Regulation Of Receptor Tyrosine Kinases Research Articles

Principles in the Management of Glioblastoma.

Glioblastoma, the most aggressive and common malignant primary brain tumour, is characterized by infiltrative growth, abundant vascularization, and aggressive clinical evolution. Patients with glioblastoma often face poor prognoses, with a median survival of approximately 15 months. Technological progress and the subsequent improvement in understanding the pathophysiology of these tumours have not translated into significant achievements in therapies or survival outcomes for patients. Progress in molecular profiling has yielded new omics data for a more refined classification of glioblastoma. Several typical genetic and epigenetic alterations in glioblastoma include mutations in genes regulating receptor tyrosine kinase (RTK)/rat sarcoma (RAS)/phosphoinositide 3-kinase (PI3K), p53, and retinoblastoma protein (RB) signalling, as well as mutation of isocitrate dehydrogenase (IDH), methylation of O6-methylguanine-DNA methyltransferase (MGMT), amplification of epidermal growth factor receptor vIII, and codeletion of 1p/19q. Certain microRNAs, such as miR-10b and miR-21, have also been identified as prognostic biomarkers. Effective treatment options for glioblastoma are limited. Surgery, radiotherapy, and alkylating agent chemotherapy remain the primary pillars of treatment. Only promoter methylation of the gene MGMT predicts the benefit from alkylating chemotherapy with temozolomide and it guides the choice of first-line treatment in elderly patients. Several targeted strategies based on tumour-intrinsic dominant signalling pathways and antigenic tumour profiles are under investigation in clinical trials. This review explores the potential genetic and epigenetic biomarkers that could be deployed as analytical tools in the diagnosis and prognostication of glioblastoma. Recent clinical advancements in treating glioblastoma are also discussed, along with the potential of liquid biopsies to advance personalized medicine in the field of glioblastoma, highlighting the challenges and promises for the future.

Identifying Transmembrane Interactions in Receptor Protein Tyrosine Phosphatase Homodimerization and Heterodimerization.

Receptor protein tyrosine phosphatases (RPTPs) are one of the key regulators of receptor tyrosine kinases (RTKs) and therefore play a critical role in modulating signal transduction. While the structure-function relationship of RTKs has been widely studied, the mechanisms modulating the activity of RPTPs still need to be fully understood. On the other hand, homodimerization has been shown to antagonize RPTP catalytic activity and appears to be a general feature of the entire family. Conversely, their documented ability to physically interact with RTKs is integral to their negative regulation of RTKs, but there is a yet-to-be proposed common model. However, specific transmembrane (TM) domain interactions and residues have been shown to be essential in regulating RPTP homodimerization, interactions with RTK substrates, and activity. Therefore, elucidating the contribution of the TM domains in RPTP regulation can provide significant insights into how these receptors function, interact, and eventually be modulated. This chapter describes the dominant-negative AraC-based transcriptional reporter (DN-AraTM) assay to identify specific TM interactions essential to homodimerization and heteroassociation with other membrane receptors, such as RTKs.

An enzyme that selectively S-nitrosylates proteins to regulate insulin signaling

Acyl-coenzyme A (acyl-CoA) species are cofactors for numerous enzymes that acylate thousands of proteins. Here, we describe an enzyme that uses S-nitroso-CoA (SNO-CoA) as its cofactor to S-nitrosylate multiple proteins (SNO-CoA-assisted nitrosylase, SCAN). Separate domains in SCAN mediate SNO-CoA and substrate binding, allowing SCAN to selectively catalyze SNO transfer from SNO-CoA to SCAN to multiple protein targets, including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1). Insulin-stimulated S-nitrosylation of INSR/IRS1 by SCAN reduces insulin signaling physiologically, whereas increased SCAN activity in obesity causes INSR/IRS1 hypernitrosylation and insulin resistance. SCAN-deficient mice are thus protected from diabetes. In human skeletal muscle and adipose tissue, SCAN expression increases with body mass index and correlates with INSR S-nitrosylation. S-nitrosylation by SCAN/SNO-CoA thus defines a new enzyme class, a unique mode of receptor tyrosine kinase regulation, and a revised paradigm for NO function in physiology and disease.

