Degradation Of Receptor Tyrosine Kinases Research Articles

Cholesterol suppresses GOLM1-dependent selective autophagy of RTKs in hepatocellular carcinoma.

Aberrant activation of receptor tyrosine kinases (RTKs) and the subsequent metabolic reprogramming play critical roles in cancer progression. Our previous study has shown that Golgi membrane protein 1 (GOLM1) promotes hepatocellular carcinoma (HCC) metastasis by enhancing the recycling of RTKs. However, how this RTK recycling process is regulated and coupled with RTK degradation remains poorly defined. Here, we demonstrate that cholesterol suppresses the autophagic degradation of RTKs in a GOLM1-dependent manner. Further mechanistic studies reveal that GOLM1 mediates the selective autophagy of RTKs by interacting with LC3 through an LC3-interacting region (LIR), which is regulated by a cholesterol-mTORC1 axis. Lowering cholesterol by statins improves the efficacy of multiple tyrosine kinase inhibitors (TKIs) invivo. Our findings indicate that cholesterol serves as a signal to switch GOLM1-RTK degradation to GOLM1-RTK recycling and suggest that lowering cholesterol by statin may be a promising combination strategy to improve the TKI efficiency in HCC.

The Oncogenic Signaling Disruptor, NDRG1: Molecular and Cellular Mechanisms of Activity.

NDRG1 is an oncogenic signaling disruptor that plays a key role in multiple cancers, including aggressive pancreatic tumors. Recent studies have indicated a role for NDRG1 in the inhibition of multiple tyrosine kinases, including EGFR, c-Met, HER2 and HER3, etc. The mechanism of activity of NDRG1 remains unclear, but to impart some of its functions, NDRG1 binds directly to key effector molecules that play roles in tumor suppression, e.g., MIG6. More recent studies indicate that NDRG1s-inducing drugs, such as novel di-2-pyridylketone thiosemicarbazones, not only inhibit tumor growth and metastasis but also fibrous desmoplasia, which leads to chemotherapeutic resistance. The Casitas B-lineage lymphoma (c-Cbl) protein may be regulated by NDRG1, and is a crucial E3 ligase that regulates various protein tyrosine and receptor tyrosine kinases, primarily via ubiquitination. The c-Cbl protein can act as a tumor suppressor by promoting the degradation of receptor tyrosine kinases. In contrast, c-Cbl can also promote tumor development by acting as a docking protein to mediate the oncogenic c-Met/Crk/JNK and PI3K/AKT pathways. This review hypothesizes that NDRG1 could inhibit the oncogenic function of c-Cbl, which may be another mechanism of its tumor-suppressive effects.

Calix[6]arene diminishes receptor tyrosine kinase lifespan in pancreatic cancer cells and inhibits their migration and invasion efficiency.

Pancreatic cancer is a challenging malignancy, mainly due to aggressive regional involvement, early systemic dissemination, high recurrence rate, and subsequent low patient survival. Scientific advances have contributed in particular by identification of molecular targets as well as the definition of the mechanism of action of the drug candidate in the cellular microenvironment. Previously, we have reported the identification of the molecular mechanisms by which calix[6]arene (CLX6) reduces the viability and proliferation of pancreatic cancer cells. Now, we show the biochemical mechanisms by which CLX6 decreases the aggressiveness of Panc-1 cells, focusing specifically on receptor tyrosine kinases (RTK). The results show that clathrin-mediated endocytosis is involved in CLX6-induced AXL receptor tyrosine kinase degradation in Panc-1 cells. This response may be related to the interaction of CLX6 with the tyrosine kinase receptor binding site (such as AXL). As a result, RTK is internalized and degraded by endocytosis, a condition that negatively impacts events dependent on its signaling. Additionally, CLX6 inhibits migration and invasion of Panc-1 cells by downregulating FAK (downstream mediator of AXL) activity and reducing expression levels of MMP2 and MMP9, directly related to the metastatic profile of these cells. It is noteworthy that according to the mechanism proposed here, CLX6 appears as a candidate to be used in therapeutic protocols of patients that display high expression of AXL and consequently, poor diagnosis.

