EGF-stimulated Epidermal Growth Factor Receptor Phosphorylation Research Articles

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation invitro and invivo.

Discovery of Cyclic Peptidomimetic Ligands Targeting the Extracellular Domain of EGFR.

It is very promising to target the extracellular domain of epidermal growth factor receptor (EGFR) for developing novel and selective anticancer therapies. Herein, we report the discovery of a novel small molecule, M-2-5, from a one-bead-two-compound (OBTC) cyclic γ-AApeptide library. The molecule was found to bind tightly to the extracellular domain of EGFR. Intriguingly, this molecule could also effectively antagonize EGF-stimulated EGFR phosphorylation and downstream signal transduction. Furthermore, together with its remarkable resistance to proteolytic degradation, M-2-5 was shown to effectively inhibit cell proliferation and migration in vitro and suppresses the growth of tumor in the A549 xenograft model in vivo, highlighting its potential therapeutic application for cancer treatment.

Enediyne-activated, EGFR-targeted human β-defensin 1 has therapeutic efficacy against non-small cell lung carcinoma

Human β-defensins contain an oncolytic motif that binds to tumor cell membranes and mediate permeabilization, rapid induction of cytolysis, and apoptosis. Previous studies have indicated that a fragment of the mature human β-defensin-1 (HBD1) peptide (DF) has antitumor properties. While targeted drug treatments using fusion proteins have been shown to increase drug efficacy, this phenomenon has not been studied for this defensin. Thus, in this study, we designed and prepared a fusion protein containing this HBD1 fragment and an epidermal growth factor receptor (EGFR)-targeting oligopeptide (Ec) as well as lidamycin (LDM), an extremely potent cytotoxic antitumor antibiotic, which consists of an apoprotein (LDP) and a highly active enediyne (AE). The fusion protein (Ec-LDP-DF) and its enediyne-integrated fusion protein (Ec-LDP(AE)-DF) were then purified and used to treat lung carcinoma cells in culture as well as lung carcinoma xenograft mouse models. The multifunctional fusion protein Ec-LDP-DF was shown to effectively bind to EGFR-expressing tumor cells. Furthermore, the enediyne-energized Ec-LDP(AE)-DF analog exhibited extremely potent cytotoxicity in NSCLC cell lines and an IC50 less than 10−10 mol/L. Ec-LDP(AE)-DF also significantly inhibited the growth of human carcinoma A549 and H460 xenografts in athymic mice at well-tolerated doses. Treatment resulted in cell cycle arrest and apoptosis in a dose-dependent manner. EGF-stimulated EGFR phosphorylation was also abolished by Ec-LDP(AE)-DF. In summary, our understanding of the role of defensins in cancer development and progression is continually expanding, and Ec-LDP(AE)-DF is a promising cancer cell-targeting agent for NSCLC.In this study, a novel epidermal growth factor receptor (EGFR)-targeted, human β-defensin 1-tailored fusion protein, Ec-LDP-DF, and its enediyne-integrated analogue, Ec-LDP(AE)-DF, have been prepared by genetic engineering and molecular reconstitution. The fusion protein Ec-LDP(AE)-DF displays extremely potent cytotoxicity and might be highly effective for non-small cell lung cancer therapy and useful for other EGFR-targeted therapeutics.

Selenoprotein W regulates epidermal growth factor receptor trafficking, expression, and activation (374.4)

Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that control growth, proliferation, differentiation, apoptosis, and cell migration. Selenoprotein W (SEPW1) is a highly conserved, ubiquitously expressed 9 kDa selenocysteine‐containing protein involved in cell cycle control. SEPW1 siRNA inhibited EGFR activation in RWPE‐1, MCF‐10A and MCF‐7 cells, while SEPW1 overexpression increased it. SEPW1 siRNA inhibited EGF‐induced activation of downstream pathways (MAPK, PI3K/Akt, JAK/STAT, and p53), and blocked EGF‐induced cell cycle entry. SEPW1 was associated with peripheral punctuate structures that partially co‐localized with EGFR. Ligand‐bound EGFR failed to traffic towards the nucleus in SEPW1‐depleted cells, indicating that SEPW1 has a role in EGFR trafficking. Consequently, the half‐life, expression, dimerization, and phosphorylation of EGFR were also decreased in SEPW1‐depleted cells. SEPW1 siRNA also inhibited hydrogen peroxide‐induced EGFR phosphorylation. However, SEPW1 siRNA inhibition of EGF‐stimulated EGFR phosphorylation was not decreased by N‐acetylcysteine or sodium orthovanadate, indicating SEPW1 regulation of EGFR does not involve reactive oxygen species or inhibition of protein tyrosine phosphatase. Glutathione peroxidase siRNA partially rescued EGF‐induced cell cycle entry in SEPW1‐depleted cells, showing the effect of SEPW1 depletion is not due to loss of antioxidant protection. Knowledge of the mechanisms underlying SEPW1 regulation of EGFR trafficking may be important to understanding the relationship between dietary selenium and cancer.Grant Funding Source: Supported by USDA CRIS Project 5306‐51530‐018‐00D

Extracellular epimorphin modulates epidermal differentiation signals mediated by epidermal growth factor receptor

BackgroundThe epidermal stratification/differentiation program is initiated in keratinocytes by a basement membrane-detachment cue and subsequently controlled by spatially and temporally regulated signaling molecules. The vital signals for the developmental behavior of the epidermis include those mediated by epidermal growth factor receptor (EGFR); however, regulatory elements responsible for activation have not yet been fully elucidated. ObjectiveThe objectives of this study were (1) to assess the effects of EGFR activation on epidermal differentiation, (2) to study the effects of epimorphin on the level of EGFR signaling degree dependent on matrix engagement, and (3) to address the impact of epimorphin modulation on EGFR-driven epidermal differentiation in a three-dimensional (3D) organotypic skin model. MethodsWe constructed skin-equivalent models with a well-stratified differentiated epidermis and utilized them to evaluate the epidermal behaviors. ResultsExtracellularly secreted epimorphin was identified as a strong candidate for signaling pathway involvement. In a 3D epidermis model, EGF stimulation was sufficient for epidermal stratification. However, overactivation of EGFR led to irregular multicellular arrangements with an abnormal differentiation profile, which appeared to be reorganized back to the normal epidermal phenotype by extracellular epimorphin. Extracellular epimorphin interestingly attenuated EGF-stimulated EGFR phosphorylation, cell growth, and migration in adherent cells. In contrast to the results of adhesion culture, extracellular epimorphin reinforced EGFR activation in suspended cells. ConclusionsThese results demonstrate that epimorphin modulates the signaling pathways mediated by EGFR for epidermal tissue organization.

Nucleobindin-2 Is a Positive Modulator of EGF-Dependent Signals Leading to Enhancement of Cell Growth and Suppression of Adipocyte Differentiation

Nucleobindin-2 is a 420-amino-acid EF-hand calcium-binding protein that undergoes proteolytic processing to generate an 82-amino-acid amino-terminal peptide termed nesfatin-1. To determine whether nucleobindin-2 has any biological function, nucleobindin-2 was either overexpressed or knocked down by short hairpin RNA in cultured CHO cells expressing the human insulin and epidermal growth factor (EGF) receptors (CHO/IE) and in 3T3-L1 cells. Reduction in nucleobindin-2 expression inhibited EGF-stimulated MAPK kinase (S217/S221) and Erk phosphorylation (T202/Y204). In contrast, there was no significant effect on EGF-stimulated EGF receptor phosphorylation, EGF receptor internalization, or 52-kDa Shc and c-Raf phosphorylation. Although kinase suppressor of Ras-1 and protein phosphatase 2A expression was not changed, intracellular calcium concentrations and PP2A activity was significantly increased in nucleobindin-2 knocked-down cells. Concomitant with these alterations in EGF-stimulated signaling, cell proliferation was significantly reduced in nucleobindin-2 knocked-down cells. Moreover, reduced nucleobindin-2 expression in 3T3-L1 preadipocytes resulted in a greater extent of 3T3-L1 cell adipocyte differentiation. Taken together, these data indicate that nucleobindin-2 regulates EGF-stimulated MAPK kinase/Erk signaling, cell proliferation, and adipocyte differentiation.

EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation

Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

S100A2 promoter-driven conditionally replicative adenovirus targets non-small-cell lung carcinoma

S100A2, a member of the S100 family of calcium-binding proteins, has been implicated in carcinogenesis as both a tumor suppressor and stimulator. Here, we characterized promoter activity of S100A2, generated an S100A2 promoter-driven conditionally replicative adenovirus (Ad/SA), and evaluated its anti-tumor activity in vitro and in vivo. Promoter activity of S100A2 was greatly restricted to tumor cells, and the S100A2 promoter bound with typical nuclear targets of epidermal growth factor receptor (EGFR) signaling. EGF-stimulated EGFR phosphorylation induced S100A2 expression and further activated E1A expression of Ad/SA, which was restored by EGFR signal inhibition in a concentration-dependent manner in non-small-cell lung carcinoma (NSCLC). In two EGFR-activated tumor xenograft animal models, Ad/SA exhibited potent anti-tumor activity, whereas cetuximab, an EGFR-targeting anticancer drug, was active transiently or ineffective. Combined treatment with cetuximab or cisplatin plus Ad/SA resulted in enhanced anti-tumor activity. Immunohistochemical analysis of tumor sections showed moderate-to-high grade signals for EGFR and adenovirus, and a reduction in viable cells in Ad/SA-treated tumors. Collectively, these results demonstrate that the S100A2 promoter-driven adenovirus is a potent inhibitor of cancers, and further suggest that S100A2 is a target gene of EGFR signaling pathway in NSCLC.

Abstract 1935: EBP50 inhibits breast cancer cell proliferation by blocking EGFR phosphorylation

Abstract EBP50 (Ezrin-radixin-moesin-binding phosphoprotein-50, also known as NHERF, NHERF1) has been reported to interact with epidermal growth factor receptor (EGFR) and enhance EGFR activity and its downstream ERK signaling in a cervical cancer cell line HeLa. However, our previous results demonstrated that EBP50 could suppress breast cancer cell proliferation, suggesting its inhibitory effect on EGFR signaling in breast cancer cell. So we detected the effect of EBP50 expression on EGF-induced cell proliferation and the activation of EGFR signaling in breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells which expressed low level of EBP50, EBP50 over-expression inhibited EGF-induced cell proliferation. In MCF-7 cells which expressed high level of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation. EBP50 was reported to interact with EGFR via its carboxyl terminal regulatory domain, which is adjacent to the autophosphorylation domain of the receptor, so it is possible that the association of EBP50 and EGFR mask the phosphorylation site of EGFR so as to prevent EGFR activation. To test this hypothesis, we detected the phosphorylation status of EGFR in the presence or absence of the EBP50 expression. Over-expression of EBP50 in MDA-MB-231 inhibits EGF-stimulated EGFR phosphorylation, and knockdown of EBP50 in MCF-7 cells led to the enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, the total expression levels of EGFR were not affected by the different expression level of EBP50 during EGF stimulation. In the case of EBP50 expression on the EGFR downstream signaling, EBP50 over-expression inhibited EGF-stimulated ERK activity in MDA-MB-231 cells, and EBP50 knockdown resulted in the enhanced EGF-stimulated ERK activity in MCF-7 cells. These data are consistent with results of MDA-MB-231 and MCF-7 cell lines pretreated with EGFR tyrosine kinase inhibitor-AG1478, that EGF-stimulated ERK activity was inhibited when EGFR phosphorylation was blocked. Taken together, our results demonstrated that EBP50 can suppress EGF-induced proliferation of breast cancer cells by retarding EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insights into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer. 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 1935. doi:10.1158/1538-7445.AM2011-1935

Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2(-/-), but not TNFR1(-/-), mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1(-/-) MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases.

