Loss of Tankyrase-mediated destruction of 3BP2 is the underlying pathogenic mechanism of cherubism.

Cherubism is an autosomal-dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone-remodeling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK, and VAV signaling pathways.

Hallucination Rates and Reference Accuracy of ChatGPT and Bard for Systematic Reviews: Comparative Analysis.

BACKGROUND: Large language models (LLMs) have raised both interest and concern in the academic community. They offer the potential for automating literature search and synthesis for systematic reviews but raise concerns regarding their reliability, as the tendency to generate unsupported (hallucinated) content persist. OBJECTIVE: The aim of the study is to assess the performance of LLMs such as ChatGPT and Bard (subsequently rebranded Gemini) to produce references in the context of scientific writing. METHODS: The performance of ChatGPT and Bard in replicating the results of human-conducted systematic reviews was assessed. Using systematic reviews pertaining to shoulder rotator cuff pathology, these LLMs were tested by providing the same inclusion criteria and comparing the results with original systematic review references, serving as gold standards. The study used 3 key performance metrics: recall, precision, and F1-score, alongside the hallucination rate. Papers were considered "hallucinated" if any 2 of the following information were wrong: title, first author, or year of publication. RESULTS: In total, 11 systematic reviews across 4 fields yielded 33 prompts to LLMs (3 LLMs×11 reviews), with 471 references analyzed. Precision rates for GPT-3.5, GPT-4, and Bard were 9.4% (13/139), 13.4% (16/119), and 0% (0/104) respectively (P<.001). Recall rates were 11.9% (13/109) for GPT-3.5 and 13.7% (15/109) for GPT-4, with Bard failing to retrieve any relevant papers (P<.001). Hallucination rates stood at 39.6% (55/139) for GPT-3.5, 28.6% (34/119) for GPT-4, and 91.4% (95/104) for Bard (P<.001). Further analysis of nonhallucinated papers retrieved by GPT models revealed significant differences in identifying various criteria, such as randomized studies, participant criteria, and intervention criteria. The study also noted the geographical and open-access biases in the papers retrieved by the LLMs. CONCLUSIONS: Given their current performance, it is not recommended for LLMs to be deployed as the primary or exclusive tool for conducting systematic reviews. Any references generated by such models warrant thorough validation by researchers. The high occurrence of hallucinations in LLMs highlights the necessity for refining their training and functionality before confidently using them for rigorous academic purposes.

Forkhead box O3 (FOXO3) transcription factor mediates apoptosis in BCG-infected macrophages.

Enhanced apoptosis of BCG-infected macrophages has been shown to induce stronger dendritic cell-mediated cross-priming of T cells, leading to higher protection against tuberculosis (TB). Uncovering host effectors underlying BCG-induced apoptosis may then prove useful to improve BCG efficacy through priming macrophage apoptosis. Her we report that BCG-mediated apoptosis of human macrophages relies on FOXO3 transcription factor activation. BCG induced a significant apoptosis of THP1 (TDMs) and human monocytes (MDMs)-derived macrophages when a high moi was used, as shown by annexin V/7-AAD staining. BCG-induced apoptosis was associated with dephosphorylation of the prosurvival activated threonine kinase (Akt) and its target FOXO3. Cell fractionation and immunofluorescence microscopy showed translocation of FOXO3 to the nucleus in BCG-infected cells, concomitantly with an increase of FOXO3 transcriptional activity. Moreover, FOXO3 expression knock-down by small interfering RNA (siRNA) partially inhibited the BCG-induced apoptosis. Finally, real-time quantitative PCR (qRT-PCR) analysis of the expression profile of BCG-infected macrophages showed an upregulation of two pro-apoptotic targets of FOXO3, NOXA and p53 upregulated modulator of apoptosis (PUMA). Our results thus indicate that FOXO3 plays an important role in BCG-induced apoptosis of human macrophages and may represent a potential target to improve vaccine efficacy through enhanced apoptosis-mediated cross-priming of T cells.

The caspase-cleaved form of LYN mediates a psoriasis-like inflammatory syndrome in mice.

