PCBP1 regulates LIFR through FAM3C to maintain breast cancer stem cell self-renewal and invasiveness.

The poly(rC) binding protein 1 gene (PCBP1) encodes the heterogeneous nuclear ribonucleoprotein E1 (hnRNPE1), a nucleic acid-binding protein that plays a tumor-suppressive role in the mammary epithelium by regulating phenotypic plasticity and cell fate. Following the loss of PCBP1 function, the FAM3C gene (encoding the Interleukin-like EMT inducer, or "ILEI" protein) and the leukemia inhibitory factor receptor (LIFR) gene are upregulated. Interaction between FAM3C and LIFR in the extracellular space induces phosphorylation of signal transducer and activator of transcription 3 (pSTAT3). Overexpression and/or hyperactivity of STAT3 has been detected in 40% of breast cancer cases and is associated with a poor prognosis. Herein, we characterize feed-forward regulation of LIFR expression in response to FAM3C/LIFR/STAT3 signaling in mammary epithelial cells. We show that PCBP1 upregulates LIFR transcription through activity at the LIFR promoter, and that FAM3C participates in transcriptional regulation of LIFR. Additionally, our bioinformatic analysis reveals a signature of transcriptional regulation associated with FAM3C/LIFR interaction and identifies the TWIST1 transcription factor as a downstream effector that participates in the maintenance of LIFR expression. Finally, we characterize the effect of LIFR expression in cell-based experiments that demonstrate the promotion of invasion, migration, and self-renewal of breast cancer stem cells (BCSCs), consistent with previous studies linking LIFR expression to tumor initiation and metastasis in mammary epithelial cells.

The ubiquitin E3 ligase ARIH1 regulates hnRNP E1 protein stability, EMT and breast cancer progression.

The epithelial to mesenchymal transition (EMT), a process that is aberrantly activated in cancer and facilitates metastasis to distant organs, requires coordinated transcriptional and post-transcriptional control of gene expression. The tumor-suppressive RNA binding protein, hnRNP-E1, regulates splicing and translation of EMT-associated transcripts and it is thought that it plays a major role in the control of epithelial cell plasticity during cancer progression. We have utilized yeast 2 hybrid screening to identify novel hnRNP-E1 interactors that play a role in regulating hnRNP-E1; this approach led to the identification of the E3 ubiquitin ligase ARIH1. Here, we demonstrate that hnRNP-E1 protein stability is increased upon ARIH1 silencing, whereas, overexpression of ARIH1 leads to a reduction in hnRNP-E1. Reduced ubiquitination of hnRNP-E1 detected in ARIH1 knockdown (KD) cells compared to control suggests a role for ARIH1 in hnRNP-E1 degradation. The identification of hnRNP-E1 as a candidate substrate of ARIH1 led to the characterization of a novel function for this ubiquitin ligase in EMT induction and cancer progression. We demonstrate a delayed induction of EMT and reduced invasion in mammary epithelial cells silenced for ARIH1. Conversely, ARIH1 overexpression promoted EMT induction and invasion. ARIH1 silencing in breast cancer cells significantly attenuated cancer cell stemness in vitro and tumor formation in vivo. Finally, we utilized miniTurboID proximity labeling to identify novel ARIH1 interactors that may contribute to ARIH1's function in EMT induction and cancer progression.

TGFß-induced expression of long noncoding lincRNA Platr18 controls breast cancer axonogenesis.

Metastasis is the leading driver of cancer-related death. Tumor cell plasticity associated with the epithelial-mesenchymal transition (EMT), an embryonic program also observed in carcinomas, has been proposed to explain the colonization of distant organs by the primary tumor cells. Many studies have established correlations between EMT marker expression in the primary tumor and metastasis in vivo. However, the longstanding model of EMT-transitioned cells disseminating to secondary sites is still actively debated and hybrid states are presently considered as more relevant during tumor progression and metastasis. Here, we describe an unexplored role of EMT on the tumor microenvironment by controlling tumor innervation. Using in vitro and in vivo breast tumor progression models, we demonstrate that TGFß-mediated tumor cell EMT triggers the expression of the embryonic LincRNA Platr18 those elevated expression controls the expression of the axon guidance protein semaphorin-4F and other neuron-related molecules such as IGSF11/VSIG-3. Platr18/Sema4F axis silencing abrogates axonogenesis and attenuates metastasis. Our observations suggest that EMT-transitioned cells are also locally required in the primary tumor to support distant dissemination by promoting axonogenesis, a biological process known for its role in metastatic progression of breast cancer.

Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity.

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.

TGFß promotes breast cancer stem cell self-renewal through an ILEI/LIFR signaling axis.

FAM3C/Interleukin-like EMT Inducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFß signaling pathway by 3'-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFß stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates with enhanced invasion and migration. Recently, EMT has been linked to the formation of breast cancer stem cells (BCSCs) that confer both tumor cell heterogeneity as well as chemoresistant properties. Herein, we demonstrate that hnRNP E1 knockdown significantly shifts normal mammary epithelial cells to mesenchymal BCSCs in vitro and in vivo. We further validate that modulating ILEI protein levels results in the abrogation of these phenotypes, promoting further investigation into the unknown mechanism of ILEI signaling that drives tumor progression. We identify LIFR as the receptor for ILEI, which mediates signaling through STAT3 to drive both EMT and BCSC formation. Reduction of either ILEI or LIFR protein levels results in reduced tumor growth, fewer tumor initiating cells and reduced metastasis within the hnRNP E1 knock-down cell populations in vivo. These results reveal a novel ligand-receptor complex that drives the formation of BCSCs and represents a unique target for the development of metastatic breast cancer therapies.

TGF-beta signaling in cancer: post-transcriptional regulation of EMT via hnRNP E1.

The TGFß signaling pathway is a critical regulator of cancer progression in part through induction of the epithelial to mesenchymal transition (EMT). This process is aberrantly activated in cancer cells, facilitating invasion of the basement membrane, survival in the circulatory system, and dissemination to distant organs. The mechanisms through which epithelial cells transition to a mesenchymal state involve coordinated transcriptional and post-transcriptional control of gene expression. One such mechanism of control is through the RNA binding protein hnRNP E1, which regulates splicing and translation of a cohort of EMT and stemness-associated transcripts. A growing body of evidence indicates a major role for hnRNP E1 in the control of epithelial cell plasticity, especially in the context of carcinoma progression. Here, we review the multiple mechanisms through which hnRNP E1 functions to control EMT and metastatic progression.

Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1).

Interleukin-like EMT inducer (ILEI, FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell-biological process that confers metastatic properties to a tumor cell. However, very little is known about how ILEI is regulated. Here we demonstrate that ILEI is an in vivo regulator of melanoma invasiveness and is transcriptionally up-regulated by the upstream stimulatory factor-1 (USF-1), an E-box-binding, basic-helix-loop-helix family transcription factor. shRNA-mediated knockdown of ILEI in melanoma cell lines attenuated lung colonization but not primary tumor formation. We also identified the mechanism underlying ILEI transcriptional regulation, which was through a direct interaction of USF-1 with the ILEI promoter. Of note, stimulation of endogenous USF-1 by UV-mediated activation increased ILEI expression, whereas shRNA-mediated USF-1 knockdown decreased ILEI gene transcription. Finally, we report that knocking down USF-1 decreases tumor cell migration. In summary, our work reveals that ILEI contributes to melanoma cell invasiveness in vivo without affecting primary tumor growth and is transcriptionally up-regulated by USF-1.

A CREB3-regulated ER-Golgi trafficking signature promotes metastatic progression in breast cancer.

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 upregulation 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 upregulated 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.

Fbxo4-mediated degradation of Fxr1 suppresses tumorigenesis in head and neck squamous cell carcinoma.

