(B) Beyond regulating splicing, LIN28, independently or in conjunction with hnRNP A1, can influence mRNA steady-state levels

(B) Beyond regulating splicing, LIN28, independently or in conjunction with hnRNP A1, can influence mRNA steady-state levels. to identify differentially spliced gene isoforms in LIN28 and hnRNP A1 small interfering RNA (siRNA)-treated cells. The results reveal that these proteins regulate alternative splicing and steady-state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of the ENAH exon 11a isoform. The expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. Intriguingly, analysis of publicly available array data from the Cancer Genome Atlas (TCGA) reveals that LIN28 expression in the HER2 subtype is significantly different from that in other breast cancer subtypes. Collectively, our data suggest that LIN28 may regulate splicing and gene expression programs that drive breast cancer subtype phenotypes. INTRODUCTION LIN28A is Col18a1 an evolutionarily conserved RNA-binding protein that plays important and widespread roles in development and disease ML604086 (1, 2). LIN28A was first identified in a screen of mutants of the nematode displaying defects in developmental timing (3). Subsequent studies have identified two homologs, LIN28A and LIN28B, in mammals, including humans and mice (4). LIN28A (here referred to as LIN28) is highly expressed during development and in human and mouse embryonic stem (ES) cells (5, 6). Conversely, LIN28 is rarely expressed in normal adult tissues except when reactivated in cancer (7,C10). Abnormal LIN28 expression has been observed in a number of human malignancies, suggesting that LIN28 is important in cancer and most likely functions as an oncogene (7, 8). Overexpression of LIN28 promotes tumor cell migration and cellular transformation, which are associated with advanced stages of poorly differentiated human cancers, including liver cancer, ovarian cancer, and myeloid leukemia (8, 11). Mechanistically, the effects of LIN28 on multiple unrelated biological and pathological processes have been attributed to the ability of LIN28 to block the biogenesis of the Let-7 family of microRNAs (miRNAs) (12,C14). Members of the Let-7 family ML604086 of miRNAs act as tumor suppressors by inhibiting the expression of oncogenes and key regulators of mitogenic pathways, including c-myc, K-Ras, and ML604086 HMGA2 (15,C17). Consistent with this idea, low levels of Let-7 and high levels of LIN28 are strongly associated with increased tumorigenesis and poor disease prognosis (8, 18). On the other hand, recent studies indicate that LIN28 can change gene regulatory networks independent of Let-7, suggesting that LIN28 may contribute to tumor progression through Let-7-independent mechanisms (5, 19,C23). LIN28 directly binds and stimulates the translation of several mRNAs that encode proteins involved in multiple cellular processes that drive cancer progression (21, 24,C26). As an example, LIN28 regulates the translation and expression of several cell cycle regulatory mRNAs that encode factors controlling the G2/S-to-M-phase transition, consistent with a role for LIN28 in cell growth and tumor promotion (22, 25). Beyond regulating the cell cycle, LIN28 also binds and regulates the translation of mRNAs encoding cell metabolic enzymes driving glycolysis and mitochondrial respiration (5, 23, 24). This would be consistent with the reprogrammed glucose metabolism needed to support the energetic requirements for proliferation and increased cell mass characteristic of tumor cells (1, 27). Despite the reactivation of LIN28 in many cancers, knowledge of the molecular mechanisms by which LIN28 functions to promote specific types of cancer, including breast cancer, is lacking. LIN28 is expressed in breast cancer tumors, and recent studies have shown that LIN28 is a powerful predictor of poor prognoses and patient clinical outcomes (8, 28, 29). With this in mind, we were interested in identifying novel LIN28 mRNA targets that could provide insights into the function of LIN28 in breast cancer. We performed RNA-protein immunoprecipitation (RIP) coupled with genome-wide sequencing (RIP-Seq) to identify endogenous LIN28 mRNA targets (30). Several studies have described LIN28 gene regulatory networks, but less is known about transacting factors that mediate its functions. We have used protein immunoprecipitation (IP) combined with mass spectrophotometry (MS) analyses to identify transacting factors that could modulate LIN28 regulatory functions in breast cancer cells (31). Our MS analyses reveal that the nucleocytoplasmic shuttling protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a protein associate of LIN28 in breast cancer cells. Furthermore, transcriptome sequencing (RNA-Seq) data suggest that LIN28 regulates the expression of a unique set of mRNAs independent of LIN28 binding, indicating that LIN28 regulates gene expression via.