Tumor-derived exosomes antagonize innate antiviral immunity.

Malignancies can compromise innate immunity, but the mechanisms of this are largely unknown. Here we found that, via tumor-derived exosomes (TEXs), cancers were able to transfer activated epidermal growth factor receptor (EGFR) to host macrophages and thereby suppress innate antiviral immunity. Screening of the human kinome identified the kinase MEKK2 in macrophages as an effector of TEX-delivered EGFR that negatively regulated the antiviral immune response. In the context of experimental tumor implantation, MEKK2-deficient mice were more resistant to viral infection than were wild-type mice. Injection of TEXs into mice reduced innate immunity, increased viral load and increased morbidity in an EGFR- and MEKK2-dependent manner. MEKK2 phosphorylated IRF3, a transcription factor crucial for the production of type I interferons; this triggered poly-ubiquitination of IRF3 and blocked its dimerization, translocation to the nucleus and transcriptional activity after viral infection. These findings identify a mechanism by which cancer cells can dampen host innate immunity and potentially cause patients with cancer to become immunocompromised.

Corrigendum: YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKɛ-mediated phosphorylation.

This corrects the article DOI: 10.1038/ni.3744.

YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKɛ-mediated phosphorylation.

The transcription regulator YAP controls organ size by regulating cell growth, proliferation and apoptosis. However, whether YAP has a role in innate antiviral immunity is largely unknown. Here we found that YAP negatively regulated an antiviral immune response. YAP deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in vivo. YAP blocked dimerization of the transcription factor IRF3 and impeded translocation of IRF3 to the nucleus after viral infection. Notably, virus-activated kinase IKKɛ phosphorylated YAP at Ser403 and thereby triggered degradation of YAP in lysosomes and, consequently, relief of YAP-mediated inhibition of the cellular antiviral response. These findings not only establish YAP as a modulator of the activation of IRF3 but also identify a previously unknown regulatory mechanism independent of the kinases Hippo and LATS via which YAP is controlled by the innate immune pathway.

OTUB2 Promotes Cancer Metastasis via Hippo-Independent Activation of YAP and TAZ.

The transcriptional regulators YAP and TAZ play important roles in development, physiology, and tumorigenesis and are negatively controlled by the Hippo pathway. It is yet unknown why the YAP/ TAZ proteins are frequently activated in human malignancies in which the Hippo pathway is still active. Here, by a gain-of-function cancer metastasis screen, we discovered OTUB2 as a cancer stemness and metastasis-promoting factor that deubiquitinates and activates YAP/TAZ. We found OTUB2 to be poly-SUMOylated on lysine 233, and this SUMOylation enables it to bind YAP/TAZ. We also identified a yet-unknown SUMO-interacting motif (SIM) in YAP and TAZ required for their association with SUMOylated OTUB2. Importantly, EGF and oncogenic KRAS induce OTUB2 poly-SUMOylation and thereby activate YAP/TAZ. Our results establish OTUB2 as an essential modulator of YAP/TAZ and also reveal a novel mechanism via which YAP/TAZ activity is induced by oncogenic KRAS.

USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

A Pro-Fluorescent Ubiquitin-Based Probe to Monitor Cysteine-Based E3 Ligase Activity.

Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity.

Inflammation induces endothelial-to-mesenchymal transition and promotes vascular calcification through downregulation of BMPR2.

Endothelial-to-mesenchymal transition (EndMT) has been unveiled as a common cause for a multitude of human pathologies, including cancer and cardiovascular disease. Vascular calcification is a risk factor for ischemic vascular disorders and slowing calcification may reduce mortality in affected patients. The absence of early biomarkers hampers the identification of patients at risk. EndMT and vascular calcification are induced upon cooperation between distinct stimuli, including inflammatory cytokines and transforming growth factor beta (TGF-ß) family members. However, how these signaling pathways interplay to promote cell differentiation and eventually vascular calcification is not well understood. Using in vitro and ex vivo analysis in animal models and patient-derived tissues, we have identified that the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) induce EndMT in human primary aortic endothelial cells, thereby sensitizing them for BMP-9-induced osteogenic differentiation. Downregulation of the BMP type II receptor BMPR2 is a key event in this process. Rather than compromising BMP canonical signal transduction, loss of BMPR2 results in decreased JNK signaling in ECs, thus enhancing BMP-9-induced mineralization. Altogether, our results point at the BMPR2-JNK signaling axis as a key pathway regulating inflammation-induced EndMT and contributing to calcification. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation.

TGF-ß members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-ß family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-ß signaling. Depletion of RNF12 dramatically reduced TGF-ß/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-ß family signaling.

JUNB governs a feed-forward network of TGFß signaling that aggravates breast cancer invasion.

