Ubiquitination Of EGFR Research Articles

Cbl interacts with multiple E2s in vitro and in cells

Many receptor tyrosine kinases (RTKs, such as EGFR, MET) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins, a family of RING finger (RF) ubiquitin ligases (E3s). Loss of Cbl protein function is associated with malignant transformation driven by increased RTK activity. RF E3s, such as the Cbl proteins, interact with a ubiquitin-conjugating enzyme (E2) to confer specificity to the ubiquitination process and direct the transfer of ubiquitin from the E2 to one or more lysines on the target proteins. Using in vitro E3 assays and yeast two-hybrid screens, we found that Ube2d, Ube2e families, Ube2n/2v1, and Ube2w catalyze autoubiquitination of the Cbl protein and Ube2d2, Ube2e1, and Ube 2n/2v1 catalyze Cbl-mediated substrate ubiquitination of the EGFR and SYK. Phosphorylation of the Cbl protein by by Src resulted in increased E3 activity compared to unphosphorylated cbl or Cbl containing a phosphomimetic Y371E mutation. Ubiquitin chain formation depended on the E2 tested with Cbl with Ube2d2 forming both K48 and K63 linked chains, Ube2n/2v1 forming only K63 linked chains, and Ube2w inducing monoubiquitination. In cells, the Ube2d family, Ube2e family, and Ube2n/2v1 contributed to EGFR ubiquitination. Our data suggest that multiple E2s can interact with Cbl and modulate its E3 activity in vitro and in cells.

Abstract 2506: Flotillin 2 negatively regulates EGFR phosphorylation and Cbl-mediated ubiquitination and degradation

Abstract EGF receptor (EGFR) signaling is dysregulated by gene amplification or activating mutations of the EGFR in a variety of human malignancies. Activation of the EGFR by EGF leads to recruitment of the ubiquitin ligase Cbl, which ubiquitinates EGFR targeting it for degradation and acts as an adaptor, recruiting additional proteins required for EGFR internalization and trafficking to the lysosome. However, the detailed molecular mechanisms remain elusive. Complete characterization of proteins involved in EGFR trafficking and degradation may uncover additional mechanisms of EGFR dysregulation in cancer. To characterize the EGFR degradative pathway, we investigated protein complexes formed on Cbl with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry. By labeling proteins with stable isotopes in culture, we compared complexes in 3 groups: 1) complexes formed on Cbl without EGF stimulation (control), 2) complexes formed on Cbl with EGF stimulation for 30 min and 3) complexes formed on Cbl with EGF stimulation in the presence of YM201636, a trafficking inhibitor, which was added to prevent degradation of the Cbl/EGFR complex and to enrich it for other proteins important for EGFR trafficking. This approach identified over a hundred novel Cbl interactors, whose abundance in the complex with Cbl changed significantly with the treatments. Fifty of these proteins were selected for RNAi knockdown to examine their effects on EGFR degradation. We found that knockdown of Flotillin 2 (FLOT2) protein, a major scaffold protein within caveolar lipid rafts, led to increased phosphorylation and accelerated degradation of EGFR upon EGF stimulation compared to negative control siRNA in HeLa cells. Conversely, overexpression of FLOT2 in HEK293T inhibited EGFR phosphorylation and formation of the EGFR/Cbl complex upon EGF stimulation and led to decreased EGFR ubiquitination and degradation. CRISPR knockout of FLOT2 in HeLa cells led to decreased steady-state levels of EGFR, increased phosphorylation of EGFR and ERK1/2 compared to non-targeting CRISPR control. Stable overexpression of FLOT2 in HeLa cells increased steady-state levels of EGFR, but decreased EGFR phosphorylation and ubiquitination upon EGF stimulation. FLOT2 is frequently amplified and overexpressed in breast, endometrial and lung cancers. In a lung adenocarcinoma cell line H441, in which FLOT2 gene is amplified, knockdown of FLOT2 increased EGFR phosphorylation and ubiquitination, as well as EGFR signaling, as detected by the phosphorylation of ERK1/2. Our data indicate that FLOT2 negatively regulates EGFR phosphorylation, ubiquitination and degradation. Although it is not clear how FLOT2 amplification could contribute to cancer pathogenesis, we speculate that studying how FLOT2 regulates EGFR signaling could lead to a better understanding of its role in normal physiology and cancer. Citation Format: Mariya Liyasova, Xu Zhang, Udayan Guha, Stanley Lipkowitz. Flotillin 2 negatively regulates EGFR phosphorylation and Cbl-mediated ubiquitination and degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2506.