A comprehensive review of SHP2 and its role in cancer

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase ubiquitously expressed mainly in the cytoplasm of several tissues. SHP2 modulates diverse cell signaling events that control metabolism, cell growth, differentiation, cell migration, transcription and oncogenic transformation. It interacts with diverse molecules in the cell, and regulates key signaling events including RAS/ERK, PI3K/AKT, JAK/STAT and PD-1 pathways downstream of several receptor tyrosine kinases (RTKs) upon stimulation by growth factors and cytokines. SHP2 acts as both a phosphatase and a scaffold, and plays prominently oncogenic functions but can be tumor suppressor in a context-dependent manner. It typically acts as a positive regulator of RTKs signaling with some inhibitory functions reported as well. SHP2 expression and activity is regulated by such factors as allosteric autoinhibition, microRNAs, ubiquitination and SUMOylation. Dysregulation of SHP2 expression or activity causes many developmental diseases, and hematological and solid tumors. Moreover, upregulated SHP2 expression or activity also decreases sensitivity of cancer cells to anticancer drugs. SHP2 is now considered as a compelling anticancer drug target and several classes of SHP2 inhibitors with different mode of action are developed with some already in clinical trial phases. Moreover, novel SHP2 substrates and functions are rapidly growing both in cell and cancer. In view of this, we comprehensively and thoroughly reviewed literatures about SHP2 regulatory mechanisms, substrates and binding partners, biological functions, roles in human cancers, and different classes of small molecule inhibitors target this oncoprotein in cancer.

Negative regulation of receptor tyrosine kinases by ubiquitination: Key roles of the Cbl family of E3 ubiquitin ligases.

Receptor tyrosine kinases (RTKs) serve as transmembrane receptors that participate in a broad spectrum of cellular processes including cellular growth, motility, differentiation, proliferation, and metabolism. Hence, elucidating the regulatory mechanisms of RTKs involved in an assortment of diseases such as cancers attracts increasing interest from researchers. Members of the Cbl family ubiquitin ligases (c-Cbl, Cbl-b and Cbl-c in mammals) have emerged as negative regulators of activated RTKs. Upon activation of RTKs by growth factors, Cbl binds to RTKs via its tyrosine kinase binding (TKB) domain and targets them for ubiquitination, thus facilitating their degradation and negative regulation of RTK signaling. RTKs such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGF), fibroblast growth factor receptor (FGFR) and hepatocyte growth factor receptor (HGFR) undergo ubiquitination upon interaction with Cbl family members. In this review, we summarize the current knowledge related to the negative regulation of RTKs by Cbl family proteins.

A general approach for engineering RTKs optically controlled with far-red light

Regulation of receptor tyrosine kinase (RTK) activity is necessary for studying cell signaling pathways in health and disease. We developed a generalized approach for engineering RTKs optically controlled with far-red light. We targeted the bacterial phytochrome DrBphP to the cell surface and allowed its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. Systematic optimization of these constructs has resulted in optically regulated epidermal growth factor receptor, HER2, TrkA, TrkB, FGFR1, IR1, cKIT and cMet, named eDrRTKs. eDrRTKs induced downstream signaling in mammalian cells in tens of seconds. The ability to activate eDrRTKs with far-red light enabled spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays. We validated eDrTrkB performance in mice and found that minimally invasive stimulation in the neocortex with penetrating via skull far-red light-induced neural activity, early immediate gene expression and affected sleep patterns.

