MANCR lncRNA modulates cell-cycle progression and metastasis in breastcancer cells by regulating the isoform-specific expression of nuclear Rho-GEF incis
A significant number of the genetic alterations observed in cancer patients lie within the non-protein-coding segments of the genome, including in regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in cancer, including Breast cancer (BrCa), but the implication of their altered expression in BrCa progression remains to be elucidated. By performing a genome-wide transcriptome screen in a Triple negative BrCa (TNBC) progression isogenic 2D and 3D spheroid model, we identify >1000 lncRNAs that display aberrant expression during BrCa progression. MANCR, a chromatin-associated lncRNA, shows elevated expression in tumorigenic and metastatic TNBC. MANCR is up regulated in response to cellular stress, including DNA damage and modulates DNA repair and cell proliferation. In addition, MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion and wound healing in vitro, and defects in lung colonization in Xenograft experiments in vivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint. At the mechanistic level, MANCR promotes the transcription ofNet1A, a Rho-GEF and regulator DNA damage checkpoint and metastasis incis,by differential promoter usage. Further experiments suggest that MANCR regulates the expression of cancer-associated genes by titrating the levels of various chromatin and RNA-binding proteins. Thus, our results identified the metastasis-promoting activities of a MANCR in TNBC by regulating the expression of genes incis. Overall design: Stable knockdown of MANCR was done using shRNA (shRNA1 and shRNA2) and CRISPRi. A Dox-inducible MANCR construct was stably expressed in M1 cells and overexpressed using Doxycycline. Isogenic M1, M3, and M4 cell lines were cultured at 37 degrees Celsius with 5% carbon dioxide. RNA was extracted using Trizol and cleaned up using Qiagen RNeasy column.