E3 Ubiquitin Ligase Cbl-b Prevents Tumor Metastasis by Maintaining the Epithelial Phenotype in Multiple Drug-Resistant Gastric and Breast Cancer Cells

Multiple drug resistance (MDR) and metastasis are two major factors that contribute to the failure of cancer treatment. However, the relationship between MDR and metastasis has not been characterized. Additionally, the role of the E3 ubiquitin ligase Cbl-b in metastasis of MDR gastric and breast cancer is not well known. In the present study, we found that MDR gastric and breast cancer cells possess a typical mesenchymal phenotype and enhanced cell migration capacity. Additionally, Cbl-b is poorly expressed in MDR gastric and breast cancer cells. In MDR gastric adenocarcinoma tissues, gastric cancer patients with low Cbl-b expression were more likely to have tumor invasion (P=.016) and lymph node metastasis (P=.007). Moreover, overexpression of Cbl-b reduced cell migration in MDR cell cultures both in vitro and in vivo. Cbl-b overexpression also prevented EMT by inducing ubiquitination and degradation of EGFR, leading to inhibition of the EGFR-ERK/Akt-miR-200c-ZEB1 axis. However, further overexpression of EGFR on a background of Cbl-b overexpression restored both the mesenchymal phenotype and cell migration capacity of MDR gastric and breast cancer cells. These results suggest that Cbl-b is an important factor for maintenance of the epithelial phenotype and inhibition of cell migration in MDR gastric and breast cancer cells.

Piccolo mediates EGFR signaling and acts as a prognostic biomarker in esophageal squamous cell carcinoma.

The presynaptic cytomatrix protein Piccolo, encoded by PCLO, is frequently mutated and amplified in esophageal squamous cell carcinoma (ESCC), but its exact roles in ESCC remain unclear. Here we report that Piccolo expression correlates significantly with clinical stage, patient survival and tumor embolus. Functional studies demonstrate that PCLO knockdown remarkably attenuates ESCC malignancy in vitro and in vivo, and ectopic EGFR expression partially compensates for Piccolo loss. PCLO knockdown promotes ubiquitination and degradation of EGFR, which is associated with the negative regulatory effect of Piccolo on E3 ligase Siah1. An anti-Piccolo monoclonal antibody inhibited tumor proliferation in a mouse model of ESCC. These results demonstrate that Piccolo contributes to tumor aggressiveness in ESCC, likely by stabilizing EGFR and promoting EGFR-dependent signaling. Our results further suggest that Piccolo may represent a novel prognostic biomarker and therapeutic target for patients with ESCC.

Antiglioma effects of N6-isopentenyladenosine, an endogenous isoprenoid end product, through the downregulation of epidermal growth factor receptor.

Malignant gliomas are highly dependent on the isoprenoid pathway for the synthesis of lipid moieties critical for cell proliferation. The isoprenoid derivative N6-isopentenyladenosine (iPA) displays pleiotropic biological effects, including a direct anti-tumor activity in several tumor models. The antiglioma effects of iPA was then explored in U87MG cells both in vitro and grafted in mice and the related molecular mechanism confirmed in primary derived patients' glioma cells. iPA powerfully inhibited tumor cell growth and induced caspase-dependent apoptosis through a mechanism involving a marked accumulation of the pro-apoptotic BIM protein and inhibition of EGFR. Indeed, activating AMPK following conversion into its iPAMP active form, iPA stimulated EGFR phosphorylation and ubiquitination along a proteasome-mediated pathway which was responsible for receptor degradation and its downstream signaling pathways inhibition, including the STAT3, ERK and AKT cascade. The inhibition of AMPK by compound C prevented iPA-mediated phosphorylation of EGFR, known to precede receptor loss. As expected the block of EGFR degradation, by exposure to the proteasome inhibitor MG132, significantly reduced iPA-induced cell death. Given the importance of receptor degradation in iPA-mediated cytotoxicity, we also documented that the EGFR expression levels in a panel of primary glioma cells confers them a high sensitivity to iPA treatment. In conclusion our study provides the first evidence of iPA antiglioma effect. Indeed, as glioma is driven by aberrant signaling of growth factor receptors, particularly the EGFR, iPA, alone or in association with EGFR targeted therapies, might be a promising therapeutic tool to achieve a potent anti-tumoral effect.