Development of Tumor‐selective Transmembrane Peptides to Promote the Activity of the Receptor Protein Tyrosine Phosphatase J by Disrupting its Homodimerization

Receptor Protein Tyrosine Phosphatases (RPTPs) are one of the most important regulators of receptor tyrosine kinases (RTKs) and therefore play a crucial role in mammalian signal transduction. But our still‐incomplete understanding of the structural aspects of their regulation and the lack of selective agonists have hampered efforts to understand their roles in downstream signaling regulation, and to pursue them as potential therapeutic targets. However, the reported ability of RPTP homodimerization to antagonize their catalytic activity presents potential opportunities to develop unique strategies to modulate the activity of RPTPs.We recently reported (Block et al., JBC, 2019; doi:10.1074/jbc.RA119.010229) that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ; also known as DEP1) is regulated by specific transmembrane (TM) residues, and that these interactions are essential in regulating its enzymatic activity and substrate access in cells.Building on these new insights and in response to the lack of selective agonists, we present here the design and testing of a novel class of tumor‐selective peptide capable of binding to the TM domain of PTPRJ and disrupting its homodimerization. By doing so, it promotes PTPRJ TM‐mediated access to EGFR, reduces the phosphorylation of EGFR (a known substrate) and other downstream signaling effectors, and antagonizes EGFR‐driven cancer cell phenotypes.This class of peptide represents a novel allosteric and a possibly orthogonal way to target the activity of PTPRJ and RTKs phosphorylation. It could therefore be used not only as probes to tease out PTPRJ regulating mechanisms but also for therapeutic purposes via the attenuation of signaling by dysregulated RTKs.

Aberrant promoter methylation contributes to LRIG1 silencing in basal/triple-negative breast cancer

BackgroundLRIG1, the founding member of the LRIG (leucine-rich repeat and immunoglobulin-like domain) family of transmembrane proteins, is a negative regulator of receptor tyrosine kinases and a tumour suppressor. Decreased LRIG1 expression is consistently observed in cancer, across diverse tumour types, and is linked to poor patient prognosis. However, mechanisms by which LRIG1 is repressed are not fully understood. Silencing of LRIG1 through promoter CpG island methylation has been reported in colorectal and cervical cancer but studies in breast cancer remain limited.MethodsIn silico analysis of human breast cancer patient data were used to demonstrate a correlation between DNA methylation and LRIG1 silencing in basal/triple-negative breast cancer, and its impact on patient survival. LRIG1 gene expression, protein abundance, and methylation enrichment were examined by quantitative reverse-transcription PCR, immunoblotting, and methylation immunoprecipitation, respectively, in breast cancer cell lines in vitro. We examined the impact of global demethylation on LRIG1 expression and methylation enrichment using 5-aza-2’-deoxycytidine. We also examined the effects of targeted demethylation of the LRIG1 CpG island, and transcriptional activation of LRIG1 expression, using the RNA guided deadCas9 transactivation system.ResultsAcross breast cancer subtypes, LRIG1 expression is lowest in the basal/triple-negative subtype so we investigated whether differential methylation may contribute to this. Indeed, we find that LRIG1 CpG island methylation is most prominent in basal/triple-negative cell lines and patient samples. Use of the global demethylating agent 5-aza-2’-deoxycytidine decreases methylation leading to increased LRIG1 transcript expression in basal/triple-negative cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines. Using a CRISPR/deadCas9 (dCas9)-based targeting approach, we demonstrate that TET1-mediated demethylation (Tet1-dCas9) along with VP64-mediated transcriptional activation (VP64-dCas9) at the CpG island, increased endogenous LRIG1 expression in basal/triple-negative breast cancer cells, without transcriptional upregulation at predicted off-target sites. Activation of LRIG1 by the dCas9 transactivation system significantly increased LRIG1 protein abundance, reduced site-specific methylation, and reduced cancer cell viability. Our findings suggest that CRISPR-mediated targeted activation may be a feasible way to restore LRIG1 expression in cancer.ConclusionsOur study contributes novel insight into mechanisms which repress LRIG1 in triple-negative breast cancer and demonstrates for the first time that targeted de-repression of LRIG1 in cancer cells is possible. Understanding the epigenetic mechanisms associated with repression of tumour suppressor genes holds potential for the advancement of therapeutic approaches.

C-Met and EPHA7 Receptor Tyrosine Kinases Are Related to Prognosis in Clear Cell Renal Cell Carcinoma: Focusing on the Association with Myoferlin Expression.