We showed previously that Lyn is a substrate for caspases, a family of cysteine proteases, involved in the regulation of apoptosis and inflammation. Here, we report that expression of the caspase-cleaved form of Lyn (LynDeltaN), in mice, mediates a chronic inflammatory syndrome resembling human psoriasis. Genetic ablation of TNF receptor 1 in a LynDeltaN background rescues a normal phenotype, indicating that LynDeltaN mice phenotype is TNF-alpha-dependent. The predominant role of T cells in the disease occurring in LynDeltaN mice was highlighted by the distinct improvement of LynDeltaN mice phenotype in a Rag1-deficient background. Using pan-genomic profiling, we also established that LynDeltaN mice show an increased expression of STAT-3 and inhibitory members of the NFkappaB pathway. Accordingly, LynDeltaN alters NFkappaB activity underlying a link between inhibition of NFkappaB and LynDeltaN mice phenotype. Finally, analysis of Lyn expression in human skin biopsies of psoriatic patients led to the detection of Lyn cleavage product whose expression correlates with the activation of caspase 1. Our data identify a new role for Lyn as a regulator of psoriasis through its cleavage by caspases.

The mechanical phenotypic plasticity of melanoma cell: an emerging driver of therapy cross-resistance.

Advanced cutaneous melanoma is the deadliest form of skin cancer and one of the most aggressive human cancers. Targeted therapies (TT) against BRAF mutated melanoma and immune checkpoints blockade therapies (ICB) have been a breakthrough in the treatment of metastatic melanoma. However, therapy-driven resistance remains a major hurdle in the clinical management of the metastatic disease. Besides shaping the tumor microenvironment, current treatments impact transition states to promote melanoma cell phenotypic plasticity and intratumor heterogeneity, which compromise treatment efficacy and clinical outcomes. In this context, mesenchymal-like dedifferentiated melanoma cells exhibit a remarkable ability to autonomously assemble their own extracellular matrix (ECM) and to biomechanically adapt in response to therapeutic insults, thereby fueling tumor relapse. Here, we review recent studies that highlight mechanical phenotypic plasticity of melanoma cells as a hallmark of adaptive and non-genetic resistance to treatment and emerging driver in cross-resistance to TT and ICB. We also discuss how targeting BRAF-mutant dedifferentiated cells and ECM-based mechanotransduction pathways may overcome melanoma cross-resistance.

Sphingosine-1 Phosphate Receptor Modulators Increase In Vitro Melanoma Cell Line Proliferation at Therapeutic Doses Used in Patients with Multiple Sclerosis.

INTRODUCTION: S1P1 receptor modulators (S1P1-RM) are oral disease-modifying therapies (DMTs) for multiple sclerosis (MS). Several authorities have raised doubts that S1P1-RM are responsible for an increased risk of melanoma in patients with MS. We studied the in vitro effects of S1P1-RM on different melanoma cell lines to compare the effect of available S1P1-RM on the proliferation of human melanoma cells. METHODS: Four S1P1-RM were studied which are currently approved for managing MS, namely fingolimod (Gilenya®), siponimod (Mayzent®), ozanimod (Zeposia®), and ponesimod (Ponvory®). We tested these four drugs at different concentrations, including therapeutic doses (0.5, 1.6, 5.5, 18, and 60 µM), on human melanoma cell lines (501Mel cells, 1205LU cells, and M249R cells) to analyze in vitro cell proliferation monitored with the IncuCyte ZOOM live cell microscope (Essen Bioscience). RESULTS: At therapeutic doses, median confluence increased overall for all lineages: + 122% for ozanimod (p < 0.001), + 71% for ponesimod (p < 0.001), + 67% for siponimod (NS), and + 41% for fingolimod (p = 0.094). Ozanimod- and ponesimod-treated cells increased confluency in 501Mel, 1205LU, and M249R cell lines (p < 0.001). CONCLUSION: These data suggest an increased proliferation of various melanoma cell lines with S1P1-RM treatments used at therapeutic concentrations for patients with MS and should raise the question of increased dermatologic surveillance.

Emerging Role of Deubiquitinating Enzymes (DUBs) in Melanoma Pathogenesis.

Metastatic melanoma is the leading cause of death from skin cancer. Therapies targeting the BRAF oncogenic pathway and immunotherapies show remarkable clinical efficacy. However, these treatments are limited to subgroups of patients and relapse is common. Overall, the majority of patients require additional treatments, justifying the development of new therapeutic strategies. Non-genetic and genetic alterations are considered to be important drivers of cellular adaptation mechanisms to current therapies and disease relapse. Importantly, modification of the overall proteome in response to non-genetic and genetic events supports major cellular changes that are required for the survival, proliferation, and migration of melanoma cells. However, the mechanisms underlying these adaptive responses remain to be investigated. The major contributor to proteome remodeling involves the ubiquitin pathway, ubiquitinating enzymes, and ubiquitin-specific proteases also known as DeUBiquitinases (DUBs). In this review, we summarize the current knowledge regarding the nature and roles of the DUBs recently identified in melanoma progression and therapeutic resistance and discuss their potential as novel sources of vulnerability for melanoma therapy.

Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3-Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells.

Fibroblastic reticular cells (FRC) are immunologically specialized myofibroblasts that control the elasticity of the lymph node, in part through their contractile properties. Swelling of tumor-draining lymph nodes is a hallmark of lymphophilic cancers such as cutaneous melanoma. Melanoma displays high intratumoral heterogeneity with the coexistence of melanoma cells with variable differentiation phenotypes from melanocytic to dedifferentiated states. Factors secreted by melanoma cells promote premetastatic lymph node reprograming and tumor spreading. Elucidating the impact of the melanoma secretome on FRC could help identify approaches to prevent metastasis. Here we show that melanocytic and dedifferentiated melanoma cells differentially impact the FRC contractile phenotype. Factors secreted by dedifferentiated cells, but not by melanocytic cells, strongly inhibited actomyosin-dependent contractile forces of FRC by decreasing the activity of the RHOA-RHO-kinase (ROCK) pathway and the mechano-responsive transcriptional coactivator Yes1 associated transcriptional regulator (YAP). Transcriptional profiling and biochemical analyses indicated that actomyosin cytoskeleton relaxation in FRC is driven by inhibition of the JAK1-STAT3 pathway. This FRC relaxation was associated with increased FRC proliferation and activation and with elevated tumor invasion in vitro. The secretome of dedifferentiated melanoma cells also modulated the biomechanical properties of distant lymph node in premetastatic mouse models. Finally, IL1 produced by dedifferentiated cells was involved in the inhibition of FRC contractility. These data highlight the role of the JAK1-STAT3 and YAP pathways in spontaneous contractility of resting FRC. They also suggest that dedifferentiated melanoma cells specifically target FRC biomechanical properties to favor tumor spreading in the premetastatic lymph node niche. Targeting this remote communication could be an effective strategy to prevent metastatic spread of the disease. SIGNIFICANCE: Communication between dedifferentiated melanoma cells and lymph node fibroblasts reprograms the biomechanical properties of the premetastatic lymph node niche to promote tumor invasion. See related commentary by Lund, p. 1692.

SYK-3BP2 Pathway Activity in Parenchymal and Myeloid Cells Is a Key Pathogenic Factor in Metabolic Steatohepatitis.

BACKGROUND & AIMS: Spleen tyrosine kinase (SYK) signaling pathway regulates critical processes in innate immunity, but its role in parenchymal cells remains elusive in chronic liver diseases. We investigate the relative contribution of SYK and its substrate c-Abl Src homology 3 domain-binding protein-2 (3BP2) in both myeloid cells and hepatocytes in the onset of metabolic steatohepatitis. METHODS: Hepatic SYK-3BP2 pathway was evaluated in mouse models of metabolic-associated fatty liver diseases (MAFLD) and in obese patients with biopsy-proven MAFLD (n = 33). Its role in liver complications was evaluated in Sh3bp2 KO and myeloid-specific Syk KO mice challenged with methionine and choline deficient diet and in homozygous Sh3bp2KI/KI mice with and without SYK expression in myeloid cells. RESULTS: Here we report that hepatic expression of 3BP2 and SYK correlated with metabolic steatohepatitis severity in mice. 3BP2 deficiency and SYK deletion in myeloid cells mediated the same protective effects on liver inflammation, injury, and fibrosis priming upon diet-induced steatohepatitis. In primary hepatocytes, the targeting of 3BP2 or SYK strongly decreased the lipopolysaccharide-mediated inflammatory mediator expression and 3BP2-regulated SYK expression. In homozygous Sh3bp2KI/KI mice, the chronic inflammation mediated by the proteasome-resistant 3BP2 mutant promoted severe hepatitis and liver fibrosis with augmented liver SYK expression. In these mice, the deletion of SYK in myeloid cells was sufficient to prevent these liver lesions. The hepatic expression of SYK is also up-regulated with metabolic steatohepatitis and correlates with liver macrophages in biopsy-proven MAFLD patients. CONCLUSIONS: Collectively, these data suggest an important role for the SYK-3BP2 pathway in the pathogenesis of chronic liver inflammatory diseases and highlight its targeting in hepatocytes and myeloid cells as a potential strategy to treat metabolic steatohepatitis.