Abstract 2655: Oxygen tension regulates lysosomal activation and receptor tyrosine kinase degradation

Abstract Oxygen sensing is crucial for adaptation to variable habitats and physiological conditions. Low oxygen tension, or hypoxia, is a common feature of solid tumors and hypoxic tumors are often more aggressive and resistant to therapy. Here we show that, in mammalian tissue culture cells, hypoxia suppressed lysosomal acidification/activation and receptor tyrosine kinase (RTK) degradation. Hypoxia down-regulated mTORc1, reducing its ability to activate transcription factor EB (TFEB), a master regulator of v-ATPase, the lysosomal proton pump. Hypoxia prevented epidermal growth factor receptor (EGFR) degradation in tumor tissues, whereas activation of lysosomes enhanced tumor cell response to anti-EGFR treatment. Our results linkoxygen tension and lysosomal activity, provide a molecular explanation of the malignant phenotype associated with hypoxic tumors, and suggest activation of lysosomes may provide therapeutic benefit in RTK-targeted cancer therapy. Citation Format: Jaewoo Hong, P. Charles Lin, Todd Wuest, Yongfen Min. Oxygen tension regulates lysosomal activation and receptor tyrosine kinase degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2655.

Abstract 4293: Analyzing the redox/fyn/c-Cbl pathway in glioblastoma multiforme: A mechanistic approach

Abstract Glioblastoma multiforme (GBM) is the most common aggressive tumor of the central nervous system (CNS) with 10,000 newly diagnosed cases annually, and an incidence rate of 3 in 100,000 individuals in the United States. Unfortunately, the current standard of care of radiotherapy with concomitant Temozolomide (TMZ) has not been effective in consistently controlling tumor growth, as the median age of survival for patients is 14.6 months after treatment, with inevitable recurrence beyond this time. One of the difficulties that emerge when treating GBM is tumor heterogeneity. For example, GBM cells upregulate different receptor tyrosine kinases (RTKs). Signaling through RTKs enables these cells to maintain their proliferative status to ensure their survival. The E3 ubiquitin ligase c-Cbl is a negative regulator of RTKs, and our lab has found it to be physically sequestered by ARGHEF7 (Cool-1) in GBM. We identified a combination of drugs, a c-Cbl restorative agent (CRA01) and an oxidizing agent (Tamoxifen [TMX]), that decreased c-Cbl/Cool-1 complex formation and activated c-Cbl through the Redox/Fyn/c-Cbl pathway (RFC) in GBM cells. Treatment with CRA01+TMX led to degradation of RTKs. Little is known about the RFC pathway in cancer regulation or its ability to prevent cell growth in TMZ resistant GBM cells. Here, we examine ways to regulate c-Cbl in GBM cells and seek to identify the mechanism(s) resulting in c-Cbl/Cool-1 complex formation. Furthermore, we aim to use a mouse model of TMZ resistance to determine the effects of CRA01+TMX in vivo. These results will provide important knowledge on the RFC pathway that can help properly identify patients with GBM and different cancers with c-Cbl dysregulation for whom this treatment could prove beneficial. Citation Format: Neal Shah, Jennifer Stripay. Analyzing the redox/fyn/c-Cbl pathway in glioblastoma multiforme: A mechanistic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4293.

Internalized CD44s splice isoform attenuates EGFR degradation by targeting Rab7A

CD44 has been postulated as a cell surface coreceptor for augmenting receptor tyrosine kinase (RTK) signaling. However, how exactly CD44 triggers RTK-dependent signaling remained largely unclear. Here we report an unexpected mechanism by which the CD44s splice isoform is internalized into endosomes to attenuate EGFR degradation. We identify a CD44s-interacting small GTPase, Rab7A, and show that CD44s inhibits Rab7A-mediated EGFR trafficking to lysosomes and subsequent degradation. Importantly, CD44s levels correlate with EGFR signature and predict poor prognosis in glioblastomas. Because Rab7A facilitates trafficking of many RTKs to lysosomes, our findings identify CD44s as a Rab7A regulator to attenuate RTK degradation.

GCL and CUL3 Control the Switch between Cell Lineages by Mediating Localized Degradation of an RTK

The separation of germline from somatic lineages is fundamental to reproduction and species preservation. Here, we show that Drosophila Germ cell-less (GCL) is a critical component in this process by acting as a switch that turns off a somatic lineage pathway. GCL, a conserved BTB (Broad-complex, Tramtrack, and Bric-a-brac) protein, is a substrate-specific adaptor for Cullin3-RING ubiquitin ligase complex (CRL3GCL). We show that CRL3GCL promotes PGC fate by mediating degradation of Torso, a receptor tyrosine kinase (RTK) and major determinant of somatic cell fate. This mode of RTK degradation does not depend upon receptor activation but isprompted by release of GCL from the nuclear envelope during mitosis. The cell-cycle-dependent change in GCL localization provides spatiotemporal specificity for RTK degradation and sequesters CRL3GCL to prevent it from participating in excessive activities. This precisely orchestrated mechanism of CRL3GCL function and regulation defines cell fate at the single-cell level.

