Honokiol Prevents Non-Alcoholic Steatohepatitis-Induced Liver Cancer via EGFR Degradation through the Glucocorticoid Receptor-MIG6 Axis.

Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5DeltaGAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.

Disclosure: A. Ocampo: None. P.D. Bagamasbad: None. In breast cancer (BCa), glucocorticoid (GC) therapy is used in anti-emetic and palliative care. However, the effects of GC therapy in BCa are conflicting as it is beneficial for hormone-responsive luminal subtypes while it promotes tumor progression in more aggressive triple-negative BCa (TNBCs). A factor implicated in this paradoxical shift is the feedback inhibitor of epidermal growth factor receptor (EGFR) signaling, ERBB receptor feedback inhibitor 1 (ERRFI1). In BCa cell lines, ERRFI1 expression is directly regulated by GC receptor (GR). More interestingly, ERRFI1 was found to restrict the GC-enhanced proliferation in normal breast epithelia but enhances the GC-mediated pro-tumorigenic effects in highly aggressive TNBC. To identify the mechanism behind the shift in ERFFI1 function through BCa progression, ERRFI1 protein interactions in the luminal, HER2, and TNBC subtypes were analyzed by identifying differentially expressed proteins from each subtype using available proteome and interactome datasets from Integrated Interactions Database and followed by network and clustering analyses through STRING. The ERRFI1 interactome varied across the subtypes, and gene ontology analysis of the protein networks revealed progressive tumorigenic effects from the least aggressive luminal subtype to the most aggressive TNBC. The luminal BCa-ERRFI1 interactome showed metastatic potential that is countered by negative regulation of receptor tyrosine kinase (RTK) signaling. The HER2-ERRFI1 interactome highlighted the amplification of RTK signaling and its possible modulation through SRC while the TNBC-ERRFI1 interactome presents pro-oncogenic proteins implicated in drug resistance, angiogenesis, and EMT. Interestingly, the interactome also revealed a link between EGFR signaling and GR signaling possibly through ERRFI1, EGFR, and SFN. To determine the function of ERFFI1 in EGFR signaling in BCa, we generated stable ERRFI1 knockdown in different breast epithelial cell models through lentiviral transduction. Cells were then treated with EGF followed by gene expression analysis of known EGF-upregulated genes such as MYC, GSK3B, MCL1, and MMP9. We found that ERRFI1 knockdown enhanced the EGF-dependent induction of MYC and MMP9 in normal MCF10A cells and this effect was not observed in the TNBC MDA-MB-468 line, suggesting the loss of ERRFI1-mediated inhibition of EGFR signaling with BCa tumorigenic potential. Taken together, our findings suggest that the progressive loss of anti-tumorigenic function of ERRFI1 may be explained by its changing interactome in BCa molecular subtypes. Presentation: 6/3/2024

Disclosure: A. Ocampo: None. P.D. Bagamasbad: None. In breast cancer (BCa), glucocorticoid (GC) therapy is used in anti-emetic and palliative care. However, the effects of GC therapy in BCa are conflicting as it is beneficial for hormone-responsive luminal subtypes while it promotes tumor progression in more aggressive triple-negative BCa (TNBCs). A factor implicated in this paradoxical shift is the feedback inhibitor of epidermal growth factor receptor (EGFR) signaling, ERBB receptor feedback inhibitor 1 (ERRFI1). In BCa cell lines, ERRFI1 expression is directly regulated by GC receptor (GR). More interestingly, ERRFI1 was found to restrict the GC-enhanced proliferation in normal breast epithelia but enhances the GC-mediated pro-tumorigenic effects in highly aggressive TNBC. To identify the mechanism behind the shift in ERFFI1 function through BCa progression, ERRFI1 protein interactions in the luminal, HER2, and TNBC subtypes were analyzed by identifying differentially expressed proteins from each subtype using available proteome and interactome datasets from Integrated Interactions Database and followed by network and clustering analyses through STRING. The ERRFI1 interactome varied across the subtypes, and gene ontology analysis of the protein networks revealed progressive tumorigenic effects from the least aggressive luminal subtype to the most aggressive TNBC. The luminal BCa-ERRFI1 interactome showed metastatic potential that is countered by negative regulation of receptor tyrosine kinase (RTK) signaling. The HER2-ERRFI1 interactome highlighted the amplification of RTK signaling and its possible modulation through SRC while the TNBC-ERRFI1 interactome presents pro-oncogenic proteins implicated in drug resistance, angiogenesis, and EMT. Interestingly, the interactome also revealed a link between EGFR signaling and GR signaling possibly through ERRFI1, EGFR, and SFN. To determine the function of ERFFI1 in EGFR signaling in BCa, we generated stable ERRFI1 knockdown in different breast epithelial cell models through lentiviral transduction. Cells were then treated with EGF followed by gene expression analysis of known EGF-upregulated genes such as MYC, GSK3B, MCL1, and MMP9. We found that ERRFI1 knockdown enhanced the EGF-dependent induction of MYC and MMP9 in normal MCF10A cells and this effect was not observed in the TNBC MDA-MB-468 line, suggesting the loss of ERRFI1-mediated inhibition of EGFR signaling with BCa tumorigenic potential. Taken together, our findings suggest that the progressive loss of anti-tumorigenic function of ERRFI1 may be explained by its changing interactome in BCa molecular subtypes. Presentation: 6/3/2024