Simple SummaryReceptor tyrosine kinases are essential for the development, growth, and progression of clear cell renal cell carcinoma (ccRCC). Targeted therapies using receptor tyrosine kinase inhibitors are widely used in ccRCC treatment. Myoferlin is a well-known protein that regulates various receptor tyrosine kinases. We aimed to identify myoferlin-associated receptor tyrosine kinases and their prognostic implications in ccRCC. After screening with proteomic analysis, we focused on c-Met and EPHA7 receptor tyrosine kinases. c-Met expression was associated with poor prognosis in ccRCC, and there was an indication that the c-Met pathway may be regulated by myoferlin. Although the expression of EPHA7 and myoferlin was not corelated, the expression of EPHA7 was also associated with unfavorable outcomes. These results suggest that c-Met and EPHA7 might be useful prognostic biomarkers, and the presumed myoferlin/c-Met pathway could be a novel therapeutic target in ccRCC.Receptor tyrosine kinases (RTKs) are important targets for clear cell renal cell carcinoma (ccRCC) treatment. Myoferlin is a strong regulator of RTKs. To identify myoferlin-associated RTKs and their prognostic implications in ccRCC, we investigated the expression of RTKs and myoferlin using proteome-based evaluation and immunohistochemical staining in tissue microarray. Multivariate Cox analysis adjusted for TNM stage and WHO grade was performed (n = 410 and 506). Proteomic analysis suggested c-Met and EPHA7 as novel candidates for myoferlin-associated RTKs. We immunohistochemically validated the positive association between c-Met and myoferlin expression. High c-Met expression was independently associated with overall (hazard ratio (HR) = 1.153–2.919) and cancer-specific survival (HR = 1.150–3.389). The prognostic effect of high c-Met expression was also determined in an independent cohort (overall survival, HR = 1.503–3.771). Although expression of EPHA7 and myoferlin was not correlated, EPHA7 expression was independently associated with progression-free (HR = 1.237–4.319) and cancer-specific survival (HR = 1.214–4.558). In addition, network-based prioritization showed co-functional enrichment of c-Met and myoferlin, suggesting a novel regulatory function of myoferlin in c-Met signaling. This study indicates that c-Met and EPHA7 might be useful prognostic biomarkers, and the presumed myoferlin/c-Met pathway could be a novel therapeutic target in ccRCC.

Abstract P2-13-19: Statin modulation of antibody drug conjugate activity in breast cancer models and patients

Abstract Background: Membrane HER2 expression levels strongly influence the activity of anti-HER2 therapies directed at the HER2 extracellular domain (1,2). Tumoral cell-surface caveolin-1 (CAV-1) regulates receptor tyrosine kinase membrane trafficking mechanisms (1,3). In xenograft models, CAV-1 can be modulated with cholesterol-depleting drugs, such as statins. We hypothesized that preclinical and clinical use of statins might influence the expression of CAV-1 and thereby affect the efficacy of anti-HER2 antibody-drug conjugates (ADCs) in breast cancer. Methods: Preclinically, statins were given alone and in combination with HER2 antibody drug conjugates such as T-DM1 to assess HER2 levels, drug uptake, and antitumor efficacy in HER2-positive cancer models. Mice received an intravenous injection of T-DM1, oral doses of lovastatin, or a combination of T-DM1 and lovastatin, for 5 weeks. Clinically, we performed retrospective analyses of HER2-positive MBC patients at MSKCC who received T-DM1 and consented to molecular profiling and clinical data abstraction. Progression-free survival (PFS) on T-DM1 was estimated using Kaplan Meyer methods and compared using the log rank test. Univariable and multivariable Cox proportional hazards models were constructed using relevant clinical covariates. Results: Tumor models with high levels of CAV-1 exhibit low HER2 density on the cell surface and show low T-DM1 targeted immunoPET binding when compared with CAV-1-low tumors. Mechanistic studies showed that CAV-1 depletion in HER2-positive BC cells temporally stabilizes HER2 on the surface and delays T-DM1 recycling. In preclinical BC models, CAV-1 depletion induced by synthetic oligonucleotides or statins enhances T-DM1 binding and efficacy in tumors with incomplete HER2 membranous reactivity. For clinical analyses, a total of 164 patients who received T-DM1 were included in the final analysis. Twenty-one (12.8%) of these patients were recorded to be taking statins concurrently with T-DM1, as part of their routine clinical care. Compared with patients with no recorded statin use, patients receiving statins were older on average (median age 58 vs. 50 years, p = 0.007), but did not differ in their race, histologic breast cancer type, ER status, or T-DM1 treatment line. Median PFS on T-DM1 in the overall cohort was 5.5 months. Median PFS in patients who received statins was 14 months vs. 5.4 months in patients who had no recorded statin use (p = 0.1). In univariable Cox analysis, the association between statin use and PFS did not reach statistical significance (p=0.119). In a multivariable analysis including age, ER status, treatment line, and breast cancer histology, no variable reached statistical significance. However, statin use had the greatest observed degree of association with PFS (p = 0.17). Conclusions: Statins modulate surface HER2 levels and T-DM1 efficacy preclinically via CAV-1. Although only a numerical difference in outcomes was observed in the MSKCC cohort, the effect appeared meaningful and therefore assessment in larger patient cohorts is now underway. Based on the results of these forthcoming analyses, prospective trials may be justified that integrate these well-tolerated and low-cost agents into HER2 ADC treatment regimens.