The E3 ubiquitin ligase Cbl-b inhibits tumor growth in multidrug-resistant gastric and breast cancer cells.

Most receptor tyrosine kinases (RTKs) contribute to tumor growth, and their ubiquitination and degradation is related to the inhibition of tumor growth. Our previous study showed that the ubiquitin ligase Cbl-b was expressed at low levels in multidrug-resistant (MDR) gastric cancer cells compared with their parental cells. However, whether enhancement of Cbl-b expression in MDR cancer cells could prevent tumor proliferation via ubiquitination and degradation of RTK remains unclear. In the present study, Cbl-b overexpression reduced cell proliferation in MDR gastric and breast cancer cells, and effectively inhibited tumor growth in vivo. Additionally, Cbl-b overexpression reduced the total protein level of insulin-like growth factor 1 (IGF-1R), an important member of the RTK family. Moreover, Cbl-b overexpression promoted interaction of Cbl-b with IGF-1R, and induced ubiquitination and degradation of IGF-1R and inactivation of the IGF-1R pathway. These results suggest that the ubiquitin ligase Cbl-b inhibited tumor growth via ubiquitination and degradation of IGF-1R in MDR gastric and breast cancer cells.

Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Adrenocorticotropic Hormone Production and Tumorous Corticotroph Cell Growth in AtT20 Cells.

Background:Two recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 (USP8) gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor (EGFR) signaling and promote adrenocorticotropic hormone (ACTH) production. This study was to investigate whether the inhibition of USP8 activity could be a strategy for the treatment of Cushing's disease (CD).Methods:The anticancer effect of USP8 inhibitor was determined by testing cell viability, colony formation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition.Results:Inhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 (ERBB2), and Met leading to a suppression of AtT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis.Conclusions:Inhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD.

Abstract 3031: Bufalin regulates Cbl-b levels in HeLa cells through protein synthesis inhibition

Abstract Cbl-b is a member of ubiquitin ligases (E3s) family. The major function of CBL family E3s is to negatively regulate signaling through ubiquitination and degradation of receptor tyrosine kinases and kinase associated receptors. Cbl-b plays a crucial role in the regulation of the immune response, as it negatively regulates the costimulatory pathway of CD8 T cells and also suppresses natural killer cell function. Thus Cbl-b down-regulation or inhibition may lead to increased adaptive and innate antitumor immunity and can be used as a treatment strategy for a wide variety of tumors. Bufalin is a major component of Chan Su, traditional Chinese medicine derived from the toad venom. Bufalin is a C-24 steroid with the wide variety of biological activities, including cardiotonic, anesthetic and antitumor activities. Bufalin has been shown to inhibit growth and induce cell cycle arrest and apoptosis of various cancer cells. Low doses of bufalin can be safely used for prolonged periods of time without severe side effects, while high doses can cause cardiac arrhythmia, seizure and coma. The purpose of the current study was to investigate if bufalin regulates the levels of Cbl-b and establish the mechanism of Cbl-b regulation. By Western blotting of HeLa cell lysates we found that bufalin decreases protein levels of Cbl-b in a time- and dose-dependent manner without affecting the levels of Cbl. The greatest decrease was observed with 50 nM bufalin after 24 hours treatment. Doses of bufalin as low as 4 nM were effective in Cbl-b down-regulation. Real-time PCR analysis showed 2.9-fold increase in the amount of Cbl-b mRNA upon 50 nM bufalin treatment for 24 hours, suggesting that Cbl-b regulation by bufalin occurs post-transcriptionally. Proteasomal, but not lysosomal inhibitors treatment attenuated the negative effect of bufalin on Cbl-b levels. Co-treatment of HeLa cells with 50 nM bufalin and 50 μg/ml cycloheximide, protein synthesis inhibitor, did not result in any further decrease in Cbl-b levels compared to cycloheximide treatment alone. By measuring the nascent protein synthesis with Click-It metabolic labeling reagents, bufalin was shown to block protein synthesis of the majority of short-lived proteins. These data taken together indicate that bufalin treatment decreases Cbl-b levels in HeLa cells by blocking protein synthesis. We found that 50 nM bufalin treatment for up to 8 hours increased the phosphorylation of eIF2α, translation initiation factor, at Ser51. Phosphorylation of eIF2α known to block translation might explain the effect of bufalin on Cbl-b levels and other short-lived proteins. To conclude, we found that bufalin increases the phosphorylation of eIF2α, thus leading to the block of translation and decreasing protein levels of Cbl-b in HeLa cells. Our future goal is to test the hypothesis that bufalin activates antitumor immunity through down-regulation of Cbl-b and can thus be used as an antineoplastic agent. Citation Format: Mariya S. Liyasova, Stanley Lipkowitz. Bufalin regulates Cbl-b levels in HeLa cells through protein synthesis inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3031.