Activated epidermal growth factor receptor (EGFR) continues to signal in the early endosome, but how this signaling process is regulated is less well understood. Here we describe a protein complex consisting of TIP30, endophilin B1, and acyl-CoA synthetase long chain family member 4 (ACSL4) that interacts with Rab5a and regulates EGFR endocytosis and signaling. These proteins are required for the proper endocytic trafficking of EGF-EGFR. Knockdown of TIP30, ACSL4, endophilin B1, or Rab5a in human liver cancer cells or genetic knock-out of Tip30 in mouse primary hepatocytes results in the trapping of EGF-EGFR complexes in early endosomes, leading to delayed EGFR degradation and prolonged EGFR signaling. Furthermore, we show that Rab5a colocalizes with vacuolar (H(+))-ATPases (V-ATPases) on transport vesicles. The TIP30 complex facilitates trafficking of Rab5a and V-ATPases to EEA1-positive endosomes in response to EGF. Together, these results suggest that this TIP30 complex regulates EGFR endocytosis by facilitating the transport of V-ATPases from trans-Golgi network to early endosomes.

Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase delta (DGKdelta), a lipid kinase that terminates diacylglycerol signaling. In DGKdelta-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta-deficient cells. Moreover, the effects of PKCalpha were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKdelta and PKCalpha modulate the levels of ubiquitinated EGFR through Akt and USP8.

Resistance to anoikis, cell death due to matrix detachment, is acquired during tumor progression. The 14-3-3σ protein is implicated in the development of chemo- and radiation resistance, indicating a poor prognosis in multiple human cancers. However, its function in anoikis resistance and metastasis in hepatocellular carcinoma (HCC) is currently unknown.Methods: Protein expression levels of 14-3-3σ were measured in paired HCC and normal tissue samples using western blot and immunohistochemical (IHC) staining. Statistical analysis was performed to evaluate the clinical correlation between 14-3-3σ expression, clinicopathological features, and overall survival. Artificial modulation of 14-3-3σ (downregulation and overexpression) was performed to explore the role of 14-3-3σ in HCC anoikis resistance and tumor metastasis in vitro and in vivo. Association of 14-3-3σ with epidermal growth factor receptor (EGFR) was assayed by co-immunoprecipitation. Effects of ectopic 14-3-3σ expression or knockdown on EGFR signaling, ligand-induced EGFR degradation and ubiquitination were examined using immunoblotting and co-immunoprecipitation, immunofluorescence staining, and flow cytometry analysis. The levels of EGFR ubiquitination, the interaction between EGFR and 14-3-3σ, and the association of EGFR with c-Cbl after EGF stimulation, in 14-3-3σ overexpressing or knockdown cells were examined to elucidate the mechanism by which 14-3-3σ inhibits EGFR degradation. Using gain-of-function or loss-of-function strategies, we further investigated the role of the EGFR signaling pathway and its downstream target machinery in 14-3-3σ-mediated anoikis resistance of HCC cells.Results: We demonstrated that 14-3-3σ was upregulated in HCC tissues, whereby its overexpression was correlated with aggressive clinicopathological features and a poor prognosis. In vitro and in vivo experiments indicated that 14-3-3σ promoted anoikis resistance and metastasis of HCC cells. Mechanistically, we show that 14-3-3σ can interact with EGFR and significantly inhibit EGF-induced degradation of EGFR, stabilizing the activated receptor, and therefore prolong the activation of EGFR signaling. We demonstrated that 14-3-3σ downregulated ligand-induced EGFR degradation by inhibiting EGFR-c-Cbl association and subsequent c-Cbl-mediated EGFR ubiquitination. We further verified that activation of the ERK1/2 pathway was responsible for 14-3-3σ-mediated anoikis resistance of HCC cells. Moreover, EGFR inactivation could reverse the 14-3-3σ-mediated effects on ERK1/2 phosphorylation and anoikis resistance. Expression of 14-3-3σ and EGFR were found to be positively correlated in human HCC tissues.Conclusions: Our results indicate that 14-3-3σ plays a pivotal role in the anoikis resistance and metastasis of HCC cells, presumably by inhibiting EGFR degradation and regulating the activation of the EGFR-dependent ERK1/2 pathway. To our best knowledge, this is the first report of the role of 14-3-3σ in the anoikis resistance of HCC cells, offering new research directions for the treatment of metastatic cancer by targeting 14-3-3σ.