Targeting Discoidin Domain Receptors DDR1 and DDR2 overcomes matrix-mediated tumor cell adaptation and tolerance to BRAF-targeted therapy in melanoma.

Resistance to BRAF/MEK inhibitor therapy in BRAFV600 -mutated advanced melanoma remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapy; however, the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MMDR) in response to BRAFV600 pathway inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MMDR is mediated by drug-induced linear clustering of phosphorylated DDR1 and DDR2, two tyrosine kinase collagen receptors. Depletion and pharmacological targeting of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF-targeted therapy. In xenografts, targeting DDR with imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling, and delays tumor relapse. Mechanistically, DDR-dependent MMDR fosters a targetable pro-survival NIK/IKKα/NF-κB2 pathway. These findings reveal a novel role for a collagen-rich matrix and DDR in tumor cell adaptation and resistance. They also provide important insights into environment-mediated drug resistance and a preclinical rationale for targeting DDR signaling in combination with targeted therapy in melanoma.

Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma.

Lineage dedifferentiation toward a mesenchymal-like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and acquired resistance to targeted therapy in melanoma. Here, we show that the anti-fibrotic drug nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy, and delay tumor relapse in a preclinical model of melanoma. Acquisition of this resistant phenotype and its reversion by nintedanib pointed to miR-143/-145 pro-fibrotic cluster as a driver of this mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p, collaborated to mediate transition toward a drug-resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof of principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy.

3BP2 adapter protein is required for receptor activator of NFκB ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells.

The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.

Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization.

Actin networks are dynamically regulated through constant depolymerization and polymerization cycles. Although the fundamental mechanisms that govern these processes have been identified, the nature and role of post-translational modifications (PTMs) of actin and actin regulatory proteins are not completely understood. Here, we employed Actin CytoFRET, a method that we developed for real time detection of fluorescence resonance energy transfer (FRET) signals generated by actin dynamics, to screen a small library of PTM-interfering compounds on a biosensor leukemic T cell line. This strategy led to the identification of small molecule inhibitors of deubiquitinating enzymes (DUBs) as potent inducers of actin polymerization and blockers of chemotactic cell migration. The examination of the underlying mechanism further revealed that the actin depolymerizing protein cofilin represents a major effector of DUB inhibitor (DUBi)-induced actin reorganization. We found that DUB blockade results in the accumulation of polyubiquitinated proteins and ROS production, associated with cofilin oxidation and dephosphorylation on serine 3, which provokes uncontrolled actin polymerization impairing cell migration. Together, our study highlights DUBs as novel regulators of actin dynamics through ROS-dependent cofilin modulation, and shows that DUBi represent attractive novel tools to impede leukemic cell migration.

Comparison of SYK Signaling Networks Reveals the Potential Molecular Determinants of Its Tumor-Promoting and Suppressing Functions.

Spleen tyrosine kinase (SYK) can behave as an oncogene or a tumor suppressor, depending on the cell and tissue type. As pharmacological SYK inhibitors are currently evaluated in clinical trials, it is important to gain more information on the molecular mechanisms underpinning these opposite roles. To this aim, we reconstructed and compared its signaling networks using phosphoproteomic data from breast cancer and Burkitt lymphoma cell lines where SYK behaves as a tumor suppressor and promoter. Bioinformatic analyses allowed for unveiling the main differences in signaling pathways, network topology and signal propagation from SYK to its potential effectors. In breast cancer cells, the SYK target-enriched signaling pathways included intercellular adhesion and Hippo signaling components that are often linked to tumor suppression. In Burkitt lymphoma cells, the SYK target-enriched signaling pathways included molecules that could play a role in SYK pro-oncogenic function in B-cell lymphomas. Several protein interactions were profoundly rewired in the breast cancer network compared with the Burkitt lymphoma network. These data demonstrate that proteomic profiling combined with mathematical network modeling allows untangling complex pathway interplays and revealing difficult to discern interactions among the SYK pathways that positively and negatively affect tumor formation and progression.

Bad Neighborhood: Fibrotic Stroma as a New Player in Melanoma Resistance to Targeted Therapies.

