LncRNA LITATS1 suppresses TGF-ß-induced EMT and cancer cell plasticity by potentiating TßRI degradation.

Epithelial cells acquire mesenchymal phenotypes through epithelial-mesenchymal transition (EMT) during cancer progression. However, how epithelial cells retain their epithelial traits and prevent malignant transformation is not well understood. Here, we report that the long noncoding RNA LITATS1 (LINC01137, ZC3H12A-DT) is an epithelial gatekeeper in normal epithelial cells and inhibits EMT in breast and non-small cell lung cancer cells. Transcriptome analysis identified LITATS1 as a TGF-ß target gene. LITATS1 expression is reduced in lung adenocarcinoma tissues compared with adjacent normal tissues and correlates with a favorable prognosis in breast and non-small cell lung cancer patients. LITATS1 depletion promotes TGF-ß-induced EMT, migration, and extravasation in cancer cells. Unbiased pathway analysis demonstrated that LITATS1 knockdown potently and selectively potentiates TGF-ß/SMAD signaling. Mechanistically, LITATS1 enhances the polyubiquitination and proteasomal degradation of TGF-ß type I receptor (TßRI). LITATS1 interacts with TßRI and the E3 ligase SMURF2, promoting the cytoplasmic retention of SMURF2. Our findings highlight a protective function of LITATS1 in epithelial integrity maintenance through the attenuation of TGF-ß/SMAD signaling and EMT.

E3 Ubiquitin Ligases: Key Regulators of TGFß Signaling in Cancer Progression.

Transforming growth factor ß (TGFß) is a secreted growth and differentiation factor that influences vital cellular processes like proliferation, adhesion, motility, and apoptosis. Regulation of the TGFß signaling pathway is of key importance to maintain tissue homeostasis. Perturbation of this signaling pathway has been implicated in a plethora of diseases, including cancer. The effect of TGFß is dependent on cellular context, and TGFß can perform both anti- and pro-oncogenic roles. TGFß acts by binding to specific cell surface TGFß type I and type II transmembrane receptors that are endowed with serine/threonine kinase activity. Upon ligand-induced receptor phosphorylation, SMAD proteins and other intracellular effectors become activated and mediate biological responses. The levels, localization, and function of TGFß signaling mediators, regulators, and effectors are highly dynamic and regulated by a myriad of post-translational modifications. One such crucial modification is ubiquitination. The ubiquitin modification is also a mechanism by which crosstalk with other signaling pathways is achieved. Crucial effector components of the ubiquitination cascade include the very diverse family of E3 ubiquitin ligases. This review summarizes the diverse roles of E3 ligases that act on TGFß receptor and intracellular signaling components. E3 ligases regulate TGFß signaling both positively and negatively by regulating degradation of receptors and various signaling intermediates. We also highlight the function of E3 ligases in connection with TGFß's dual role during tumorigenesis. We conclude with a perspective on the emerging possibility of defining E3 ligases as drug targets and how they may be used to selectively target TGFß-induced pro-oncogenic responses.

The roles of ubiquitin modifying enzymes in neoplastic disease.

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFß, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy.

Bone morphogenetic protein receptor signal transduction in human disease.

Bone morphogenetic proteins (BMPs) are secreted cytokines that were initially discovered on the basis of their ability to induce bone. Several decades of research have now established that these proteins function in a large variety of physiopathological processes. There are about 15 BMP family members, which signal via three transmembrane type II receptors and four transmembrane type I receptors. Mechanistically, BMP binding leads to phosphorylation of the type I receptor by the type II receptor. This activated heteromeric complex triggers intracellular signaling that is initiated by phosphorylation of receptor-regulated SMAD1, 5, and 8 (also termed R-SMADs). Activated R-SMADs form heteromeric complexes with SMAD4, which engage in specific transcriptional responses. There is convergence along the signaling pathway and, besides the canonical SMAD pathway, BMP-receptor activation can also induce non-SMAD signaling. Each step in the pathway is fine-tuned by positive and negative regulation and crosstalk with other signaling pathways. For example, ligand bioavailability for the receptor can be regulated by ligand-binding proteins that sequester the ligand from interacting with receptors. Accessory co-receptors, also known as BMP type III receptors, lack intrinsic enzymatic activity but enhance BMP signaling by presenting ligands to receptors. In this review, we discuss the role of BMP receptor signaling and how corruption of this pathway contributes to cardiovascular and musculoskeletal diseases and cancer. We describe pharmacological tools to interrogate the function of BMP receptor signaling in specific biological processes and focus on how these agents can be used as drugs to inhibit or activate the function of the receptor, thereby normalizing dysregulated BMP signaling. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

USP26 regulates TGF-ß signaling by deubiquitinating and stabilizing SMAD7.