It Takes More than Two to Tango: Complex, Hierarchal, and Membrane-Modulated Interactions in the Regulation of Receptor Tyrosine Kinases.

Simple SummaryReceptor tyrosine kinases probably constitute the most important subfamily of transmembrane receptors with respect to their role in regulating the balance between cell proliferation and cell death. Their activation involves ligand-induced conformational changes followed by their dimerization. Although this simple mechanism is still known to lie at the root of the process, the picture is complicated by the involvement of several receptor domains in the dimerization and the formation of larger receptor aggregates. Both clustering and activation are influenced by lipid-mediated interactions of the plasma membrane with the receptors. The intricate regulation of receptor activation is subverted in cancer that involves not only alterations in receptor structure and expression but also changes in lipid composition of the cell membrane. This paper provides a concise overview of how these biophysical aspects of transmembrane signaling regulate this important process in health and disease.The search for an understanding of how cell fate and motility are regulated is not a purely scientific undertaking, but it can also lead to rationally designed therapies against cancer. The discovery of tyrosine kinases about half a century ago, the subsequent characterization of certain transmembrane receptors harboring tyrosine kinase activity, and their connection to the development of human cancer ushered in a new age with the hope of finding a treatment for malignant diseases in the foreseeable future. However, painstaking efforts were required to uncover the principles of how these receptors with intrinsic tyrosine kinase activity are regulated. Developments in molecular and structural biology and biophysical approaches paved the way towards better understanding of these pathways. Discoveries in the past twenty years first resulted in the formulation of textbook dogmas, such as dimerization-driven receptor association, which were followed by fine-tuning the model. In this review, the role of molecular interactions taking place during the activation of receptor tyrosine kinases, with special attention to the epidermal growth factor receptor family, will be discussed. The fact that these receptors are anchored in the membrane provides ample opportunities for modulatory lipid–protein interactions that will be considered in detail in the second part of the manuscript. Although qualitative and quantitative alterations in lipids in cancer are not sufficient in their own right to drive the malignant transformation, they both contribute to tumor formation and also provide ways to treat cancer. The review will be concluded with a summary of these medical aspects of lipid–protein interactions.

Promoting receptor protein tyrosine phosphatase activity by targeting transmembrane domain interactions

Receptor protein tyrosine phosphatases (RPTPs) are one of the most important regulators of receptor tyrosine kinases (RTKs) and therefore play a crucial role in mammalian signal transduction. But our still-incomplete understanding of the structural aspects of their regulation and the lack of selective agonists have hampered efforts to understand their roles in downstream signaling regulation, and to pursue them as potential therapeutic targets. However, the reported ability of RPTP homodimerization to antagonize their catalytic activity presents potential opportunities to develop unique strategies to modulate the activity of RPTPs.