It is well established that transforming growth factor-ß (TGFß) switches its function from being a tumor suppressor to a tumor promoter during the course of tumorigenesis, which involves both cell-intrinsic and environment-mediated mechanisms. We are interested in breast cancer cells, in which SMAD mutations are rare and interactions between SMAD and other transcription factors define pro-oncogenic events. Here, we have performed chromatin immunoprecipitation (ChIP)-sequencing analyses which indicate that the genome-wide landscape of SMAD2/3 binding is altered after prolonged TGFß stimulation. De novo motif analyses of the SMAD2/3 binding regions predict enrichment of binding motifs for activator protein (AP)1 in addition to SMAD motifs. TGFß-induced expression of the AP1 component JUNB was required for expression of many late invasion-mediating genes, creating a feed-forward regulatory network. Moreover, we found that several components in the WNT pathway were enriched among the late TGFß-target genes, including the invasion-inducing WNT7 proteins. Consistently, overexpression of WNT7A or WNT7B enhanced and potentiated TGFß-induced breast cancer cell invasion, while inhibition of the WNT pathway reduced this process. Our study thereby helps to explain how accumulation of pro-oncogenic stimuli switches and stabilizes TGFß-induced cellular phenotypes of epithelial cells.

Cancer-associated fibroblast-derived Gremlin 1 promotes breast cancer progression.

BACKGROUND: Bone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor ß (TGFß)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer. METHODS: In this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models. RESULTS: Expression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer cell lines did not express GREM1 in vitro, but breast CAFs did express GREM1 both in vitro and in vivo. Transforming growth factor ß (TGFß) secreted by breast cancer cells, and also inflammatory cytokines, stimulated GREM1 expression in CAFs. Grem1 abrogated bone morphogenetic protein (BMP)/SMAD signaling in breast cancer cells and promoted their mesenchymal phenotype, stemness, and invasion. Moreover, Grem1 production by CAFs strongly promoted the fibrogenic activation of CAFs and promoted breast cancer cell intravasation and extravasation in co-injection xenograft zebrafish models. CONCLUSIONS: Our results demonstrated that Grem1 is a pivotal factor in the reciprocal interplay between breast cancer cells and CAFs, which promotes cancer cell invasion. Targeting Grem1 could be beneficial in the treatment of breast cancer patients with high Grem1 expression.

Molecular insights into tumour metastasis: tracing the dominant events.

Metastasis of malignant cells to vital organs remains the major cause of mortality in many types of cancers. The tumour invasion-metastasis cascade is a stepwise and multistage process whereby tumour cells disseminate from primary sites and spread to colonize distant sites through the systemic haematogenous or lymphatic circulations. The general steps of metastasis may be similar in almost all tumour types, but metastasis to different tissues seems to require distinct sets of regulators and/or an 'educated' microenvironment which may facilitate the infiltration and colonization of tumour cells to specific tissues. Moreover, interactions of tumour cells with stromal cells, endothelial cells, and immune cells that they encounter will also aid them to gain survival advantages, evade immune surveillance, and adapt to the new host microenvironment. Due to the high correlation between tumour metastasis and survival rate of patients, a deeper understanding of the molecular participants and processes involved in metastasis could pave the way towards novel, more effective and targeted approaches to prevent and treat tumour metastasis. In this review, we provide an update on the regulation networks orchestrated by the dominant regulators of different stages throughout the metastatic process including, but not limited to, epithelial-mesenchymal transition in local invasion, resistance to anoikis during migration, and colonization of different distant sites. We also put forward some suggestions and problems concerning the treatment of tumour metastasis that should be solved and/or improved for better therapies in the near future. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif.

Reversible protein modification by small ubiquitin-like modifiers (SUMOs) is critical for eukaryotic life. Mass spectrometry-based proteomics has proven effective at identifying hundreds of potential SUMO target proteins. However, direct identification of SUMO acceptor lysines in complex samples by mass spectrometry is still very challenging. We have developed a generic method for the identification of SUMO acceptor lysines in target proteins. We have identified 103 SUMO-2 acceptor lysines in endogenous target proteins. Of these acceptor lysines, 76 are situated in the SUMOylation consensus site [VILMFPC]KxE. Interestingly, eight sites fit the inverted SUMOylation consensus motif [ED]xK[VILFP]. In addition, we found direct mass spectrometric evidence for crosstalk between SUMOylation and phosphorylation with a preferred spacer between the SUMOylated lysine and the phosphorylated serine of four residues. In 16 proteins we identified a hydrophobic cluster SUMOylation motif (HCSM). SUMO conjugation of RanGAP1 and ZBTB1 via HCSMs is remarkably efficient.

TGF-ß signaling in cancer metastasis.