The amplitude of transforming growth factor-ß (TGF-ß) signal is tightly regulated to ensure appropriate physiological responses. As part of negative feedback loop SMAD7, a direct transcriptional target of downstream TGF-ß signaling acts as a scaffold to recruit the E3 ligase SMURF2 to target the TGF-ß receptor complex for ubiquitin-mediated degradation. Here, we identify the deubiquitinating enzyme USP26 as a novel integral component of this negative feedback loop. We demonstrate that TGF-ß rapidly enhances the expression of USP26 and reinforces SMAD7 stability by limiting the ubiquitin-mediated turnover of SMAD7. Conversely, knockdown of USP26 rapidly degrades SMAD7 resulting in TGF-ß receptor stabilization and enhanced levels of p-SMAD2. Clinically, loss of USP26 correlates with high TGF-ß activity and confers poor prognosis in glioblastoma. Our data identify USP26 as a novel negative regulator of the TGF-ß pathway and suggest that loss of USP26 expression may be an important factor in glioblastoma pathogenesis.

Regulation of Ubiquitin Enzymes in the TGF-ß Pathway.

The transforming growth factor-ß (TGF-ß) pathway has a tumor suppressor role in normal and premalignant cells but promotes oncogenesis in advanced cancer cells. Components of the pathway are tightly controlled by ubiquitin modifying enzymes and aberrations in these enzymes are frequently observed to dysregulate the pathway causing diseases such as bone disorders, cancer and metastasis. These enzymes and their counterparts are increasingly being tested as druggable targets, and thus a deeper understanding of the enzymes is required. This review summarizes the roles of specific ubiquitin modifying enzymes in the TGF-ß pathway and how they are regulated.

MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation.

Spermatogenesis is a highly complicated metamorphosis process of male germ cells. Recent studies have provided evidence that the ubiquitin-proteasome system plays an important role in sperm head shaping, but the underlying mechanism is less understood. In this study, we localized membrane-associated RING-CH (MARCH)7, an E3 ubiquitin ligase, in rat testis. Northern blot analysis showed that March7 mRNA is expressed ubiquitously but highly in the testis and ovary. In situ hybridization of rat testis demonstrated that March7 mRNA is expressed weakly in spermatogonia and its level is gradually increased as they develop. Immunohistochemical analysis detected MARCH7 protein expression in spermiogenic cells from late round spermatids to elongated spermatids and in epididymal spermatozoa. Moreover, MARCH7 was found to be localized to the caudal end of the developing acrosome of late round and elongating spermatids, colocalizing with ß-actin, a component of the acroplaxome. In addition, MARCH7 was also detected in the developing flagella and its expression levels were prominent in elongated spermatids. We also showed that MARCH7 catalyzes lysine 48 (K48)-linked ubiquitination. Immunolocalization studies revealed that K48-linked ubiquitin chains were detected in the heads of elongating spermatids and in the acrosome/acroplaxome, neck, midpiece and cytoplasmic lobes of elongated spermatids. These results suggest that MARCH7 is involved in spermiogenesis by regulating the structural and functional integrity of the head and tail of developing spermatids.

Membrane-associated RING-CH 10 (MARCH10 protein) is a microtubule-associated E3 ubiquitin ligase of the spermatid flagella.