<div>Abstract<p>Cisplatin and its analogues are the most commonly used agents in the treatment of head and neck squamous cell carcinoma. In this study, we investigated a possible role of epidermal growth factor (EGF) receptor (EGFR) phosphorylation and degradation in cisplatin-induced cytotoxicity. Cisplatin treatment led to an increase in initial EGFR phosphorylation at Y1045, the binding site of ubiquitin ligase, Casitas B-lineage lymphoma (c-Cbl), followed by ubiquitination in the relatively cisplatin-sensitive cell lines. However, cisplatin-resistant cell lines underwent minimal EGFR phosphorylation at the Y1045 site and minimal ubiquitination. We found that EGFR degradation in response to cisplatin was highly correlated with cytotoxicity in seven head and neck cancer cell lines. Pretreatment with EGF enhanced cisplatin-induced EGFR degradation and cytotoxicity, whereas erlotinib pretreatment blocked EGFR phosphorylation, degradation, and cisplatin-induced cytotoxicity. Expression of a mutant Y1045F EGFR, which is relatively resistant to c-Cbl–mediated degradation, in Chinese hamster ovary cells and the UMSCC11B human head and neck cancer cell line protected EGFR from cisplatin-induced degradation and enhanced cell survival compared with wild-type (WT) EGFR. Transfection of WT c-Cbl enhanced EGFR degradation and cisplatin-induced cytotoxicity compared with control vector. These results show that cisplatin-induced EGFR phosphorylation and subsequent ubiquitination and degradation is an important determinant of cisplatin sensitivity. Our findings suggest that treatment with an EGFR inhibitor before cisplatin would be antagonistic, as EGFR inhibition would protect EGFR from cisplatin-mediated phosphorylation and subsequent ubiquitination and degradation, which may explain the negative results of several recent clinical trials. Furthermore, they suggest that EGFR degradation is worth exploring as an early biomarker of response and as a target to improve outcome. Cancer Res; 70(7); 2862–9</p></div>

Cisplatin and its analogues are the most commonly used agents in the treatment of head and neck squamous cell carcinoma. In this study, we investigated a possible role of epidermal growth factor (EGF) receptor (EGFR) phosphorylation and degradation in cisplatin-induced cytotoxicity. Cisplatin treatment led to an increase in initial EGFR phosphorylation at Y1045, the binding site of ubiquitin ligase, Casitas B-lineage lymphoma (c-Cbl), followed by ubiquitination in the relatively cisplatin-sensitive cell lines. However, cisplatin-resistant cell lines underwent minimal EGFR phosphorylation at the Y1045 site and minimal ubiquitination. We found that EGFR degradation in response to cisplatin was highly correlated with cytotoxicity in seven head and neck cancer cell lines. Pretreatment with EGF enhanced cisplatin-induced EGFR degradation and cytotoxicity, whereas erlotinib pretreatment blocked EGFR phosphorylation, degradation, and cisplatin-induced cytotoxicity. Expression of a mutant Y1045F EGFR, which is relatively resistant to c-Cbl-mediated degradation, in Chinese hamster ovary cells and the UMSCC11B human head and neck cancer cell line protected EGFR from cisplatin-induced degradation and enhanced cell survival compared with wild-type (WT) EGFR. Transfection of WT c-Cbl enhanced EGFR degradation and cisplatin-induced cytotoxicity compared with control vector. These results show that cisplatin-induced EGFR phosphorylation and subsequent ubiquitination and degradation is an important determinant of cisplatin sensitivity. Our findings suggest that treatment with an EGFR inhibitor before cisplatin would be antagonistic, as EGFR inhibition would protect EGFR from cisplatin-mediated phosphorylation and subsequent ubiquitination and degradation, which may explain the negative results of several recent clinical trials. Furthermore, they suggest that EGFR degradation is worth exploring as an early biomarker of response and as a target to improve outcome.