Salinomycin co-treatment enhances tamoxifen cytotoxicity in luminal A breast tumor cells by facilitating lysosomal degradation of receptor tyrosine kinases.

Luminal A breast cancer is the most common breast cancer subtype which is usually treated with selective estrogen receptor modulators (SERMS) like tamoxifen. Nevertheless, one third of estrogen receptor positive breast cancer patients initially do not respond to endocrine therapy and about 40% of luminal A breast tumors recur in five years. In this study, we investigated an alternative treatment approach by combining tamoxifen and salinomycin in luminal A breast cancer cell lines. We have found that salinomycin induces an additional cytotoxic effect by inhibiting the ligand independent activation of ERα. Thereby salinomycin increases the intracellular calcium level. This leads to a premature fusion of endosomes with lysosomes and thus to the degradation of Egfr family members. Since this process is essential for luminal A breast cancer cells to circumvent tamoxifen treatment, the combination of both drugs induces cytotoxicity in tamoxifen sensitive as well as resistant luminal A breast cancer cell lines.

Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated.

USP8 controls the trafficking and sorting of lysosomal enzymes.

The endosomal deubiquitylase USP8 has profound effects on endosomal morphology and organisation. Previous reports have proposed both positive (EGFR, MET) and negative roles in the down-regulation of receptors (Frizzled, Smoothened). Here we report an additional influence of USP8 on the retromer-dependent shuttling of ci-M6PR between the sorting endosome and biosynthetic pathway. Depletion of USP8 leads to a steady state redistribution of ci-M6PR from the Trans-Golgi Network (TGN) to endosomal compartments. Consequently we observe a defect in sorting of lysosomal enzymes, evidenced by increased levels of unprocessed Cathepsin D, which is secreted into the medium. The normal distribution of receptor can be restored by expression of siRNA-resistant USP8 but not by a catalytically inactive mutant or a truncated form, lacking a MIT domain required for endosomal localisation. We suggest that effects of USP8 depletion may reflect the loss of ESCRT-0 components which associate with retromer components Vps35 and SNX1, whilst failure to efficiently deliver lysosomal enzymes may also contribute to the observed block in receptor tyrosine kinase degradation.

Abstract 2912: Inhibition of redox/Fyn/c-Cbl pathway function by Cdc42 controls tumor initiation capacity and tamoxifen sensitivity in basal-like breast cancer cells.

Abstract Two of the substantial challenges in cancer research are to identify means overcoming resistance of malignant cells to existing therapies and to eliminate the unique small population within bulk tumors, named tumor initiating cells or cancer stem cells which have been proposed to regenerate resistant relapsed tumors. In this study, we offer a novel therapeutic target relevant to both of these goals on basal-like (triple-negative) breast cancer (BLBC) cells, one of the most challenging cancers to treat with current regimens. Our findings emerged from examining the puzzling differences in regulation of activation of the E3 ubiqutin ligase c-Cbl in normal cells and in cancer cells by low microM concentrations of tamoxifen (TMX). In normal progenitor cells, TMX causes oxidation and sequential activation of Fyn kinase and the c-Cbl ubiquitin ligase. Activation of c-Cbl via this redox/Fyn/c-Cbl (RFC) pathway leads to accelerated degradation of receptor tyrosine kinases that are critical in cell survival and division (such as the epidermal growth factor (EGFR)). In BLBC cells, in contrast, TMX does not activate c-Cbl despite making the cells more oxidized and causing activation of Fyn. Moreover, BLBC cells exposed to TMX show no reduction in levels of EGFR. We found that TMX-induced activation of c-Cbl in BLBC cells was inhibited due to expression of Cdc42. Cdc42 sequestered c-Cbl, prevented its activation and prevented EGFR from being degraded. Restoration of c-Cbl function, by inhibiting activation of Cdc42, reduced EGFR levels in these cells. More critically, restoring c-Cbl function sensitized these cells to TMX both in vitro and in vivo through estrogen receptor-independent mechanisms. Analysis of tumor growth and formation in vivo showed that reducing the levels of Cdc42 reduced the size of tumors, increased sensitivity to TMX and decreased tumor forming capacity of these cells. The results provide a novel defense mechanism that BLBC cells utilize to prevent EGFR degradation, which may have high relevance to treatment of these tumors. Of particular importance is the ability of cdc42 to confer TMX sensitivity on these otherwise resistant tumor cells. Moreover, as use of low microM levels of TMX has been attempted for over a dozen different kinds of cancers, at least some of which express increased levels of Cdc42, this strategy may be relevant to the treatment of multiple different cancers. Citation Format: Hsing-Yu Chen, Yin Yang, Brett Stevens, Mark Noble. Inhibition of redox/Fyn/c-Cbl pathway function by Cdc42 controls tumor initiation capacity and tamoxifen sensitivity in basal-like breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2912. doi:10.1158/1538-7445.AM2013-2912