Abstract 4542: Ube2d family members, Ube2e family members and Ube2w modulate the ubiquitination and degradation of EGFR by Cbl

Abstract Ubiquitin ligases (E3s) are critical component of ubiquitination. In collaboration with ubiquitin-conjugating enzymes (E2s), E3s confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the target proteins. Cbl proteins are RING finger E3s that play a significant role in regulating activity of many tyrosine kinases (e.g., EGFR, MET) by ubiquitination. In our study, we used enzymatic and yeast two-hybrid assays to characterize the E2s that can interact with Cbl proteins. Using an in vitro E3 assay, we found that only the Ube2d family of E2s mediates autoubiquitination of the Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that the three Ube2e family members and Ube2w interact with the RF domain of Cbl. This suggests that Ube2e and Ube2w are relevant to the ubiquitination and degradation of substrates by Cbl. Knockdown of Ube2w decreases ubiquitination of EGFR in Hela cells. In the in vitro E3 assay we found that Ube2w can monoubiquitinate Cbl and increase autoubiquitination of Cbl mediated by ube2d2. Surprisingly, we found that knockdown of Ube2e increases downregulation and ubiquitination of EGFR in HeLa cells. Mechanistically we found that three Ube2e members inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Further, we showed that Ube2e does not affect ubiquitin charging of Ube2d2 by the ubiquitin-activating enzyme (E1) in vitro. This suggests that Ube2e does not compete with Ube2d2 for the E1 under these conditions. Thus, our data suggest that Ube2e acts as a positive modulator of EGFR signaling by competing for Cbl with Ube2d2 and thus prevents ubiquitination and downregulation of the EGFR by Cbl in combination with Ube2d2. Together these data demonstrate that there is an E2 network which modulates the ubiquitination and downregulation of the EGFR by Cbl. Citation Format: Ke Ma, Philip Ryan, Rachel Klevit, Stanley Lipkowitz. Ube2d family members, Ube2e family members and Ube2w modulate the ubiquitination and degradation of EGFR by Cbl. [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 4542.

Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.

Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved, depicting the protein at different stages of its activation cycle and thus providing mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins-may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than random noncancer mutations. We further tested the ability of these computational models, assessing the changes in CBL stability and its binding to ubiquitin-conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL.

Assess the expression of ubiquitin specific protease USP2a for bladder cancer diagnosis.

BackgroundGiven that a deubiquitinating enzyme, ubiquitin-specific protease 2a (USP2a), regulates ubiquitination, trafficking, and degradation of EGFR, which plays a critical role in bladder cancer, in this study, we aimed to quantify the USP2a gene expression, and to determine the possibility that USP2a can be used for bladder cancer diagnosis.MethodsUsing two independent cohorts (cohort 1, n = 339 in total; cohort 2, n = 140 in total) consisting of human bladder tissues from BC patients and normal controls, we analyzed the gene expression levels of USP2a. A quantitative real-time PCR amplification was performed using a Rotor Gene 6000 instrument to quantify the expression of USP2a mRNA.ResultsA comparison of 305 bladder cancers and 34 age-matched controls showed an 81.4 % reduction in USP2a expression in bladder cancers as compared to normal bladder tissues (p < 0.001). In the independent cohort consisting of 140 BC tissues and matched adjacent normal bladder tissues, the levels of USP2a in the specimens of BC patients were reduced by 86.9 % as compared to matched surrounding normal specimens from the same patients (p < 0.001). Furthermore, there was 36.3 % reduction of USP2a gene expression in muscle invasive bladder cancer (MIBC, n = 121), compared to non muscle invasive bladder cancer (NMIBC, n = 184) (p = 0.004). Lastly, USP2a mRNA expression was significantly reduced in higher stages of MIBC patients (p = 0.024), but not in NMIBC patients.ConclusionsOur findings suggest that USP2a mRNA may be considered as a diagnostic marker candidate for bladder cancer, in particular, to stratify MIBC patients with a more invasive phenotype.