Spermiogenesis is a complex and dynamic process of the metamorphosis of spermatids into spermatozoa. There is a great deal that is still unknown regarding the regulatory mechanisms for the formation of the sperm flagellum. In this study, we determined that the membrane-associated RING-CH 10 (March10) gene is predominantly expressed in rat testis. We isolated two March10 isoforms encoding MARCH10a and MARCH10b, which are generated by alternative splicing. MARCH10a is a long RING finger protein, and MARCH10b is a short RING finger-less protein. Immunohistochemical staining revealed that the MARCH10 proteins are specifically expressed in elongating and elongated spermatids, and the expression is absent in epididymal spermatozoa. MARCH10 immunoreactivity was observed in the cytoplasmic lobes as well as the principal piece and annulus of the flagella. When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, whereas MARCH10b was distributed throughout the cytoplasm. An in vitro microtubule cosedimentation assay showed that MARCH10a is directly associated with microtubules. An in vitro ubiquitination assay demonstrated that the RING finger domain of MARCH10a exhibits an E3 ubiquitin ligase activity along with the E2 ubiquitin-conjugating enzyme UBE2B. Moreover, MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly. These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids.

OVOL1 inhibits breast cancer cell invasion by enhancing the degradation of TGF-ß type I receptor.

Ovo-like transcriptional repressor 1 (OVOL1) is a key mediator of epithelial lineage determination and mesenchymal-epithelial transition (MET). The cytokines transforming growth factor-ß (TGF-ß) and bone morphogenetic proteins (BMP) control the epithelial-mesenchymal plasticity (EMP) of cancer cells, but whether this occurs through interplay with OVOL1 is not known. Here, we show that OVOL1 is inversely correlated with the epithelial-mesenchymal transition (EMT) signature, and is an indicator of a favorable prognosis for breast cancer patients. OVOL1 suppresses EMT, migration, extravasation, and early metastatic events of breast cancer cells. Importantly, BMP strongly promotes the expression of OVOL1, which enhances BMP signaling in turn. This positive feedback loop is established through the inhibition of TGF-ß receptor signaling by OVOL1. Mechanistically, OVOL1 interacts with and prevents the ubiquitination and degradation of SMAD family member 7 (SMAD7), which is a negative regulator of TGF-ß type I receptor stability. Moreover, a small-molecule compound 6-formylindolo(3,2-b)carbazole (FICZ) was identified to activate OVOL1 expression and thereby antagonizing (at least in part) TGF-ß-mediated EMT and migration in breast cancer cells. Our results uncover a novel mechanism by which OVOL1 attenuates TGF-ß/SMAD signaling and maintains the epithelial identity of breast cancer cells.

TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling.

Patients with bladder cancer often have a poor prognosis due to the highly invasive and metastatic characteristics of bladder cancer cells. Epithelial-to-mesenchymal transition (EMT) has been causally linked to bladder cancer invasion. The E3 ubiquitin ligase, tumor necrosis factor receptor-associated factor 4 (TRAF4) has been implicated as a tumor promoter in a wide range of cancers. In contrast, here we show that low TRAF4 expression is associated with poor overall survival in patients with bladder cancer. We show that the TRAF4 gene is epigenetically silenced and that ERK mediates TRAF4 phosphorylation, resulting in lower TRAF4 protein levels in bladder cancer cells. In addition, we demonstrate that TRAF4 is inversely correlated with an EMT gene signature/protein marker expression. Functionally, by manipulating TRAF4 expression, we show that TRAF4 regulates EMT genes and epithelial and invasive properties in bladder cancer cells. Transcriptomic analysis of dysregulated TRAF4 expression in bladder cancer cell lines revealed that high TRAF4 expression enhances the bone morphogenetic protein (BMP)/SMAD and inhibits the NF-κB signaling pathway. Mechanistically, we show that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signaling, for proteasomal degradation in bladder cancer cells. This was corroborated in patient samples where TRAF4 positively correlates with phospho-SMAD1/5, and negatively correlates with phospho-NFκb-p65. Lastly, we show that genetic and pharmacologic inhibition of SMURF1 inhibits the migration of aggressive mesenchymal bladder cancer cells. IMPLICATIONS: Our findings identify E3 ubiquitin ligase TRAF4 as a potential therapeutic target or biomarker for bladder cancer progression.

OTUD4 enhances TGFß signalling through regulation of the TGFß receptor complex.

