resected early stage breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C114.