Abstract 4965: Multiple ubiquitin-conjugating enzymes modulate the ubiquitination and downregulation of the EGFR by the Cbl RING finger ubiquitin ligase

Abstract Receptor tyrosine kinases (RTKs) are known drivers of malignant transformation. Many RTKs (e.g., EGFR, MET) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins, a family of RING finger (RF) ubiquitin ligases (E3s). Loss of Cbl protein function is associated with malignant transformation driven by increased RTK activity. E3s such as the Cbl proteins confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the target proteins in collaboration with ubiquitin-conjugating enzymes (E2s). We used enzymatic and yeast two-hybrid assays to characterize the E2s that can interact with Cbl proteins. Using an in vitro E3 assay, we found that only the Ube2d family of E2s mediates autoubiquitination of the Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that Ube2e1, Ube2e2, Ube2e3, Ube2l3, Ube2u, Ube2w and Ube2z can interact with Cbl even though they do not support autoubiquitination of Cbl in the in vitro E3 assay. Among these E2s, three Ube2e family members and Ube2w bind to the RF domain of Cbl as demonstrated by the loss of interaction when the RF domain is disrupted. This suggests that Ube2e and Ube2w are relevant to the ubiquitination and degradation of substrates by Cbl. Knockdown of Ube2w decreases downregulation of EGFR in Hela cells. In the in vitro E3 assay we found that Ube2w can increase autoubiquitination of Cbl mediated by ube2d2. Surprisingly, we found that knockdown of Ube2e increases downregulation and ubiquitination of EGFR in HeLa cells. Mechanistically we found that three Ube2e members inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Further, we showed that Ube2e does not affect ubiquitin charging of Ube2d2 by the ubiquitin-activating enzyme (E1) in vitro. This suggests that Ube2e does not compete with Ube2d2 for the E1 under these conditions. Thus, our data suggest that Ube2e acts as a positive modulator of EGFR signaling by competing for Cbl with Ube2d2 and thus prevents ubiquitination and downregulation of the EGFR by Cbl in combination with Ube2d2. Together these data demonstrate that there is an E2 network which modulates the ubiquitination and downregulation of the EGFR by Cbl. Citation Format: Ke Ma, Philip Ryan, Rachel Klevit, Stanley Lipkowitz. Multiple ubiquitin-conjugating enzymes modulate the ubiquitination and downregulation of the EGFR by the Cbl RING finger ubiquitin ligase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4965. doi:10.1158/1538-7445.AM2015-4965

Abstract 4361: Efficacy of MET inhibitors in NSCLC with CBL alterations

Abstract Purpose/Objective(s): Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase and adaptor molecule that is important in cancer. It belongs in the family with CBL-B and CBL-3. Our previous studies detected CBL mutations, loss of heterozygosity, and low protein expression in non-small cell lung cancer (NSCLC). We also determined the genetic variations of CBL, their relationship to receptor tyrosine kinases (RTK) such as EGFR and MET, and their functionality in NSCLC. However, ubiquitination of EGFR showed no difference between CBL wild type (WT) and mutants (Mts). Therefore, we undertook to investigate whether MET is a potential target of CBL and better therapeutic strategy. Materials/ Methods: CBL alterations were determined in NSCLC patients with next generation sequencing. Immunoprecipitation was performed to detect the ubiquitination of MET by CBL WT and Mts. MET inhibitor SU11274 was utilized to compare cell viability of CBL WT cells with viability of CBL Mt cells. Cell motility after SU11274 treatment was examined by wound healing assay. Soft agar assay was performed to investigate colony formation of shRNA knockdown (sh-CBL) cells and SU11274 treatment. PamGene Chip analysis was used to detect and predict the RTK phosphorylation difference between CBL WT and Mt/sh-CBL cells. In vivo mouse study was performed to examine tumor growth and metastasis of sh-CBL cells. Results: CBL had 6% (8/135) mutation rate in NSCLC patient population. When CBL and MET protein expression levels were analyzed by immunoblot, CBL WT cells had lower MET protein expression than CBL Mt cells. The ubiquitination of MET was decreased in cells that transiently expressed CBL Mt relative to CBL WT cells. In addition, CBL Mt cells were more sensitive to MET inhibitor SU11274 than CBL WT cells. Wound healing assay demonstrated sh-CBL cells had an increase in cell motility and more sensitive with SU11274 migration inhibition. Furthermore, soft agar assay showed an increase in colony number and size of sh-CBL cells compared to control cells. After treatment with SU11274, sh-CBL cells showed inhibition in colony formation. In vivo study showed sh-CBL cells had lower tumor growth but more metastasis than control cells. PamGene results predicted an effective cell signaling pathway involving MET, paxillin, EPHA2, and VEGFR.. Conclusions: Ubiquitination assay result suggested MET is one of direct targets of CBL. The cell viability and motility results demonstrated that CBL mutants and sh-CBL cells are more sensitive than CBL WT cells to MET inhibitor SU11274, suggesting they have higher MET protein expression. Moreover, the PamGene results indicated that MET is involved in CBL cell signaling pathway. Thus, CBL gene status could be a potential indication for lung cancer therapy utilizing MET and other inhibitors. Citation Format: Yi-Hung Carol Tan, Cleo Rolle, Li Zhu, Minu K. Srivastava, Sherven Sharma, Ravi Salgia. Efficacy of MET inhibitors in NSCLC with CBL alterations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4361. doi:10.1158/1538-7445.AM2015-4361