The transforming growth factor (TGF)-ß signaling events are well known to control diverse processes and numerous responses, such as cell proliferation, differentiation, apoptosis, and migration. TGF-ß signaling plays context-dependent roles in cancer: in pre-malignant cells TGF-ß primarily functions as a tumor suppressor, while in the later stages of cancer TGF-ß signaling promotes invasion and metastasis. Recent studies have also suggested that the cross-talk between TGF-ß signaling and other signaling pathways, such as Hippo, Wnt, EGFR/RAS, and PI3K/AKT pathways, may substantially contribute to our current understanding of TGF-ß signaling and cancer. As a result of the wide-ranging effects of TGF-ß, blockade of TGF-ß and its downstream signaling components provides multiple therapeutic opportunities. Therefore, the outlook for anti-TGF-ß signaling therapy for numerous diseases appears bright and will provide valuable information and thinking on the drug molecular design. In this review, we focus on recent insights into the regulation of TGF-ß signaling in cancer metastasis which may contribute to the development of novel cancer-targeting therapies.

Tamoxifen Inhibits TGF-ß-Mediated Activation of Myofibroblasts by Blocking Non-Smad Signaling Through ERK1/2.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine which stimulates the differentiation of fibroblasts into myofibroblasts. Myofibroblasts are critical for normal wound healing, but also accumulate pathologically in a number of chronic inflammatory conditions where they are key contributors to aberrant tissue remodeling and fibrosis, and in cancer stroma. In the current study, we identified a role for tamoxifen as a potent inhibitor of the TGF-ß-mediated activation of primary human skin and breast fibroblasts. Our data indicate that tamoxifen does not interfere with canonical Smad signaling downstream of TGF-ß but rather blocks non-Smad signaling through ERK1/2 MAP-kinase and the AP-1 transcription factor FRA2. We further demonstrate by siRNA-mediated knockdown that FRA2 is critical for the induced expression of myogenic proteins in response to TGF-ß. Functionally, TGF-ß-stimulated fibroblast-mediated contraction of collagen gels was impaired in the presence of tamoxifen. Altogether, these data demonstrate that tamoxifen prevents myofibroblast differentiation and, therefore, may provide therapeutic benefits to patients suffering from chronic inflammatory conditions or cancer.

Genetic depletion and pharmacological targeting of αv integrin in breast cancer cells impairs metastasis in zebrafish and mouse xenograft models.

INTRODUCTION: Increased expression of αv integrins is frequently associated with tumor cell adhesion, migration, invasion and metastasis, and correlates with poor prognosis in breast cancer. However, the mechanism by which αv integrins can enhance breast cancer progression is still largely unclear. The effects of therapeutic targeting of αv integrins in breast cancer also have yet to be investigated. METHODS: We knocked down αv integrin in MDA-MB-231 and MCF10A-M4 breast cancer cells, or treated these cells with the αv antagonist GLPG0187. The effects of αv integrin depletion on mesenchymal markers, transforming growth factor-ß (TGF-ß)/Smad signaling and TGF-ß-induced target gene expression were analyzed in MDA-MB-231 cells by RNA analysis or Western blotting. The function of αv integrin on breast cancer cell migration was investigated by transwell assay in vitro, and its effect on breast cancer progression was assessed by both zebrafish and mouse xenografts in vivo. In the mouse model, GLPG0187 was administered separately, or in combination with the standard-of-care anti-resorptive agent zoledronate and the chemotherapeutic drug paclitaxel, to study the effects of combinational treatments on breast cancer metastasis. RESULTS: Genetic interference and pharmacological targeting of αv integrin with GLPG0187 in different breast cancer cell lines inhibited invasion and metastasis in the zebrafish or mouse xenograft model. Depletion of αv integrin in MDA-MB-231 cells inhibited the expression of mesenchymal markers and the TGF-ß/Smad response. TGF-ß induced αv integrin mRNA expression and αv integrin was required for TGF-ß-induced breast cancer cell migration. Moreover, treatment of MDA-MB-231 cells with non-peptide RGD antagonist GLPG0187 decreased TGF-ß signaling. In the mouse xenografts GLPG0187 inhibited the progression of bone metastasis. Maximum efficacy of inhibition of bone metastasis was achieved when GLPG0187 was combined with the standard-of-care metastatic breast cancer treatments. CONCLUSION: These findings show that αv integrin is required for efficient TGF-ß/Smad signaling and TGF-ß-induced breast cancer cell migration, and for maintaining a mesenchymal phenotype of the breast cancer cells. Our results also provide evidence that targeting αv integrin could be an effective therapeutic approach for treatment of breast cancer tumors and/or metastases that overexpress αv integrin.

Ubiquitin-specific protease 4 mitigates Toll-like/interleukin-1 receptor signaling and regulates innate immune activation.

