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.

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.

Isoflavone ME-344 Disrupts Redox Homeostasis and Mitochondrial Function by Targeting Heme Oxygenase 1.

ME-344 is a second-generation isoflavone with unusual cytotoxic properties that is in clinical testing in cancer. To identify targets that contribute to its anticancer activity and therapeutic index, we used lung cancer cell lines that are naturally sensitive or resistant to ME-344. Drug-induced apoptosis was linked with enhanced levels of reactive oxygen species and this initiated a nuclear erythroid factor 2-like 2 signaling response, downstream of which, heme oxygenase 1 (HO-1) was also found to be time-dependently inhibited by ME-344. ME-344 specifically bound to, and altered, HO-1 structure and increased HO-1 translocation from the rough endoplasmic reticulum to mitochondria, but only in drug-sensitive cells. These effects did not occur in either drug-resistant or primary lung fibroblasts with lower HO-1 basal levels. HO-1 was confirmed as a drug target by using surface plasmon resonance technology and through interaction with a clickable ME-344 compound (M2F) and subsequent proteomic analyses, showing direct binding of ME-344 with HO-1. Proteomic analysis showed that clusters of mitochondrial proteins, including voltage-dependent anion-selective channels, were also impacted by ME-344. Human lung cancer biopsies expressed higher levels of Nrf2 and HO-1 compared with normal tissues. Overall, our data show that ME-344 inhibits HO-1 and impacts its mitochondrial translocation. Other mitochondrial proteins are also affected, resulting in interference in tumor cell redox homeostasis and mitochondrial function. These factors contribute to a beneficial therapeutic index and support continued clinical development of ME-344. SIGNIFICANCE: A novel cytotoxic isoflavone is shown to inhibit heme oxygenase, a desirable yet elusive target that disrupts redox homeostasis causing cell death.

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.

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.

Directed evolution provides insight into conformational substrate sampling by SrtA.

The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme's inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.

Correction: Constitutive Association of Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and Fibronectin.

[This corrects the article DOI: 10.1371/journal.pone.0163732.].

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.

Constitutive Association of Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and Fibronectin.

Functional cross-talk between Tie2 and Integrin signaling pathways is essential to coordinate endothelial cell adhesion and migration in response to the extracellular matrix, yet the mechanisms behind this phenomenon are unclear. Here, we examine the possibility that receptor cross-talk is driven through uncharacterized Tie-integrin interactions on the endothelial surface. Using a live cell FRET-based proximity assay, we monitor Tie-integrin receptor recognition and demonstrate that both Tie1 and Tie2 readily associate with integrins α5ß1 and αVß3 through their respective ectodomains. Although not required, Tie2-integrin association is significantly enhanced in the presence of the extracellular component and integrin ligand fibronectin. In vitro binding assays with purified components reveal that Tie-integrin recognition is direct, and further demonstrate that the receptor binding domain of the Tie2 ligand Ang-1, but not the receptor binding domain of Ang-2, can independently associate with α5ß1 or αVß3. Finally, we reveal that cooperative Tie/integrin interactions selectively stimulate ERK/MAPK signaling in the presence of both Ang-1 and fibronectin, suggesting a molecular mechanism to sensitize Tie2 to extracellular matrix. We provide a mechanistic model highlighting the role of receptor localization and association in regulating distinct signaling cascades and in turn, the angiogenic switch.

Over-expression of secreted proteins from mammalian cell lines.

Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats.

Tie2 and Eph receptor tyrosine kinase activation and signaling.

The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.

Structural basis for angiopoietin-1-mediated signaling initiation.

Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.