Efficacy of MET Inhibitors in NSCLC With CBL Alterations: Novel Therapies/Experimental Therapies

Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase and adaptor molecule that is important in cancer. It belongs in the family with CBL-B and CBL-3. Our previous studies detected CBL mutations, loss of heterozygosity, and low protein expression in non-small cell lung cancer (NSCLC). We also determined the genetic variations of CBL, their relationship to receptor tyrosine kinases (RTK) such as EGFR and MET, and their functionality in NSCLC. However, ubiquitination of EGFR showed no difference between CBL wild type (WT) and mutants (Mts).

Overcoming acquired resistance to cetuximab by dual targeting HER family receptors with antibody-based therapy.

BackgroundCetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers. However, many patients who initially respond to cetuximab acquire resistance. To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (CtxR) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226. Previous studies characterizing this model revealed that: 1) EGFR was robustly overexpressed in CtxR clones due to decreased EGFR ubiquitination and degradation and 2) CtxR clones expressed increased HER2 and HER3 activation resulting in constitutive activation of the PI3K/AKT signaling axis. These findings suggest that dual targeting HER family receptors would be highly beneficial in the CtxR setting.ResultsSince HER3 has been implicated in resistance to EGFR inhibitors, the efficacy of dually targeting both EGFR and HER3 in CtxR models was evaluated. First, EGFR and HER3 expression were knocked down with siRNAs. Compared to the CtxS parental cell line (HP), all CtxR clones exhibited robust decreases in cell proliferation upon dual knockdown. Analysis of CtxR clones indicated that neuregulin-1 was highly overexpressed compared to HP cells. Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab. Next, dual treatment of CtxR clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects. Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT. Treatment with both mAbs resulted in knockdown of both signaling pathways simultaneously. HER2 was also strongly inactivated upon dual mAb therapy, suggesting that this treatment regimen can diminish signaling from three HER family receptors. De novo CtxR H226 mouse xenografts were established to determine if dual therapy could overcome acquired resistance to cetuximab in vivo. Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only. Combinatorial-treated xenograft tumors expressed decreased Ki67 and increased cleaved caspase-3 levels compared to tumors treated with either monotherapy.ConclusionsThese studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis

Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of 125I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl–CIN85–endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.

Abstract 4441: Ube2e inhibits the ubiquitination and degradation of EGFR mediated by Cbl and Ube2d

Abstract Receptor tyrosine kinases (RTKs) have a demonstrated role as drivers of malignant transformation. Many RTKs (e.g., EGFR, MET, FLT3) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins a family of RING finger (RF) ubiquitin ligases (E3s). There are three mammalian family members Cbl, Cbl-b and Cbl-c. All Cbl proteins have a highly conserved N-terminal tyrosine kinase binding domain and a C3HC4 (RF) domain. Loss of Cbl protein function has be associated with malignant transformation drive by increased RTK activity. E3s such as the Cbl proteins confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the specific target proteins in collaboration with ubiquitin-conjugating enzymes (E2s). The E2s bind to the E3 and together they mediate ubiquitination of specific substrates. There are approximately 30 human E2s. All of the E2s have a highly conserved catalytic domain (UBC domain). In this study, we characterized the interaction of Cbl with E2s. Using an in vitro E3 assay, we found that only the Ube2d family (Ube2d1, Ube2d2, and Ube2d3) of E2s mediates autoubiquitination of the three Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that Ube2e1, Ube2e2, Ube2e3, Ube2L3, Ube2u, Ube2w and Ube2z can interact with Cbl even though they do not support autoubiquitination of Cbl in vitro E3 assay. Among these E2s, three Ube2e family members and Ube2w bind to the RF domain of Cbl as demonstrated by the loss of interaction when the RF domain is disrupted. Further, we demonstrated that Ube2e family members are involved in regulation of EGFR activity mediated by Cbl in HeLa cells. Knockdown of Ube2e increases down-regulation and ubiquitination of EGFR in HeLa cells. We further showed that three Ube2e inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Together these data suggest that RTK degradation can be modulated by competition between E2s for the Cbl E3s. Citation Format: Ke Ma, Stanley Lipkowitz, Rachel Klevit, Mariya Sergeyevna Liyasova. Ube2e inhibits the ubiquitination and degradation of EGFR mediated by Cbl and Ube2d. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4441. doi:10.1158/1538-7445.AM2014-4441