E3 ubiquitin ligase-mediated regulation of bone formation and tumorigenesis

The ubiquitination–proteasome and degradation system is an essential process that regulates protein homeostasis. This system is involved in the regulation of cell proliferation, differentiation and survival, and dysregulations in this system lead to pathologies including cancers. The ubiquitination system is an enzymatic cascade that mediates the marking of target proteins by an ubiquitin label and thereby directs their degradation through the proteasome pathway. The ubiquitination of proteins occurs through a three-step process involving ubiquitin activation by the E1 enzyme, allowing for the transfer to a ubiquitin-conjugated enzyme E2 and to the targeted protein via ubiquitin-protein ligases (E3), the most abundant group of enzymes involved in ubiquitination. Significant advances have been made in our understanding of the role of E3 ubiquitin ligases in the control of bone turnover and tumorigenesis. These ligases are implicated in the regulation of bone cells through the degradation of receptor tyrosine kinases, signaling molecules and transcription factors. Initial studies showed that the E3 ubiquitin ligase c-Cbl, a multi-domain scaffold protein, regulates bone resorption by interacting with several molecules in osteoclasts. Further studies showed that c-Cbl controls the ubiquitination of signaling molecules in osteoblasts and in turn regulates osteoblast proliferation, differentiation and survival. Recent data indicate that c-Cbl expression is decreased in primary bone tumors, resulting in excessive receptor tyrosine kinase signaling. Consistently, c-Cbl ectopic expression reduces bone tumorigenesis by promoting tyrosine kinase receptor degradation. Here, we review the mechanisms of action of E3 ubiquitin ligases in the regulation of normal and pathologic bone formation, and we discuss how targeting the interactions of c-Cbl with some substrates may be a potential therapeutic strategy to promote osteogenesis and to reduce tumorigenesis.

The Casitas B Lineage Lymphoma (Cbl) Mutant G306E Enhances Osteogenic Differentiation in Human Mesenchymal Stromal Cells in Part by Decreased Cbl-mediated Platelet-derived Growth Factor Receptor α and Fibroblast Growth Factor Receptor 2 Ubiquitination

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.

Ligand-induced downregulation of TrkA is partly regulated through ubiquitination by Cbl

Nerve growth factor (NGF) binding to its receptor TrkA, which belongs to the family of receptor tyrosine kinases (RTKs), is known to induce its internalization, endosomal trafficking and subsequent lysosomal degradation. The Cbl family of ubiquitin ligases plays a major role in mediating ubiquitination and degradation of RTKs. However, it is not known whether Cbl participates in mediating ubiquitination of TrkA. Here we report that c-Cbl mediates ligand-induced ubiquitination and degradation of TrkA. TrkA ubiquitination and degradation required direct interactions between c-Cbl and phosphorylated TrkA. c-Cbl and ubiquitinated TrkA are found in a complex after NGF stimulation and are degraded in lysosomes. Taken together, our data demonstrate that c-Cbl can induce downregulation of NGF-TrkA complexes through ubiquitination and degradation of TrkA. Structured summary of protein interactionsTrkAphysically interacts with c-Cbl by anti bait coimmunoprecipitation(View interaction)c-Cblphysically interacts with TrkA by anti bait coimmunoprecipitation(View interaction)

Nbr1 Is a Novel Inhibitor of Ligand-Mediated Receptor Tyrosine Kinase Degradation

