Abstract 3047: PRC2-mediated silencing of circRNA CDR1as drives miR-7-independent melanoma metastasis
Authors: Hanniford Doug , Moubarak Rana , Imig Jochen , Ulloa Alejandro , Sendra Beatriz Sanchez , Karz Alcida , Osman Iman , Aifantis Ioannis , Hernando Eva 
Published: July 2017

Circular RNAs are a novel class of non-coding RNAs with functions that remain poorly characterized in normal and pathological conditions. CDR1as is a non-canonical circRNA observed to act as a sponge for miR-7 in brain tissues. Analysis of RNA-seq data of melanocytes and melanoma cell lines and short-term cultures revealed loss of CDR1as expression as a hallmark of melanoma cells. We confirmed silencing of CDR1as in melanoma cells and tissues by RT-qPCR using divergent primers. Clinically, we observed CDR1as loss associated with metastatic progression and poor patient outcomes in a cohort of fresh-frozen melanoma tissue samples. Depletion of CDR1as in melanoma cell lines enhanced invasion in vitro and lung metastasis in vivo, demonstrating functional significance of CDR1as silencing. Surprisingly, CDR1as depletion had no clear effect on miR-7 activity in melanoma cells, and miR-7 inhibition was insufficient to rescue CDR1as silencing-induced invasion. Moreover, GSEA analyses of proteomic profiling of melanoma cells depleted of CDR1as revealed reductions of proteins involved in oxidative phosphorylation (OXPHOS) and mitochondrial function, suggesting CDR1as loss may alter metabolism of melanoma cells. Mining of CLIP-Seq data sets and subsequent RIP-PCR revealed direct interactions of CDR1as with the IGF2BP family of proteins and TARDBP, each of which are involved in regulation of mitochondrial function. These data suggest that CDR1as could regulate the subcellular localization and/or function of proteins involved in OXPHOS and mitochondrial respiration.

To understand the upstream mechanism driving CDR1as loss in melanoma, we sought to examine the primary transcript from which CDR1as arises. Interestingly, examination of the CDR1as locus revealed an upstream long non-coding RNA, LINC00632, as a plausible primary transcript of CDR1as. CDR1as and LINC00632 expression is strikingly correlated across human tissues and in melanoma cell lines (r&gt;.65). Moreover, depletion of LINC00632 using GapmeRs reduced expression of CDR1as, suggesting they arise from the same precursor transcript. Intriguingly, from data mining of ChIP-Seq datasets, we observed that the PRC2-dependent repressive chromatin mark H3K27me3, is abundant in the genomic locus of LINC00632 in melanoma cells. We validated the presence of H3K27me3 at LINC00632 by ChIP-PCR in melanoma cells lacking CDR1as expression. Strikingly, treatment of such melanoma cell lines with the EZH2 inhibitor, GSK126, induced robust re-expression of both CDR1as and LINC00632, and removal of H3K27me3 at LINC00632.

Our data document that CDR1as is epigenetically silenced in melanoma, and its loss promotes melanoma invasion and metastasis. Moreover, CDR1as loss could contribute to metabolic adaptations during metastasis through misregulation of RNA binding proteins.

Citation Format: Doug Hanniford, Rana Moubarak, Jochen Imig, Alejandro Ulloa, Beatriz Sanchez Sendra, Alcida Karz, Iman Osman, Ioannis Aifantis, Eva Hernando. PRC2-mediated silencing of circRNA CDR1as drives miR-7-independent melanoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2017-3047