A Novel Integrated VMAT/IMRT Technique for the Treatment of Non-Small Cell Lung Cancer

Purpose/Objective(s): Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase and adaptor molecule that is important in cancer. It belongs in the family with CBL-B and CBL-3. Our previous studies detected CBL mutations, loss of heterozygosity, and low protein expression in nonsmall cell lung cancer (NSCLC). We also determined the genetic variations of CBL, their relationship to receptor tyrosine kinases (RTK) such as EGFR and MET, and their functionality in NSCLC. However, ubiquitination of EGFR showed no difference between CBL wild type (WT) and mutants (Mts). Therefore, we undertook to investigate whether MET is a potential target of CBL and better therapeutic strategy. Materials/Methods: CBL alterations were determined in NSCLC patients with next generation sequencing. Immunoprecipitation was performed to detect the ubiquitination of MET by CBL WT and Mts. MET inhibitor SU11274 was utilized to compare cell viability of CBL WT cells with viability of CBL Mt cells. Cell motility after SU11274 treatment was examined by wound healing assay. Soft agar assay was performed to investigate colony formation of shRNA knockdown (sh-CBL) cells and SU11274 treatment. PamGene Chip analysis was used to detect and predict the RTK phosphorylation difference between CBL WT and Mt/sh-CBL cells. In vivo mouse study was performed to examine tumor growth and metastasis of sh-CBL cells. Results: CBL had 6% (8/135) mutation rate in NSCLC patient population. When CBL and MET protein expression levels were analyzed by immunoblot, CBL WT cells had lower MET protein expression than CBL Mt cells. The ubiquitination of MET was decreased in cells that transiently expressed CBL Mt relative to CBL WT cells. In addition, CBL Mt cells were more sensitive toMET inhibitor SU11274 than CBLWT cells. Wound healing assay demonstrated sh-CBL cells had an increase in cell motility and more sensitive with SU11274 migration inhibition. Furthermore, soft agar assay showed an increase in colony number and size of sh-CBL cells compared to control cells. After treatment with SU11274, sh-CBL cells showed inhibition in colony formation. In vivo study showed sh-CBL cells had lower tumor growth but more metastasis than control cells. PamGene results predicted an effective cell signaling pathway involving MET, paxillin, EPHA2, and VEGFR. Conclusions: Ubiquitination assay result suggested MET is one of the direct targets of CBL. The cell viability and motility results demonstrated that CBL mutants and sh-CBL cells are more sensitive than CBLWT cells to MET inhibitor SU11274, suggesting they have higher MET protein expression. Moreover, the PamGene results indicated that MET is involved in CBL cell signaling pathway. Thus, CBL gene status could be a potential indication for lung cancer therapy utilizing MET and other inhibitors. Author Disclosure: Y.C. Tan: None. C. Rolle: None. L. Zhu: None. M.K. Srivastava: None. S. Sharma: None. R. Salgia: None.

Abstract A40: CTEN prolongs EGFR signaling by reducing ligand-induced EGFR degradation

Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase engages a vast array of signaling pathways regulating tissue development and homeostasis. The signal is normally induced by ligand binding (such as EGF) that activates the receptors and then the downstream molecules, which mediate cell fate decisions such as proliferation, migration, and differentiation. EGF binding not only turns on the EGFR activity; it also turns on the mechanisms to inactivate EGFR signaling. One way is the activation of c-Cbl, an E3 ubiquitin ligase that binds to tyrosine-phosphorylated EGFR and leads to ubiquitination of EGFR for lysosomal degradation. Dysregulated EGFR signaling either by up-regulation or aberrant activation of the receptor will trigger cellular events that contribute to tumor microenvironments and cancer formation. Focal adhesions connect extracellular matrix to cytoskeletal networks and play critical roles in cell adhesion, migration, proliferation, and survival. They also crosstalk with growth factor receptors, such as EGFR to elicit a wider range of cellular responses, and may function as biosensors for microenvironments. Cten (C-terminal tensin like) is a focal adhesion molecule known to regulate cell adhesion and migration. Previously, we had shown that cten was up-regulated by EGF and mediated EGF induced cell migration. To further investigate cten's role in EGFR signaling pathway, we examined cten's effect on EGFR protein stability. Cten significantly reduced ligand-induced EGFR degradation through decreasing EGFR ubiquitination. This was accomplished by binding to c-Cbl in a phosphotyrosine-SH2 dependent fashion. A functional SH2 domain of cten was essential for its activities in promoting tumor cell migration, invasion, and colony formation. In addition to cell culture systems, EGFR levels were markedly enhanced in a cten tissue-specific knockin mouse model. By analysis of cten and EGFR expressions in human cancer samples, we found a positive association between cten and EGFR protein levels. Since cten regulates the activated EGFR level and plays roles in tumor migration/invasion, targeting of cten might have a beneficial effect on EGFR targeted therapies. Citation Format: Shiao-Ya Hong, Yi-Ping Shih, Tianhong Li, Kermit Carraway, Su Hao Lo. CTEN prolongs EGFR signaling by reducing ligand-induced EGFR degradation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A40.

