The contribution of lncRNAs to tumor progression and regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleo-cytoplasmic translocation or in response to TGFβ, allows alternative splicing and generates a non-coding isoform of PNUTS. Functionally the lncRNA-PNUTS serves as a competitive sponge for miR-205 during epithelial-mesenchymal transition. In mesenchymal breast tumor cells and in breast tumor samples, the expression of lncRNA-PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS-mRNA and lncRNA-PNUTS each eliciting distinct biological functions. While PNUTS-mRNA is ubiquitously expressed, lncRNA-PNUTS appears to be tightly regulated dependent on hnRNP E1’s status and tumor context.

The mitotic checkpoint maintains genomic stability by ensuring that chromosomes are accurately segregated during mitosis. When the checkpoint is activated, the mitotic checkpoint complex (

A major challenge in the clinical management of human cancers is to accurately stratify patients according to risk and likelihood of a favorable response. Stratification is confounded by significant phenotypic heterogeneity in some tumor types, often without obvious criteria for subdivision. Despite intensive transcriptional array analyses, the identity and validation of cancer specific ‘signature genes’ remains elusive, partially because the transcriptome does not mirror the proteome. The simplification associated with transcriptomic profiling does not take into consideration changes in the relative expression among transcripts that arise due to post-transcriptional regulatory events. We have previously shown that TGFβ post-transcriptionally regulates epithelial-mesenchymal transition (EMT) by causing increased expression of two transcripts, Dab2 and ILEI, by modulating hnRNP E1 phosphorylation. Using a genome-wide combinatorial approach involving expression profiling and RIP-Chip analysis, we have identified a cohort of translationally regulated mRNAs that are induced during TGFβ-mediated EMT. Coordinated translational regulation by hnRNP E1 constitutes a post-transcriptional regulon inhibiting the expression of related EMT-facilitating genes, thus enabling the cell to rapidly and coordinately regulate multiple EMT-facilitating genes.

In order to better understand the process of breast cancer metastasis, we have generated a mammary epithelial progression series of increasingly aggressive cell lines that metastasize to lung. Here, we demonstrate that up-regulation of an Endoplasmic Reticulum (ER) to Golgi trafficking gene signature in metastatic cells enhances transport kinetics, which promotes malignant progression. We observe increased ER-Golgi trafficking, an altered secretome and sensitivity to the retrograde transport inhibitor brefeldin A (BFA) in cells that metastasize to lung. CREB3 was identified as a transcriptional regulator of up-regulated ER-Golgi trafficking genes ARF4, COPB1 and USO1, and silencing of these genes attenuated the metastatic phenotype in vitro and lung colonization in vivo. Furthermore, high trafficking gene expression significantly correlated with increased risk of distant metastasis and reduced relapse-free and overall survival in breast cancer patients, suggesting that modulation of ER-Golgi trafficking plays an important role in metastatic progression.

The epithelial-to-mesenchymal transition (EMT) is a cellular process that functions during embryonic development and tissue regeneration, thought to be aberrantly activated in epithelial-derived cancer and play an important role in the process of metastasis. The TGFβ signaling pathway is a key inducer of EMT and we have elucidated a post-transcriptional mechanism by which TGFβ modulates expression of select transcripts via the RNA binding protein hnRNP E1 during EMT. One such transcript inhibin βA is a member of the TGFβ superfamily. Here, we show by polysome profiling that inhibin βA is translationally regulated by TGFβ via hnRNP E1. TGFβ treatment or knockdown of hnRNP E1 relieves silencing of the inhibin βA transcript, resulting in increased protein expression and secreted levels of the inhibin βA homodimer, activin A. Our data indicates that the translational up-regulation of inhibin βA enhances the migration and invasion of cells that have undergone an EMT and promotes cancer progression in vivo.

Cdc27 is a core component of the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase whose oscillatory activity is responsible for the metaphase-to-anaphase transition and mitotic exit. Here, in normal murine mammary gland epithelial cells (NMuMG), Cdc27 expression is controlled post-transcriptionally through the RNA binding protein heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1/PCBP1). shRNA-mediated knockdown of hnRNP E1 abrogates translational silencing of the Cdc27 transcript resulting in constitutive expression of Cdc27. Dysregulated expression of Cdc27 leads to premature activation of the G2/M-APC/C-Cdc20 complex resulting in the aberrant degradation of Cdh1/Fzr1, a co-factor of the G1 and late G2/M-APC/C and a substrate normally reserved for the SCF-βTRCP ligase. Loss of Cdh1 expression and of APC/C-Cdh1 activity, upon constitutive expression of Cdc27, results in mitotic aberrations and aneuploidy in NMuMG cells. Further, tissue microarray (TMA) of breast cancer patient tumor samples reveals high Cdc27 levels compared to non-neoplastic breast tissue and a significant correlation between disease recurrence and Cdc27 expression. These results suggest that dysregulation of hnRNP E1-mediated translational regulation of Cdc27 leads to chromosomal instability and aneuploidy, and that Cdc27 expression represents a significant predictor of breast cancer recurrence. Implication Statement The RNA binding protein hnRNP E1 mediates translational regulation of the cell cycle regulator Cdc27 and that dysregulation of Cdc27 leads to aneuploidy. In addition, high Cdc27 expression in breast cancer patient tumor specimens significantly predicts disease recurrence, suggesting a novel role for Cdc27 as a predictor of relapse.

In this Addendum, the authors include western blot data using a C-terminal PNUTS antibody. This is important in that an annotation of the alternative spliced form of PNUTS, denoted in the UCSC genome browser (https://genome.ucsc.edu/), depicts it as a non-coding RNA. However, downstream of the alternative splice site is an alternative AUG located in frame in the PNUTS ORF at position 1039. The potential for a protein product of ~61 kDa being generated from this AUG was examined experimentally using a C-terminal raised antibody to PNUTS to exclude the possibility that the N-terminal deletion of the splice isoform was not the reason that the predicted 61-kDa protein was not detected in cells using an N-terminal generated antibody. The results presented here confirm our previous results using the N-terminal PNUTS antibody and originally presented in Supplementary Fig. 2b of the Article; namely, that this predicted ~61-kDa product is not detectable in cells under the conditions used, even under conditions of overexpression. (1 day) is shown. The C-terminal antibody used was EPR11706 (Abcam: Ab173285; clone PPP1R10; 1/1000 dilution) raised against the C-terminal region of the PNUTS protein (amino acids 550-650). The western blot protocol and extracts used in this experiment were identical to those described in Supplementary Fig. 2 An#--PPP1R10 an#body [EPR11706]NATURE CELL BIOLOGY VOLUME 19 | NUMBER 12 | DECEMBER 2017 1443 © 2 0 1 7 M a c m i l l a n P u b l i s h e r s L i m i t e d , p a r t o f S p r i n g e r N a t u r e . A l l r i g h t s r e s e r v e d .

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