<div>Abstract<p>Cisplatin and its analogues are the most commonly used agents in the treatment of head and neck squamous cell carcinoma. In this study, we investigated a possible role of epidermal growth factor (EGF) receptor (EGFR) phosphorylation and degradation in cisplatin-induced cytotoxicity. Cisplatin treatment led to an increase in initial EGFR phosphorylation at Y1045, the binding site of ubiquitin ligase, Casitas B-lineage lymphoma (c-Cbl), followed by ubiquitination in the relatively cisplatin-sensitive cell lines. However, cisplatin-resistant cell lines underwent minimal EGFR phosphorylation at the Y1045 site and minimal ubiquitination. We found that EGFR degradation in response to cisplatin was highly correlated with cytotoxicity in seven head and neck cancer cell lines. Pretreatment with EGF enhanced cisplatin-induced EGFR degradation and cytotoxicity, whereas erlotinib pretreatment blocked EGFR phosphorylation, degradation, and cisplatin-induced cytotoxicity. Expression of a mutant Y1045F EGFR, which is relatively resistant to c-Cbl–mediated degradation, in Chinese hamster ovary cells and the UMSCC11B human head and neck cancer cell line protected EGFR from cisplatin-induced degradation and enhanced cell survival compared with wild-type (WT) EGFR. Transfection of WT c-Cbl enhanced EGFR degradation and cisplatin-induced cytotoxicity compared with control vector. These results show that cisplatin-induced EGFR phosphorylation and subsequent ubiquitination and degradation is an important determinant of cisplatin sensitivity. Our findings suggest that treatment with an EGFR inhibitor before cisplatin would be antagonistic, as EGFR inhibition would protect EGFR from cisplatin-mediated phosphorylation and subsequent ubiquitination and degradation, which may explain the negative results of several recent clinical trials. Furthermore, they suggest that EGFR degradation is worth exploring as an early biomarker of response and as a target to improve outcome. Cancer Res; 70(7); 2862–9</p></div>

The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.

Interdependent epidermal growth factor receptor signalling and trafficking

Ligand-mediated endocytosis is an intricate regulatory mechanism for epidermal growth factor receptor (EGFR) signal transduction. Coordinated trafficking of EGFR ensures its temporal and spatial communication with downstream signaling effectors. We focused our work on Rab5, a monomeric GTPase shown to participate in early stages of the endocytic pathway. Rab5 has three isoforms (A, B, and C) sharing more than 90% of sequence identity. We individually ablated endogenous isoforms in HeLa cells with short interfering RNAs and examined the loss-of-function phenotypes. We found that suppression of Rab5A or 5B hampered the degradation of EGFR, whereas Rab5C depletion had very little effect. The differential delay of EGFR degradation also corresponds with retarded progression of EGFR from early to late endosomes. We investigated the activators/effectors of Rab5A that can potentially separate its potency in EGFR degradation from other isoforms and found that Rin1, a Rab5 exchange factor, preferably associated with Rab5A. Moreover, Rab5A activation is sensitive to EGF stimulation, and suppression of Rin1 diminished this sensitivity. Based on our results together with previous work showing that Rin1 interacts with signal transducing adapter molecule to facilitate the degradation of EGFR (Kong, C., Su, X., Chen, P. I., and Stahl, P. D. (2007) J. Biol. Chem. 282, 15294-15301), we hypothesize that the selective association of Rab5A and Rin1 contributes to the dominance of Rab5A in EGFR trafficking, whereas the other isoforms may have major functions unrelated to the EGFR degradation pathway.