Neighbor of BRCA1 (Nbr1) is a highly conserved multidomain scaffold protein with proposed roles in endocytic trafficking and selective autophagy. However, the exact function of Nbr1 in these contexts has not been studied in detail. Here we investigated the role of Nbr1 in the trafficking of receptor tyrosine kinases (RTKs). We report that ectopic Nbr1 expression inhibits the ligand-mediated lysosomal degradation of RTKs, and this is probably done via the inhibition of receptor internalization. Conversely, the depletion of endogenous NBR1 enhances RTK degradation. Analyses of truncation mutations demonstrated that the C terminus of Nbr1 is essential but not sufficient for this activity. Moreover, the C terminus of Nbr1 is essential but not sufficient for the localization of the protein to late endosomes. We demonstrate that the C terminus of Nbr1 contains a novel membrane-interacting amphipathic α-helix, which is essential for the late endocytic localization of the protein but not for its effect on RTK degradation. Finally, autophagic and late endocytic localizations of Nbr1 are independent of one another, suggesting that the roles of Nbr1 in each context might be distinct. Our results define Nbr1 as a negative regulator of ligand-mediated RTK degradation and reveal the interplay between its various regions for protein localization and function.

Molecular Dissection of Receptor Tyrosine Kinase Regulation by Cbl Proteins

Aberrations of receptor tyrosine kinases (RTKs) have been implicated in the etiology of human cancers. Members of the Casitas B‐lineage Lymphoma (Cbl) family of ubiquitin ligases play a role as negative regulators of RTK function and mutations of Cbl proteins are involved in human cancers such as leukemia. The purpose of studies here is to determine the molecular mechanisms of oncogenic signaling and to explore possible defects in negative regulation of RTK signaling by Cbl proteins. We hypothesize that Cbl and Cbl‐b represent redundant yet essential components to facilitate lysosomal degradation of RTKs. In initial studies, we have derived wildtype, Cbl‐null, Cbl‐b‐null and cbl/cbl‐b‐null mouse embryonic fibroblast (MEF) cell lines to demonstrate an important role of both Cbl and Cbl‐b proteins in RTK ubiquitination and degradation. Absence of Cbl and Cbl‐b expression dramatically alters actin cytoskeleton reorganization upon attachment to extracellular matrix and markedly enhances growth factor‐dependent cell migration indicating that Cbl proteins provide functionally‐important negative regulatory feedback upon RTK activation. Further studies are underway to express specific RTKs (such as human EGFR) in the characterized MEFs together with reconstitution with wildtype or mutant Cbl proteins to dissect the fundamental steps in endocytic traffic where Cbl proteins play an essential role. These studies will focus on the role of Cbl proteins in initial endocytosis vs. later endosomal sorting steps as the consequences of these mechanisms are distinct. This work was supported by: the NIH grant CA99163 to HB.

Targeting heat shock protein 90 with CUDC-305 overcomes erlotinib resistance in non–small cell lung cancer

CUDC-305 is a heat shock protein 90 (HSP90) inhibitor of the novel imidazopyridine class. Here, we report its activities in non-small cell lung cancer (NSCLC) cell lines with gene deregulations conferring primary or secondary resistance to epidermal growth factor receptor (EGFR) inhibitors. We show that CUDC-305 binds strongly to HSP90 extracted from erlotinib-resistant NSCLC cells (IC50 70 nmol/L). This result correlates well with the potent antiproliferative activity in erlotinib-resistant NSCLC cell lines (IC50 120-700 nmol/L) reported previously. Furthermore, it exhibits durable inhibition of multiple oncoproteins and induction of apoptosis in erlotinib-resistant NSCLC cells. CUDC-305 potently inhibits tumor growth in subcutaneous xenograft models of H1975 and A549, which harbor EGFR T790M mutation or K-ras mutations conferring acquired and primary erlotinib resistance, respectively. In addition, CUDC-305 significantly prolongs animal survival in orthotopic lung tumor models of H1975 and A549, which may be partially attributed to its preferential exposure in lung tissue. Furthermore, CUDC-305 is able to extend animal survival in a brain metastatic model of H1975, further confirming its ability to cross the blood-brain barrier. Correlating with its effects in various tumor models, CUDC-305 induces degradation of receptor tyrosine kinases and downstream signaling molecules of the PI3K/AKT and RAF/MEK/ERK pathways simultaneously, with concurrent induction of apoptosis in vivo. In a combination study, CUDC-305 enhanced the antitumor activity of a standard-of-care agent in the H1975 tumor model. These results suggest that CUDC-305 holds promise for the treatment of NSCLC with primary or acquired resistance to EGFR inhibitor therapy.