Mitotically-Associated lncRNA (MANCR) Affects Genomic Stability and Cell Division in Aggressive Breast Cancer
Abstract
Aggressive breast cancer is difficult to treat as it is unresponsive to many hormone-based therapies; therefore, it is imperative to identify novel, targetable regulators of progression. Long noncoding RNAs (lncRNA) are important regulators in breast cancer and have great potential as therapeutic targets; however, little is known about how the majority of lncRNAs function within breast cancer. This study characterizes a novel lncRNA, MANCR (mitotically-associated long noncoding RNA; LINC00704), which is upregulated in breast cancer patient specimens and cells. Depletion of MANCR in triple-negative breast cancer cells significantly decreases cell proliferation and viability, with concomitant increases in DNA damage. Transcriptome analysis, based on RNA sequencing, following MANCR knockdown reveals significant differences in the expression of >2,000 transcripts, and gene set enrichment analysis identifies changes in multiple categories related to cell-cycle regulation. Furthermore, MANCR expression is highest in mitotic cells by both RT-qPCR and RNA in situ hybridization. Consistent with a role in cell-cycle regulation, MANCR-depleted cells have a lower mitotic index and higher incidences of defective cytokinesis and cell death. Taken together, these data reveal a role for the novel lncRNA, MANCR, in genomic stability of aggressive breast cancer, and identify it as a potential therapeutic target.
Introduction
Breast cancer is a major health concern worldwide; it is the most frequently diagnosed cancer and the leading cause of cancerrelated death in women (1). There are several subtypes of breast cancer, defined by their expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). The most common subtypes, luminal A and B are both ERþ/PRþ and have the best prognosis, as they are generally low grade and responsive to hormone therapy (2). The Her2 overexpression subtype grows faster, but is responsive to targeted drug treatments such as Herceptin (3). A third subtype, basal or triple negative breast cancer (TNBC), is characterized as being ER/PR/Her2. Although TNBC is diagnosed in only 15% of breast cancer patients, it is the most aggressive subtype, unresponsive to treatment, and usually has a poor prognosis (4). As such, it remains critical to identify and characterize novel, targetable regulators of breast cancer. One novel class of regulators that hold great clinical potential are the epigenetic regulators, long noncoding RNAs (lncRNA).
lncRNAs encompass a broad class of transcripts loosely defined as being greater than 200 nucleotides in length and lacking protein-coding potential. They are transcribed by RNA polymerase II, can have multiple splice variants, and their expression is regulated by transcription factors. Expression of lncRNAs is more cell type and tissue specific than their mRNA protein-coding counterparts (5). Once dismissed as transcriptional noise, lncRNAs are involved in a variety of biological processes, such as X-chromosome inactivation by Xist (6, 7), genomic imprinting by H19 (8, 9), and mammary epithelial differentiation by Zfas1 (10).
lncRNAs are often deregulated in cancer (11). HOTAIR is a wellstudied lncRNA that is overexpressed in a variety of cancers (12). It promotes cancer invasiveness and metastasis by interacting with polycomb repressive complex 2 (PRC2), altering histone H3K27me3, and silencing anti-metastatic genes (13). In addition to pan-cancer lncRNAs, several studies have identified signatures of lncRNAs associated with specific breast cancer stages and subtypes (14–17). Although these studies have identified hundreds of cancer-associated lncRNAs, such as DSCAM-AS1, an ERregulated lncRNA that has been implicated in luminal breast cancer and tamoxifen resistance (18, 19), relatively few have been functionally studied.
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