High-fat diet-induced hyperinsulinemia promotes the development of prostate adenocarcinoma in prostate-specific Pten-/- mice.

Metabolic syndrome (MetS) and its four clinical entities, central obesity, insulin resistance, hypertension and dyslipidemia, are implicated in increasing the risk and mortality of cancer in several organs. However, it is unclear how they are associated with increased risk of prostate cancer. To elucidate the mechanistic link between MetS and prostate carcinogenesis, we characterized the development of MetS and prostate adenocarcinoma in prostate-specific Pten-/- (Ptenp-/-) mice fed a high-fat (HF) diet. We found that male Ptenp-/- mice on an HF diet gained excess body weight and elevated blood glucose, insulin and insulin-like growth factor 1 (IGF1) levels at 20 weeks of age and were obese at 40 weeks. Prostate adenocarcinoma multiplicity at 40 weeks was significantly higher in the mice on an HF diet, suggesting that the HF diet promotes the development of prostate adenocarcinoma. Increased cell proliferation and enhanced AKT activation were found in the prostates of mice on an HF diet. Further transcriptome study revealed that receptor tyrosine kinase regulation, which mediates insulin/IGF1 signaling, was one of the top enriched pathways by HF diet-induced transcriptome changes. Together, our results suggest that HF diet-induced hyperinsulinemia leads to increased activation of insulin/IGF1/AKT signaling in lesioned prostates, promoting the development of adenocarcinoma.

Cell surface integrin α5ß1 clustering negatively regulates receptor tyrosine kinase signaling in colorectal cancer cells via glycogen synthase kinase 3.

As a key process within the tissue microenvironment, integrin signaling can influence cell functional responses to growth factor stimuli. We show here that clustering of integrin α5ß1 at the plasma membrane of colorectal cancer-derived epithelial cells modulates their ability to respond to stimulation by receptor tyrosine kinase (RTK)-activating growth factors EGF, NRG and HGF, through GSK3-mediated suppression of Akt pathway. We observed that integrin α5ß1 is lost from the membrane of poorly organized human colorectal tumors and that treatment with the integrin-clustering antibody P4G11 is sufficient to induce polarity in a mouse tumor xenograft model. While adding RTK growth factors (EGF, NRG and HGF) to polarized colorectal cancer cells induced invasion and loss of monolayer formation in 2D and 3D, this pathological behavior could be blocked by P4G11. Phosphorylation of ErbB family members as well as MET following EGF, NRG and HGF treatment was diminished in cells pretreated with P4G11. Focusing on EGFR, we found that blockade of integrin α5ß1 increased EGFR phosphorylation. Since activity of multiple downstream kinase pathways were altered by these various treatments, we employed computational machine learning techniques to ascertain the most important effects. Partial least-squares discriminant analysis identified GSK3 as a major regulator of EGFR pathway activities influenced by integrin α5ß1. Moreover, we used partial correlation analysis to examine signaling pathway crosstalk downstream of EGF stimulation and found that integrin α5ß1 acts as a negative regulator of the AKT signaling cascade downstream of EGFR, with GSK3 acting as a key mediator. We experimentally validated these computational inferences by confirming that blockade of GSK3 activity is sufficient to induce loss of polarity and increase of oncogenic signaling in the colonic epithelial cells.

Promoting Receptor Protein Tyrosine Phosphatase Activity by Targeting Transmembrane Domain Interactions