Mechanism of action of a novel “combi-triazene” engineered to possess a polar functional group on the alkylating moiety: Evidence for enhancement of potency

Previous studies showed that SMA41, a 3-methyltriazene termed “combi-molecules” possessing a dual epidermal growth factor receptor (EGFR)/DNA targeting properties induced potent antiproliferative activity against alkylating-agent-resistant cells expressing EGFR in vitro. However, despite its marked potency, its antitumour activity in vivo was significantly hampered by its poor hydrosolubility and the moderate reactivity of its alkylating moiety. To circumvent this problem, we designed the quinazolinotriazene ZRBA1 to contain a N, N-dimethylaminoethyl group grafted to the 3-position of the triazene chain where it could serve both as a water soluble and a more potent alkylating moiety. ZRBA1 exhibited five-fold stronger EGFR tyrosine kinase (TK) inhibitory activity (IC 50 = 37 nM) than SMA41, decomposed into a 6-amino-quinazoline FD105 (IC 50 = 200 nM) and preferentially blocked EGF- over platelet-derived growth factor (PDGF)-or serum-induced cell growth. ZRBA1 induced DNA damage, concomitantly blocked EGF-stimulated EGFR phosphorylation by a partially irreversible mechanism in MDA-MB-468 breast cancer cells, and induced partially irreversible antiproliferative activity. It also prevented EGFR-mediated MAP kinase activation and, in contrast to FD105 and SMA41, induced high levels of apoptosis. Furthermore, ZRBA1 showed significantly greater antitumor activity ( p < 0.05) than SMA41 in the human MDA-MB-468 breast cancer xenograft model. The results in toto indicate that the appendage of N, N-dimethylaminoethyl to combi-triazenes may be an alternative to the reduced hydrosolubility and also to the lack of potency of monofunctional combi-triazenes against resistant tumours.

Insulin-like Growth Factor-binding Protein-3 Potentiates Epidermal Growth Factor Action in MCF-10A Mammary Epithelial Cells: INVOLVEMENT OF p44/42 AND p38 MITOGEN-ACTIVATED PROTEIN KINASES

Insulin-like growth factor-binding protein-3 (IGFBP-3) is inhibitory to the growth of many breast cancer cells in vitro; however, a high level of expression of IGFBP-3 in breast tumors correlates with poor prognosis, suggesting that IGFBP-3 may be associated with growth stimulation in some breast cancers. We have shown previously in MCF-10A breast epithelial cells that chronic activation of Ras-p44/42 mitogen-activated protein (MAP) kinase confers resistance to the growth-inhibitory effects of IGFBP-3 (Martin, J. L., and Baxter, R. C. (1999) J. Biol. Chem. 274, 16407-16411). Here we show that, in the same cell line, IGFBP-3 potentiates DNA synthesis and cell proliferation stimulated by epidermal growth factor (EGF), a potent activator of Ras. A mutant of IGFBP-3, which fails to translocate to the nucleus and has reduced ability to cell-associate, similarly enhanced EGF action in these cells. By contrast, the structurally related IGFBP-5, which shares many functional features with IGFBP-3, was slightly inhibitory to DNA synthesis in the presence of EGF. IGFBP-3 primes MCF-10A cells to respond to EGF because pre-incubation caused a similar degree of EGF potentiation as co-incubation. In IGFBP-3-primed cells, EGF-stimulated EGF receptor phosphorylation at Tyr-1068 was increased relative to unprimed cells, as was phosphorylation and activity of p44/42 and p38 MAP kinases, but not Akt/PKB. Partial blockade of the p44/42 and p38 MAP kinase pathways abolished the potentiation by IGFBP-3 of EGF-stimulated DNA synthesis. Collectively, these findings indicate that IGFBP-3 enhances EGF signaling and proliferative effects in breast epithelial cells via increased EGF receptor phosphorylation and activation of p44/42 and p38 MAP kinase signaling pathways.

Epidermal growth factor receptor activation in androgen-independent but not androgen-stimulated growth of human prostatic carcinoma cells

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.