Abstract 1479: Hypoxia upregulates the long noncoding RNA MALAT1 through increased chromatin looping

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA that is overexpressed in many solid tumors and promotes proliferation, angiogenesis, drug resistance, invasion and metastasis. MALAT1 contributes to oncogenesis through increased expression and splicing of cell cycle and motility-associated transcripts. While the role of MALAT1 in cancer progression is well understood, the mechanisms underlying MALAT1 overexpression in cancer are poorly characterized. Hypoxia is a known contributor to MALAT1 upregulation in multiple cancers, but the exact molecular mechanism is unknown. Here, we report that hypoxia causes changes in long-range chromatin interactions at the MALAT1 locus in breast cancer and upregulates MALAT1 in a hypoxia inducible factor (HIF)-independent manner.To determine the mechanism of hypoxic-mediated upregulation, we first cultured a panel of cell lines including one non-tumorigenic, immortalized mammary cell line (MCF10A) and eight breast cancer cell lines in normoxia (21% O2) or hypoxia (1% O2) for 24 hours. This demonstrated MALAT1 was upregulated under hypoxia in only the cancer cell lines. Chromatin immunoprecipitation and qPCR (ChIP-qPCR) demonstrated both HIF-1α and HIF-2α enrichment increased at the MALAT1 promoter in the cancer lines MCF7 and MDA-MB-231 under hypoxia, but HIF knockdown had no effect on MALAT1 expression. Through an extensive genomic data analysis, we then identified two putative enhancer elements at -21kb and -11kb upstream of the transcription start site and one downstream of the transcription termination site (+9kb enhancer). ChIP-qPCR showed an increased in active promoter mark H3K4me3 at the promoter, active enhancer mark H3K4me1 at the +9 and -11kb enhancers, and increase in RNA polymerase II occupancy at the promoter, +9, -11, and -21kb enhancers under hypoxia. To investigate whether these enhancers regulate MALAT1 expression through long-range chromatin interactions, we performed chromosome conformation capture and qPCR (3C-qPCR). The results demonstrated a hypoxic-specific increase in chromatin interaction between the enhancer elements and promoter in the cancer lines MCF7 and MDA-MB-231, which were notably absent in the non-tumorigenic line MCF10A. In conclusion, our results demonstrate a novel mechanism of MALAT1 upregulation in hypoxia specific to cancer cells. Increased chromatin looping and contacts between enhancer elements allow upregulation of MALAT1, which does not depend on HIF, suggesting other cancer-specific factor(s) guide loop formation or take advantage of this novel structure for transcription initiation. Citation Format: Joshua Kenneth Stone, Jung-Hyun Kim, Ming Tan, Eun-Young Erin Ahn. Hypoxia upregulates the long noncoding RNA MALAT1 through increased chromatin looping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1479.