The Toll-like receptor (TLR)/IL-1 receptor (IL-1R) signaling pathway is essential for innate immune responses and immune homeostasis. Lys-63-polyubiquitinated TRAF6 mediates its downstream signaling activation. In a gain-of-expression screen of 66 different deubiquitinating enzymes, we identified USP4 as a potent negative regulator of TLR/IL-1R signaling and TRAF6-interacting protein. USP4 deubiquitinates TRAF6 and thereby prevents the activation of NF-κB and AP-1 transcription factors and subsequent proinflammatory responses. LPS-treated usp4-depleted zebrafish larvae expressed higher levels of proinflammatory cytokines and were more susceptible to endotoxic challenge. Taken together, our results demonstrate that USP4 plays an essential role in negative regulation of the TLR/IL-1R signaling-mediated innate immune response.

Fas-associated factor 1 is a scaffold protein that promotes ß-transducin repeat-containing protein (ß-TrCP)-mediated ß-catenin ubiquitination and degradation.

FAS-associated factor 1 (FAF1) antagonizes Wnt signaling by stimulating ß-catenin degradation. However, the molecular mechanism underlying this effect is unknown. Here, we demonstrate that the E3 ubiquitin ligase ß-transducin repeat-containing protein (ß-TrCP) is required for FAF1 to suppress Wnt signaling and that FAF1 specifically associates with the SCF (Skp1-Cul1-F-box protein)-ß-TrCP complex. Depletion of ß-TrCP reduced FAF1-mediated ß-catenin polyubiquitination and impaired FAF1 in antagonizing Wnt/ß-catenin signaling. FAF1 was shown to act as a scaffold for ß-catenin and ß-TrCP and thereby to potentiate ß-TrCP-mediated ß-catenin ubiquitination and degradation. Data mining revealed that FAF1 expression is statistically down-regulated in human breast carcinoma compared with normal breast tissue. Consistent with this, FAF1 expression is higher in epithelial-like MCF7 than mesenchymal-like MDA-MB-231 human breast cancer cells. Depletion of FAF1 in MCF7 cells resulted in increased ß-catenin accumulation and signaling. Importantly, FAF1 knockdown promoted a decrease in epithelial E-cadherin and an increase in mesenchymal vimentin expression, indicative for an epithelial to mesenchymal transition. Moreover, ectopic FAF1 expression reduces breast cancer cell migration in vitro and invasion/metastasis in vivo. Thus, our studies strengthen a tumor-suppressive function for FAF1.

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.

Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.

Key signaling nodes in mammary gland development and cancer: Smad signal integration in epithelial cell plasticity.

Smad proteins are the key intermediates of transforming growth factor-beta (TGF-ß) signaling during development and in tissue homeostasis. Pertubations in TGF-ß/Smad signaling have been implicated in cancer and other diseases. In the cell nucleus, Smad complexes trigger cell type- and context-specific transcriptional programs, thereby transmitting and integrating signals from a variety of ligands of the TGF-ß superfamily and other stimuli in the cell microenvironment. The actual transcriptional and biological outcome of Smad activation critically depends on the genomic integrity and the modification state of genome and chromatin of the cell. The cytoplasmic and nuclear Smads can also modulate the activity of other signal transducers and enzymes such as microRNA-processing factors. In the case of breast cancer, the role of Smads in epithelial plasticity, tumor-stroma interactions, invasion, and metastasis seems of particular importance.

In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo.

Genetic and biochemical studies have shown that selective interactions between the Jun, Fos, and activating transcription factor (ATF) components of transcription factor activating protein 1 (AP-1) exhibit specific and critical functions in the regulation of cell proliferation, differentiation, and survival. For instance, the ratio between c-Jun/c-Fos and c-Jun/ATF2 dimers in the cell can be a determining factor in the cellular response to oncogenic or apoptotic stimuli. Until recently, no methods were available to detect endogenous AP-1 complexes in cells and tissues in situ. Here, we validated the proximity ligation assay (PLA) for its ability to specifically visualize and quantify changes in endogenous c-Jun/c-Fos, c-Jun/ATF2, and c-Jun/Fra1 complexes by using, among others, partner-selective c-Jun mutants. Furthermore, we examined the levels of c-Jun/AP-1 dimers in cell lines representing different types of human breast cancer and found that aggressive basal-like breast cancer cells can be discriminated from much less invasive luminal-like cells by PLA detection of c-Jun/Fra1 rather than of c-Jun/ATF2 and c-Jun/c-Fos. Also in tumor tissue derived from highly metastatic basal-like MDA-MB231 cells, high levels of c-Jun/Fra1 complexes were detected. Together, these results demonstrate that in situ PLA is a powerful diagnostic tool to analyze and quantify the amounts of biologically critical AP-1 dimers in fixed cells and tissue material.