The Fbxo4 tumour suppressor is a component of an Skp1-Cul1-F-box E3 ligase for which two substrates are known. Here we show purification of SCFFbxo4 complexes results in the identification of fragile X protein family (FMRP, Fxr1 and Fxr2) as binding partners. Biochemical and functional analyses reveal that Fxr1 is a direct substrate of SCFFbxo4. Consistent with a substrate relationship, Fxr1 is overexpressed in Fbxo4 knockout cells, tissues and in human cancer cells, harbouring inactivating Fbxo4 mutations. Critically, in head and neck squamous cell carcinoma, Fxr1 overexpression correlates with reduced Fbxo4 levels in the absence of mutations or loss of mRNA, suggesting the potential for feedback regulation. Direct analysis reveals that Fbxo4 translation is attenuated by Fxr1, indicating the existence of a feedback loop that contributes to Fxr1 overexpression and the loss of Fbxo4. Ultimately, the consequence of Fxr1 overexpression is the bypass of senescence and neoplastic progression.

Interleukin-like EMT inducer regulates partial phenotype switching in MITF-low melanoma cell lines.

ILEI (FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell biological process that confers metastatic properties to a tumor cell. Initially, we found that ILEI mRNA is highly expressed in melanoma metastases but not in primary tumors, suggesting that ILEI contributes to the malignant properties of melanoma. While melanoma is not an epithelial cell-derived tumor and does not undergo a traditional EMT, melanoma undergoes a similar process known as phenotype switching in which high (micropthalmia-related transcription factor) MITF expressing (MITF-high) proliferative cells switch to a low expressing (MITF-low) invasive state. We observed that MITF-high proliferative cells express low levels of ILEI (ILEI-low) and MITF-low invasive cells express high levels of ILEI (ILEI-high). We found that inducing phenotype switching towards the MITF-low invasive state increases ILEI mRNA expression, whereas phenotype switching towards the MITF-high proliferative state decreases ILEI mRNA expression. Next, we used in vitro assays to show that knockdown of ILEI attenuates invasive potential but not MITF expression or chemoresistance. Finally, we used gene expression analysis to show that ILEI regulates several genes involved in the MITF-low invasive phenotype including JARID1B, HIF-2α, and BDNF. Gene set enrichment analysis suggested that ILEI-regulated genes are enriched for JUN signaling, a known regulator of the MITF-low invasive phenotype. In conclusion, we demonstrate that phenotype switching regulates ILEI expression, and that ILEI regulates partial phenotype switching in MITF-low melanoma cell lines.

Membrane-organizing protein moesin controls Treg differentiation and antitumor immunity via TGF-ß signaling.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that are important for organizing membrane domains and receptor signaling and regulating the migration of effector T cells. Whether moesin plays any role during the generation of TGF-ß-induced Tregs (iTregs) is unknown. Here, we have discovered that moesin is translationally regulated by TGF-ß and is also required for optimal TGF-ß signaling that promotes efficient development of iTregs. Loss of moesin impaired the development and function of both peripherally derived iTregs and in vitro-induced Tregs. Mechanistically, we identified an interaction between moesin and TGF-ß receptor II (TßRII) that allows moesin to control the surface abundance and stability of TßRI and TßRII. We also found that moesin is required for iTreg conversion in the tumor microenvironment, and the deletion of moesin from recipient mice supported the rapid expansion of adoptively transferred CD8+ T cells against melanoma. Our study establishes moesin as an important regulator of the surface abundance and stability of TßRII and identifies moesin's role in facilitating the efficient generation of iTregs. It also provides an advancement to our understanding about the role of the ERM proteins in regulating signal transduction pathways and suggests that modulation of moesin is a potential therapeutic target for Treg-related immune disorders.

PCBP1/HNRNP E1 Protects Chromosomal Integrity by Translational Regulation of CDC27.

UNLABELLED: CDC27 is a core component of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit 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 posttranscriptionally through the RNA binding protein poly(rC) binding protein 1 (PCBP1)/heterogeneous nuclear ribonucleoprotein E1 (HNRNP E1). 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 FZR1/CDH1, a cofactor of the G1 and late G2-M-APC/C and a substrate normally reserved for the SCF-ßTRCP 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. Furthermore, tissue microarray of breast cancer patient tumor samples reveals high CDC27 levels compared with nonneoplastic 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. IMPLICATIONS: 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. Mol Cancer Res; 14(7); 634-46. ©2016 AACR.

Establishment of a TGFß-induced post-transcriptional EMT gene signature.

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 TGFß 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 TGFß-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.