Current treatments for metastatic cutaneous melanoma include immunotherapies and drugs targeting key molecules of the mitogen-activated protein kinase (MAPK) pathway, which is often activated by BRAF driver mutations. Overall responses from patients with metastatic BRAF mutant melanoma are better with therapies combining BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors. However, most patients that initially respond to therapies develop drug resistance within months. Acquired resistance to targeted therapies can be due to additional genetic alterations in melanoma cells and to non-genetic events frequently associated with transcriptional reprogramming and a dedifferentiated cell state. In this second scenario, it is possible to identify pro-fibrotic responses induced by targeted therapies that contribute to the alteration of the melanoma tumor microenvironment. A close interrelationship between chronic fibrosis and cancer has been established for several malignancies including breast and pancreatic cancers. In this context, the contribution of fibrosis to drug adaptation and therapy resistance in melanoma is rapidly emerging. In this review, we summarize recent evidence underlining the hallmarks of fibrotic diseases in drug-exposed and resistant melanoma, including increased remodeling of the extracellular matrix, enhanced actin cytoskeleton plasticity, high sensitivity to mechanical cues, and the establishment of an inflammatory microenvironment. We also discuss several potential therapeutic options for manipulating this fibrotic-like response to combat drug-resistant and invasive melanoma.

A Feed-Forward Mechanosignaling Loop Confers Resistance to Therapies Targeting the MAPK Pathway in BRAF-Mutant Melanoma.

Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM. Analyses revealed that therapy-resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and force-mediated ECM remodeling through activation of actin-dependent mechanosensors Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF). Short-term inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP- and MRTF-dependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacologic targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCE: These findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing, and therapy resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1927/F1.large.jpg.

Targeting the Proteasome-Associated Deubiquitinating Enzyme USP14 Impairs Melanoma Cell Survival and Overcomes Resistance to MAPK-Targeting Therapies.

Advanced cutaneous melanoma is one of the most challenging cancers to treat because of its high plasticity, metastatic potential, and resistance to treatment. New targeted therapies and immunotherapies have shown remarkable clinical efficacy. However, such treatments are limited to a subset of patients and relapses often occur, warranting validation of novel targeted therapies. Posttranslational modification of proteins by ubiquitin coordinates essential cellular functions, including ubiquitin-proteasome system (UPS) function and protein homeostasis. Deubiquitinating enzymes (DUB) have been associated to multiple diseases, including cancer. However, their exact involvement in melanoma development and therapeutic resistance remains poorly understood. Using a DUB trap assay to label cellular active DUBs, we have observed an increased activity of the proteasome-associated DUB, USP14 (Ubiquitin-specific peptidase 14) in melanoma cells compared with melanocytes. Our survey of public gene expression databases indicates that high expression of USP14 correlates with melanoma progression and with a poorer survival rate in metastatic melanoma patients. Knockdown or pharmacologic inhibition of USP14 dramatically impairs viability of melanoma cells irrespective of the mutational status of BRAF, NRAS, or TP53 and their transcriptional cell state, and overcomes resistance to MAPK-targeting therapies both in vitro and in human melanoma xenografted mice. At the molecular level, we find that inhibition of USP14 rapidly triggers accumulation of poly-ubiquitinated proteins and chaperones, mitochondrial dysfunction, ER stress, and a ROS production leading to a caspase-independent cell death. Our results provide a rationale for targeting the proteasome-associated DUB USP14 to treat and combat melanomas. Mol Cancer Ther; 17(7); 1416-29. ©2018 AACR.

Abl-SH3 binding protein 2, 3BP2, interacts with CIN85 and HIP-55.

The adapter 3BP2 is involved in leukocyte signaling downstream Src/Syk-kinases coupled immunoreceptors. Here, we show that 3BP2 directly interacts with the endocytic scaffold protein CIN85 and the actin-binding protein HIP-55. 3BP2 co-localized with CIN85 and HIP-55 in T cell rafts and at the T cell/APC synapse, an active zone of receptors and proteins recycling. A binding region of CIN85 SH3 domains on 3BP2 was mapped to a PVPTPR motif in the first proline-rich region of 3BP2, whereas the C-terminal SH3 domain of HIP-55 bound a more distal proline-rich domain of 3BP2. Together, our data suggest an unexpected role of 3BP2 in endocytic and cytoskeletal regulation through its interaction with CIN85 and HIP-55.

HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1.

The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.