Abstract MIP-057: TARGETING THE LONG–NONCODING RNA MALAT1 IN OVARIAN CANCER

Abstract

Abstract PURPOSE: Ovarian cancer metastasizes via direct seeding, whereby cells must resist anoikis (detachment-induced cell death) in the peritoneal cavity before invading through the mesothelium to metastasize into peritoneal organs. The molecular mechanisms that facilitate these processes are not fully understood. Using an unbiased screen, we identified the long non-coding RNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) as being a potential regulator of anoikis resistance. We hypothesized that MALAT1 promotes progression through the early steps of metastasis, including anoikis resistance, peritoneal invasion, and metastatic outgrowth, and that therapeutic inhibition of MALAT1 will reduce ovarian tumor burden and metastatic spread. EXPERIMENTAL PROCEDURES: Modified anti-sense oligonucleotides (ASO) targeting MALAT1 were used to inhibit MALAT1 expression in ovarian cancer cell lines (OVCA420, HEY, OV1847). Transfections were performed using lipofectamine as well as novel lipid-based nanoparticles that are minimally toxic and have potential in vivo applications. Transcriptome profiling was performed on Affymetrix arrays. Migration and invasion assays were performed on an XCelligence Real Time Cell Analyzer, and proliferation was measured by crystal violet. Anoikis was measured using Caspase 3/7 assay. Knockdown of RBFOX2 and KIF1B-beta was performed using shRNAs. Gene expression was measured by RT-qPCR. Publicly available datasets including the Cancer Genome Atlas were analyzed for association of MALAT1 gene expression and DNA copy number with clinicopathologic variables. RESULTS: MALAT1 gene expression significantly increased after 24 hours in forced suspension culture versus attached culture in multiple anoikis-resistant ovarian cancer cell lines. MALAT1 knockdown via ASO resulted in decreased proliferation, migration, invasion, anchorage-independent growth, and increased anoikis. Lipid nanoparticle-mediated delivery of MALAT1-targeted ASO resulted in effective gene knockdown with minimal off-target toxicity. Transcriptome profiling of MALAT1-knockdown cells revealed significant changes in splicing patterns, including alternative splicing of the pro-apoptotic gene KIF1B (Kinesin Family Member 1B). MALAT1 knockdown resulted in decreased gene expression of the splicing factor RBFOX2 (RNA Binding Protein Fox-1, Homolog 2), which is associated with epithelial-to-mesenchymal transition (EMT). KIF1B contains RBFOX2 binding sites within spliced regions, suggesting that MALAT1 may control alternative splicing through regulation of RBFOX2. Indeed, RBFOX2 knockdown resulted in increased expression of the pro-apoptotic isoform of KIF1B (KIF1B-beta), as well as increased anoikis. Furthermore, knockdown of KIF1B-beta reversed the apoptotic effects of MALAT1-knockdown. In ovarian cancer patients, high MALAT1 gene expression is associated with higher stage disease, increased risk of recurrence, and reduced overall survival. Additionally, MALAT1 is amplified in 3% of ovarian cancers, and amplification is associated with increased MALAT1 gene expression and reduced overall survival. CONCLUSIONS: The lncRNA MALAT1 facilitates a pro-metastatic phenotype in ovarian cancer cells by promoting alternative splicing and differential expression of anti-apoptotic and EMT-related genes. In vivo studies to determine the efficacy of MALAT1-knockdown via lipid-mediated delivery to inhibit progression of ovarian cancer cell lines and patient derived xenografts are ongoing. Citation Format: Michael A Gordon, Dawn R Cochrane, Jamie Betker, Tom Anchordoquy, Jennifer K Richer. TARGETING THE LONG–NONCODING RNA MALAT1 IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-057.