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

Approximately half of all basal type breast cancers show evidence of autocrine activation of the epidermal growth factor receptor (EGFR) by amphiregulin (AREG). In addition, AREG expression is associated with aggressive and chemoresistant forms of breast cancer. Studies in our laboratory show that when AREG is the activating ligand, EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To investigate the effect of AREG-mediated activation of EGFR on the phenotype of AREG-expressing cancers, AREG expression was knocked-down in SUM149 cells, a highly tumorigenic and metastatic cell line isolated from a basal subtype inflammatory breast cancer, which expresses high levels of AREG and overexpresses EGFR. Microarray analysis of AREG knockdown cells revealed a number of relevant pathways affected by AREG-EGFR signaling. These pathways include Notch signaling, MAPK activation, IL-1 signaling and multiple components of the Wnt signaling pathway. In particular, knockdown of AREG or inhibition of EGFR signaling in AREG stimulated cells resulted in the upregulation of DKK-1, an antagonist of canonical Wnt signaling, and the expression of several WNT target genes. Flow cytometric analysis of these cells demonstrated that AREG knockdown resulted in a significant reduction in CD44+ cells while simultaneously increasing the non-stem cell fraction of CD44-/CD24+ cells. Because CD44hi/CD24lo is characteristic of undifferentiated cells that have the potential to initiate tumor formation, the tumorigenicity of these cells was investigated by transplanting parental and AREG knock-down SUM149 cells into the mammary fat pads of NOD-SCID mice. The tumorigenic activity of the AREG knock-down derivatives was significantly reduced compared to the parental SUM149 cells. In particular, the AREG knockdown clone that expressed the lowest level of AREG expression and the highest level of DKK-1 expression failed to form tumors in NOD-SCID mice. To investigate the link between AREG-mediated activation of EGFR, receptor trafficking, and downstream signaling pathways that are responsible for changes in expression of WNT pathway genes, we are investigating differences in EGFR phosphorylation and downstream signaling stimulated by AREG versus EGF. In conjunction with our previous findings that AREG-mediated EGFR signaling results in activation of NF-κβ and the upregulation of IL1α/β, our current results suggest a link between these pathways, WNT signaling, and tumorigenicity in SUM-149 cells. Citation Format: Christiana S. Kappler, Stephen P. Ethier. Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in 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 530. doi:10.1158/1538-7445.AM2013-530

Studies on the differential routing of internalized epidermal growth factor receptors (EGFRs) induced by EGF, TGF alpha, and the superagonist EGF-TGF alpha chimera E4T suggested a correlation between receptor recycling and their mitogenic potency. EGFR sorting to lysosomes depends on its kinase domain and its ubiquitination by Cbl proteins. Proteasomes have also been proposed to regulate EGFR degradation, but the underlying mechanism remains obscure. Here we evaluated EGFR activation, Cbl recruitment, EGFR ubiquitination and degradation in response to EGF, TGF alpha, and E4T. We also determined the fate of activated EGFRs and Cbl proteins by using v-ATPase (bafilomycin A1) and proteasome (lactacystin) inhibitors. Our results demonstrate that E4T and TGF alpha provoke decreased Cbl recruitment, EGFR ubiquitination and EGFR degradation compared with EGF. Furthermore, bafilomycin treatment blocks EGFR but not c-Cbl degradation. In contrast, lactacystin treatment blocks EGF-induced c-Cbl degradation but does not block EGFR degradation, even though lactacystin causes a minor delay in EGFR degradation. Surprisingly, even though bafilomycin completely blocks EGFR degradation, it does not prevent EGFR de-ubiquitination upon prolonged EGF stimulation. Strikingly, when combined with bafilomycin, lactacystin treatment stabilizes the ubiquitinated EGFR and prevents its de-ubiquitination. We conclude that the enhanced EGFR recycling that has been observed in HER-14 cells following TGF alpha or E4T stimulation correlates with decreased EGFR ubiquitination and EGFR degradation, and that proteasomal activity is required for de-ubiquitination of the EGFR prior to its lysosomal degradation.