Abstract 196: A SNP in the 3′-untranslated region of FZD4 linked to lung cancer survival modulates a miRNA-mediated FZD4 transcript binding, cleavage, expression, and Wnt-signaling in NSCLC cells

Single nucleotide polymorphisms (SNPs) in miRNA genes and miRNA-associated pathways (miR-SNPs) are predicted to have significant effects on gene expression and cellular processes by disrupting miRNA biogenesis and modulating miRNA-mRNA interactions. We identified several germline SNPs at potential miRNA-binding sites in the 3′ untranslated regions (3′UTRs) of genes in the wingless (Wnt) signaling pathway to be significantly associated with overall survival in NSCLC patients. SNP (rs713065 with a C allele) in the 3′UTR of the FZD4 gene in the Wnt canonical pathway displayed a significant association with decreased risk of death in early NSCLC patients. We aimed to determine the biological function and clinical relevance of this novel epidemiological FZD4-miR-SNP (rs713065) in NSCLC by identifying specific miRNAs that could directly interact with the FZD4-miR-SNP locus and assessing the effect of such a SNP and associated miRNAs on target gene regulation and NSCLC prognosis. We used a novel stem-loop array-reverse transcription-PCR (SLA-RT-PCR) assay to assess miRNA:target mRNA interaction at the specific SNP locus and detected miRNA-mediated FZD4 mRNA cleavage and 3′-uridylation activities in FZD4-SNP (C, rs713065) allele-bearing NSCLC cells H1299 and H322, but not in FZD4-WT (T) allele-containing A549 and normal human bronchial epithelial cells. A significant down regulation of GFP and Luciferase (Luc) gene and protein expression in the presence of FZD4-SNP was detected in NSCLC cell lines using both GFP-SNP and Dual-Luc-SNP reporter systems. The presence of the FZD4-SNP in the 3′UTR also down-regulated the ectopic expression of the host FZD4 gene and protein and inhibited tumor colony formation and tumor cell mobility in NSCLC cells. We identified miR-204 as a potential miRNA candidate that differentially interacts with and targets allelic variants at FZD4-miR-SNP (rs713065) loci. Target mRNA cleavage was detected at the SNP C allele site but not at the WT-allele T site in H1299 cells after co-transfection with a GFP-FZD4-SNP reporter and miR-204 expression constructs by SLA-RT-PCR. A significant correlation of miR-204 with FZD4 gene expression was also detected in NSCLC cell lines and primary tumor specimens in various miRNA expression profiling databases. Our findings suggest that the miR-204 binding site at the SNP (rs713065) loci in the 3′UTR of FZD4 may influence survival in NSCLC by modulating FZD4 expression and the Wnt/FZD4-signaling driven tumor cell proliferation and progression. (This study is partially supported by NIH/NCI grants Lung SPORE 5P50CA070907 and R01CA176568, a CPRIT Grant and a MDACC Moonshot Program Grant) Citation Format: Jing Lin, Roza Zandi, Jian Gu, Yuanqin Ye, Alexander Pertsemlidis, Xifeng Wu, Jack A. Roth, Lin Ji. A SNP in the 3′-untranslated region of FZD4 linked to lung cancer survival modulates a miRNA-mediated FZD4 transcript binding, cleavage, expression, and Wnt-signaling in NSCLC cells. [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 196. doi:10.1158/1538-7445.AM2015-196