Abstract 5646: Overcoming acquired resistance to cetuximab by dual targeting of HER family members using antibody based therapy.

Abstract The EGFR is recognized as a key mediator of proliferation and progression in many human tumors and strategies to inhibit EGFR signaling have emerged as highly promising cancer therapy approaches. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to treatment. In an effort to examine mechanisms of acquired resistance to anti-EGFR monoclonal antibody we developed a series of cetuximab-resistant clones using the NSCLC line NCI-H226. During efforts to elucidate molecular pathways that are involved in acquired resistance to cetuximab we found that 1) EGFR ubiquitination and degradation is altered in cetuximab-resistant cells resulting in increased steady-state expression of the EGFR and 2) enhanced signaling from the EGFR to HER3 and the PI(3)K/Akt pathway indicating that activation of HER3 may lead to escape from cetuximab therapy. To investigate the role of HER3 in acquired resistance to cetuximab we used both RNAi and targeted approaches using the anit-HER3 antibody U3-1287/AMG888. siRNA directed against HER3 indicated that resistant cells had a strong dependency on HER3. Further, targeting HER3 using U3-1287/AMG888 showed a dose-dependent anti-proliferative response in resistant cells. In studies using siRNA against the EGFR and HER3 resulted in robust inhibition of cell proliferation than did silencing of either receptor alone in resistant cells. Comparable effects on cell proliferation were observed after combinatorial treatment of cells by the EGFR antibody cetuximab and the HER3 antibody U3-1287/AMG888. In addition, combined EGFR and HER3 inhibition by either siRNAs or antibodies decreased the phosphorylation of downstream signaling pathways AKT and MAPK. Annexin-V binding assays showed a significant increase in apoptosis in U3-1287/AMG888 plus cetuximab treated cells as compared to either agent alone. To determine if U3-1287/AMG888 could have therapeutic benefit as a combination treatment with cetuximab in vivo, mice harboring NCI-H226 xenografts will be treated with cetuximab for approximately 70-100 days until cetuximab-resistant tumors develop. At this time mice with resistant tumors will be divided into two groups that will be treated with both cetuximab and U3-1287/AMG888 or cetuximab alone. The work presented herein suggests that cells with acquired resistance to cetuximab maintain their dependence on EGFR and HER3 signaling via the AKT and MAPK pathways. Citation Format: Mari Iida, Toni M. Brand, Megan M. Starr, Neha Luthar, Matthew J. Wleklinski, Deric L. Wheeler. Overcoming acquired resistance to cetuximab by dual targeting of HER family members using antibody based therapy. [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 5646. doi:10.1158/1538-7445.AM2013-5646

Aneuploid human colonic epithelial cells are sensitive to AICAR-induced growth inhibition through EGFR degradation

Trisomy for chromosome 7 is frequently observed as an initiating event in sporadic colorectal cancer. Although unstable chromosome numbers and recurrent aneuploidies drive a large fraction of human cancers, targeted therapies selective to pre-neoplastic trisomic cells are non-existent. We have previously characterized a trisomy 7 cell line (1CT+7) spontaneously derived from normal diploid human colonic epithelial cells that aberrantly expresses the epidermal growth factor receptor (EGFR, chromosome 7p11). Recent studies identified AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) as a pharmacological inhibitor of aneuploid murine fibroblast proliferation. Here, we report that AICAR induces profound cytostatic and metabolic effects on 1CT+7 cells, but not on their isogenic diploid counterpart. Dose-response experiments indicate that 1CT+7 cells are fourfold preferentially sensitive to AICAR compared to diploid cells. Unexpectedly, treatment of 1CT+7 cells with AICAR led to a reversible 3.5-fold reduction (P=0.0025) in EGFR overexpression. AICAR-induced depletion of EGFR protein can be abrogated through inhibition of the proteasome with MG132. AICAR also heavily promoted EGFR ubiquitination in cell-based immunoprecipitation assays, suggesting enhanced degradation of EGFR protein mediated by the proteasome. Moreover, treatment with AICAR reduced EGFR protein levels in a panel of human colorectal cancer cells in vitro and in xenograft tumors in vivo. Our data collectively support the pharmacological compound AICAR as a novel inhibitor of EGFR protein abundance and as a potential anticancer agent for aneuploidy-driven colorectal cancer.