Receptor Protein Tyrosine Phosphatases (RPTPs) are one of the most important regulators of receptor tyrosine kinases (RTKs) and therefore play a crucial role in mammalian signal transduction. But our still-incomplete understanding of the structural aspects of their regulation and the lack of selective agonists have hampered efforts to understand their roles in downstream signaling regulation, and to pursue them as potential therapeutic targets. However, the reported ability of RPTP homodimerization to antagonize their catalytic activity presents potential opportunities to develop unique strategies to modulate the activity of RPTPs. We recently reported (10.1074/jbc.RA119.010229) that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ; also known as DEP1) is regulated by specific transmembrane (TM) residues, and that these interactions are essential in regulating its enzymatic activity and substrate access in cells. Building on these new insights and in response to the lack of selective agonists, we report here the design and testing of a tumor-selective peptide capable of binding to the TM domain of PTPRJ and disrupting its homodimerization. By doing so, it promotes PTPRJ TM-mediated access to EGFR, reduces the phosphorylation of EGFR (a known substrate) and other downstream signaling effectors, and antagonizes EGFR-driven cancer cell phenotypes. This peptide represents a novel allosteric and a possibly orthogonal way to target the activity of PTPRJ and RTKs phosphorylation. It could therefore be used not only as probes to tease out PTPRJ regulating mechanisms but also for therapeutic purposes via the attenuation of signaling by dysregulated RTKs in cancers. We also expect that the basic framework developed here can be extended to other RPTPs.

USP8 is a Novel Therapeutic Target in Melanoma Through Regulating Receptor Tyrosine Kinase Levels.

IntroductionThe hyperactivation of receptor tyrosine kinase (RTK)-mediated pathways plays an important role in melanoma progression and resistance to therapy. The ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme and its inhibition induces degradation of RTKs. This work explored the expression and role of USP8 in melanoma.MethodsELISA and qPCR were performed to assess USP8 expression in melanoma tissues and cells, as well as their normal counterparts. Cellular proliferation, migration and apoptosis assays were performed to determine USP8 functions in three melanoma cell lines. Western blot was performed to analyze RTK signaling in melanoma cells after USP8 inhibition.ResultsmRNA and protein level of USP8 were higher in melanoma cells than normal melanocytes. Higher USP8 expression was also found in tumors in the majority of melanoma patients. USP8 expression was not associated with clinicopathological features, such as age, disease stage, histology, ulceration and BRAF status. Functional analysis demonstrated that USP8 overexpression promoted melanoma cell activities and alleviated the inhibitory effects of therapeutic drugs. In contrast, USP8 knockdown suppressed melanoma cell growth, survival and migration, and augmented the inhibitory effects of therapeutic drugs. Mechanism studies revealed that USP8 inhibition remarkably reduced the expression level of multiple oncogenic RTKs, including c-Met, Kit, EGFR and GPCR. Consistently, RTK-mediated downstream pathways were disrupted in USP8-depleted cells, leading to the increased level of pro-apoptotic proteins and decreased level of anti-apoptotic proteins.ConclusionInhibition of USP8 activity is a novel sensitizing strategy to overcome therapy resistance in melanoma.

Regulation of Phosphoinositide Levels in the Retina by Protein Tyrosine Phosphatase 1B and Growth Factor Receptor-Bound Protein 14.

Protein tyrosine kinases and protein phosphatases play a critical role in cellular regulation. The length of a cellular response depends on the interplay between activating protein kinases and deactivating protein phosphatases. Protein tyrosine phosphatase 1B (PTP1B) and growth factor receptor-bound protein 14 (Grb14) are negative regulators of receptor tyrosine kinases. However, in the retina, we have previously shown that PTP1B inactivates insulin receptor signaling, whereas phosphorylated Grb14 inhibits PTP1B activity. In silico docking of phosphorylated Grb14 and PTP1B indicate critical residues in PTP1B that may mediate the interaction. Phosphoinositides (PIPs) are acidic lipids and minor constituents in the cell that play an important role in cellular processes. Their levels are regulated by growth factor signaling. Using phosphoinositide binding protein probes, we observed increased levels of PI(3)P, PI(4)P, PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 in PTP1B knockout mouse retina and decreased levels of these PIPs in Grb14 knockout mouse retina. These observations suggest that the interplay between PTP1B and Grb14 can regulate PIP metabolism.

SHP2 Nuclear/Cytoplasmic Trafficking in Granulosa Cells Is Essential for Oocyte Meiotic Resumption and Maturation.