Systematic control of the transforming growth factor-ß (TGFß) pathway is essential to keep the amplitude and the intensity of downstream signalling at appropriate levels. Ubiquitination plays a crucial role in the general regulation of this pathway. Here we identify the deubiquitinating enzyme OTUD4 as a transcriptional target of the TGFß pathway that functions through a positive feedback loop to enhance overall TGFß activity. Interestingly we demonstrate that OTUD4 functions through both catalytically dependent and independent mechanisms to regulate TGFß activity. Specifically, we find that OTUD4 enhances TGFß signalling by promoting the membrane presence of TGFß receptor I. Furthermore, we demonstrate that OTUD4 inactivates the TGFß negative regulator SMURF2 suggesting that OTUD4 regulates multiple nodes of the TGFß pathway to enhance TGFß activity.

Deubiquitinase Activity Profiling Identifies UCHL1 as a Candidate Oncoprotein That Promotes TGFß-Induced Breast Cancer Metastasis.

PURPOSE: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies. EXPERIMENTAL DESIGN: Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations. RESULTS: Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFß signaling-induced metastasis by protecting TGFß type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell-conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression. CONCLUSIONS: Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFß-induced breast cancer metastasis and may provide a potential target for TNBC treatment.

Epigenetic Reprogramming of TGF-ß Signaling in Breast Cancer.

The Transforming Growth Factor-ß (TGF-ß) signaling pathway has a well-documented, context-dependent role in breast cancer development. In normal and premalignant cells, it acts as a tumor suppressor. By contrast, during the malignant phases of breast cancer progression, the TGF-ß signaling pathway elicits tumor promoting effects particularly by driving the epithelial to mesenchymal transition (EMT), which enhances tumor cell migration, invasion and ultimately metastasis to distant organs. The molecular and cellular mechanisms that govern this dual capacity are being uncovered at multiple molecular levels. This review will focus on recent advances relating to how epigenetic changes such as acetylation and methylation control the outcome of TGF-ß signaling and alter the fate of breast cancer cells. In addition, we will highlight how this knowledge can be further exploited to curb tumorigenesis by selective targeting of the TGF-ß signaling pathway.

c-Met activation leads to the establishment of a TGFß-receptor regulatory network in bladder cancer progression.

Treatment of muscle-invasive bladder cancer remains a major clinical challenge. Aberrant HGF/c-MET upregulation and activation is frequently observed in bladder cancer correlating with cancer progression and invasion. However, the mechanisms underlying HGF/c-MET-mediated invasion in bladder cancer remains unknown. As part of a negative feedback loop SMAD7 binds to SMURF2 targeting the TGFß receptor for degradation. Under these conditions, SMAD7 acts as a SMURF2 agonist by disrupting the intramolecular interactions within SMURF2. We demonstrate that HGF stimulates TGFß signalling through c-SRC-mediated phosphorylation of SMURF2 resulting in loss of SMAD7 binding and enhanced SMURF2 C2-HECT interaction, inhibiting SMURF2 and enhancing TGFß receptor stabilisation. This upregulation of the TGFß pathway by HGF leads to TGFß-mediated EMT and invasion. In vivo we show that TGFß receptor inhibition prevents bladder cancer invasion. Furthermore, we make a rationale for the use of combinatorial TGFß and MEK inhibitors for treatment of high-grade non-muscle-invasive bladder cancers.

Author Correction: c-Met activation leads to the establishment of a TGFß-receptor regulatory network in bladder cancer progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling. Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members. Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models. Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients. Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors. Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.

USP15 regulates SMURF2 kinetics through C-lobe mediated deubiquitination.

Ubiquitin modification of the TGF-ß pathway components is emerging as a key mechanism of TGF-ß pathway regulation. To limit TGF-ß responses, TGF-ß signaling is regulated through a negative feedback loop whereby the E3 ligase SMURF2 targets the TGF-ß receptor (TßR) complex for ubiquitin-mediated degradation. Counteracting this process, a number of deubiquitinating (DUBs) enzymes have recently been identified that deubiquitinate and stabilize the TßR. However the precise mechanism by which these DUBs act on TßR function remains poorly defined. Here, we demonstrate that apart from targeting the TßR complex directly, USP15 also deubiquitinates SMURF2 resulting in enhanced TßR stability and downstream pathway activation. Through proteomic analysis, we show that USP15 modulates the ubiquitination of Lys734, a residue required for SMURF2 catalytic activity. Our results show that SMURF2 is a critical target of USP15 in the TGF-ß pathway and may also explain how USP15 and SMURF2 target multiple complementary protein complexes in other pathways.