Abstract 3863: Aneuploid human colonic epithelial cells are sensitive to AICAR-induced growth inhibition through EGFR degradation

Abstract Trisomy for chromosome 7 is frequently observed as an initiating event in sporadic colorectal cancer. Although unstable chromosome numbers and recurrent aneuploidies drive a large fraction of human cancers, targeted therapies selective to pre-neoplastic trisomic cells are non-existent. We have previously characterized a trisomy 7 cell line (1CT+7) spontaneously derived from normal diploid human colonic epithelial cells that aberrantly express the epidermal growth factor receptor (EGFR, located on 7p11). Recent studies identified AICAR as a pharmacological inhibitor of aneuploid murine fibroblast proliferation. Here, we report that AICAR induces profound cytostatic and metabolic effects on 1CT+7 cells but not their isogenic diploid counterpart. Dose-response assays demonstrate that 1CT+7 cells are four-fold preferentially sensitive to AICAR compared to diploid cells. Unexpectedly, treatment of 1CT+7 cells with AICAR led to a reversible 3.5-fold reduction (p=0.0025) in EGFR overexpression. AICAR-induced depletion of EGFR protein can be abrogated through inhibition of the proteasome with MG132. AICAR also heavily promoted EGFR ubiquitination in cell-based immunoprecipitation assays, suggesting enhanced degradation of EGFR protein mediated by the proteasome. Moreover, treatment with AICAR reduced EGFR protein levels in a panel of human colorectal cancer cells in vitro and in xenograft tumors in vivo. Our data collectively supports the pharmacological compound AICAR as a novel inhibitor of EGFR protein abundance and as a potential therapeutic agent for aneuploidy-driven colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3863. doi:1538-7445.AM2012-3863

Abstract 1220: Enigma blocks Cbl-c-mediated RET ubiquitination and degradation

Abstract RET (rearranged during transfection) is an RTK that is activated by glial cell-derived neurotrophic factors (GDNF) and is critical to neuronal and kidney development. RET mutations causing loss of function are associated with Hirschsprung's disease, while gain of function mutations are associated with thyroid carcinomas and mutiple endocrine neoplasias (MEN2A&amp;B). Cbl proteins are a family of ubiquitin protein ligases that mediate RTK degradation and thereby serve as negative regulators of RTK signaling. Cbl-c (a.k.a. Cbl-3), the most recently identified of the human Cbl proteins, is restricted to epithelial cells and most resembles the Cbl proteins found in lower organisms. Cbl-c can ubiquitinate and downregulate RTKs including activated RET and EGFR. Through yeast two-hybrid screening, we identified the LIM protein Enigma (a.k.a. PDLIM7) as a Cbl-c interacting protein. Enigma is an adapter protein for kinase anchoring to the cytoskeleton and is required for the mitogenic signaling of the RET/PTC2 oncogene. The interaction was confirmed in mammalian cells through co-immunoprecipitation of transfected and endogenous Cbl-c and Enigma. Enigma did not co-immunoprecipitate with c-Cbl or Cbl-b indicating that this interaction is specific to Cbl-c. Cbl-c can ubiquitinate and downregulate an activated form of RET, RET-MEN2A and we show that Enigma prevents Cbl-c-mediated RET ubiquitination and downregulation. This effect is specific to RET as EGFR ubiquitination was unaffected by the presence of Enigma. An Enigma mutant, lacking the RET binding site, does not block Cbl-c recruitment and subsequent ubiquitination and downregulation of RET. Our data suggest that Enigma blocks Cbl-c binding to the RET receptor and prevents its ubiquitination. This is further supported by microscopy data which demonstrates maintained surface expression of RET in the presence of Enigma. Together these data demonstrate a novel interaction between a positive and negative regulator or RET signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1220. doi:10.1158/1538-7445.AM2011-1220