Src-homology-2-containing phosphotyrosine phosphatase (SHP2), a classic cytoplasmic protein and a major regulator of receptor tyrosine kinases and G protein-coupled receptors, plays a significant role in preimplantation embryo development. In this study, we deciphered the role of SHP2 in the somatic compartment of oocytes during meiotic maturation. SHP2 showed nuclear/cytoplasmic localization in bovine cumulus and human granulosa (COV434) cells. Follicle-stimulating hormone (FSH) treatment significantly enhanced cytoplasmic SHP2 localization, in contrast to the E2 treatment, which augmented nuclear localization. Enhanced cytoplasmic SHP2 was found to negatively regulate the expression of the ERα-transcribed NPPC and NPR2 mRNAs, which are vital for oocyte meiotic arrest. The co-immunoprecipitation results revealed the presence of the SHP2/ERα complex in the germinal vesicle-stage cumulus–oocyte complexes, and this complex significantly decreased with the progression of meiotic maturation. The complex formation between ERα and SHP2 was also confirmed by using a series of computational modeling methods. To verify the correlation between SHP2 and NPPC/NPR2, SHP2 was knocked down via RNA interference, and NPPC and NPR2 mRNAs were analyzed in the control, E2, and FSH-stimulated COV434 cells. Furthermore, phenyl hydrazonopyrazolone sulfonate 1, a site-directed inhibitor of active SHP2, showed no significant effect on the ERα-transcribed NPPC and NPR2 mRNAs. Taken together, these findings support a novel nuclear/cytoplasmic role of SHP2 in oocyte meiotic resumption and maturation.

Heparan Sulfate Synthesized by Ext1 Regulates Receptor Tyrosine Kinase Signaling and Promotes Resistance to EGFR Inhibitors in GBM.

Signaling from multiple receptor tyrosine kinases (RTK) contributes to therapeutic resistance in glioblastoma (GBM). Heparan sulfate (HS), present on cell surfaces and in the extracellular matrix, regulates cell signaling via several mechanisms. To investigate the role for HS in promoting RTK signaling in GBM, we generated neural progenitor cells deficient for HS by knockout of the essential HS-biosynthetic enzyme Ext1, and studied tumor initiation and progression. HS-null cells had decreased proliferation, invasion, and reduced activation of multiple RTKs compared with control. In vivo tumor establishment was significantly decreased, and rate of tumor growth reduced with HS-deficient cells implanted in an HS-poor microenvironment. To investigate if HS regulates RTK activation through platelet-derived growth factor receptor α (PDGFRα) signaling, we removed cell surface HS in patient-derived GBM lines and identified reduced cell surface PDGF-BB ligand. Reduced ligand levels were associated with decreased phosphorylation of PDGFRα, suggesting HS promotes ligand-receptor interaction. Using human GBM tumorspheres and a murine GBM model, we show that ligand-mediated signaling can partially rescue cells from targeted RTK inhibition and that this effect is regulated by HS. Indeed, tumor cells deficient for HS had increased sensitivity to EGFR inhibition in vitro and in vivo. IMPLICATIONS: Our study shows that HS expressed on tumor cells and in the tumor microenvironment regulates ligand-mediated signaling, promoting tumor cell proliferation and invasion, and these factors contribute to decreased tumor cell response to targeted RTK inhibition.

LRIG1-Mediated Inhibition of EGF Receptor Signaling Regulates Neural Precursor Cell Proliferation in the Neocortex

Here, we ask how neural stem cells (NSCs) transition in the developing neocortex from a rapidly to a slowly proliferating state, a process required to maintain lifelong stem cell pools. We identify LRIG1, known to regulate receptor tyrosine kinase signaling in other cell types, as a negative regulator of cortical NSC proliferation. LRIG1 is expressed in murine cortical NSCs as they start to proliferate more slowly during embryogenesis and then peaks postnatally when they transition to give rise to a portion of adult NSCs. Constitutive or acute loss of Lrig1 in NSCs over this developmental time frame causes stem cell expansion due to increased proliferation. LRIG1 controls NSC proliferation by associating with and negatively regulating the epidermal growth factor receptor (EGFR). These data support a model in which LRIG1 dampens the stem cell response to EGFR ligands within the cortical environment to slow their proliferation as they transition to postnatal adult NSCs.