Epidermal Growth Factor Receptor Degradation Research Articles

PDZK1 suppresses TNBC development and sensitizes TNBC cells to erlotinib via the EGFR pathway

Epidermal growth factor receptor (EGFR)-targeted drugs (erlotinib, etc.) are used to treat multiple types of tumours. EGFR is highly expressed in most triple-negative breast cancer (TNBC) patients. However, only a small proportion of TNBC patients benefit from EGFR-targeted drugs in clinical trials, and the resistance mechanism is unclear. Here, we found that PDZ domain containing 1 (PDZK1) is downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation is related to erlotinib resistance in TNBC. PDZK1 binds to EGFR. Through this interaction, PDZK1 promotes EGFR degradation by enhancing the binding of EGFR to c-Cbl and inhibits EGFR phosphorylation by hindering EGFR dimerisation. We also found that PDZK1 is specifically downregulated in TNBC tissues and correlated with a poor prognosis in TNBC patients. In vitro and in vivo functional assays showed that PDZK1 suppressed TNBC development. Restoration of EGFR expression or kinase inhibitor treatment reversed the degree of cell malignancy induced by PDZK1 overexpression or knockdown, respectively. PDZK1 overexpression sensitised TNBC cells to erlotinib both in vitro and in vivo. In conclusion, PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for reversing erlotinib resistance in TNBC cells.

Hedgehog ligand and receptor cooperatively regulate EGFR stability and activity in non-small cell lung cancer.

The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth. Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting. Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth. Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease.

Targeting EGFR degradation by autophagosome degraders

Several generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been developed for the treatment of non-small cell lung cancer (NSCLC) in clinic. However, emerging drug resistance mediated by new EGFR mutations or activations by pass, leads to malignant progression of NSCLC. Proteolysis targeting chimeras (PROTACs) have been utilized to overcome the drug resistance acquired by mutant EGFR, newly potent and selective degraders are still need to be developed for clinical applications. Herein, we developed autophagosome-tethering compounds (ATTECs) in which EGFR can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of the LC3 ligand GW5074. A series of EGFR-ATTECs have been designed and synthesized. Biological evaluations showed that these compounds could degrade EGFR and exhibited moderate inhibitory effects on certain NSCLC cell lines. The ATTEC 12c potently induced the degradation of EGFR with a DC50 value of 0.98 μM and a Dmax value of 81% in HCC827 cells. Mechanistic exploration revealed that the lysosomal pathway was mainly involved in this degradation. Compound 12c also exhibited promising inhibitory activity, as well as degradation efficiency in vivo. Our study highlights that EGFR-ATTECs could be developed as a new expandable EGFR degradation tool and also reveals a novel potential therapeutic strategy to prevent drug resistance acquired EGFR mutations.

Abstract 549: PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation

Abstract Background: Triple-negative breast cancer (TNBC) is lack of effective targeted therapies. Epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. However, TNBC is resistant to EGFR-targeted drugs in clinical trials, and the tolerance mechanism remains unclear. This study aimed to elucidate the possible resistance mechanism for TNBC to EGFR-targeted therapy and how sensitize TNBC cells to erlotinib. Methods: PDZ domain containing 1 (PDZK1) correlated with TNBC development was screened out by bioinformatics analysis and its correlation with resistance to erlotinib was identified by western blotting (WB). Its interaction with EGFR and structural basis were determined by GST-Pull down and coimmunoprecipitation. Its regulation on EGFR signaling activation and expression level were detected by WB in PDZK1 overexpression and knockdown TNBC cells, xenograft tissues and correlation analyses. Correlation was performed by gene set enrichment analysis (GSEA) and immunohistochemistry of tissue microarray followed by Pearson analysis. TNBC-suppressing effects of PDZK1 and its sensitizing effects on erlotinib were investigated using cell viability assay, colony-forming assay, wound healing assay, Matrigel invasion assay and xenograft model in combination with PDZK1 wild type/mutant transfection and rescue experiment. Results: Here we found that PDZK1 was correlated with TNBC development and its level was downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation was related to erlotinib resistance in TNBC. PDZK1 bound with EGFR. Through the interaction, PDZK1 promoted EGFR degradation by enhancing the binding of EGFR with c-Cbl and inhibited EGFR phosphorylation by hindering EGFR dimerization. PDZK1 was specifically downregulated in TNBC tissues and was correlated with poor prognosis of TNBC. Functional assays in vitro and in vivo showed that PDZK1 suppressed TNBC development. Restoring EGFR expression and kinase inhibitor treatment reversed the malignancy caused by PDZK1 overexpression and knockdown, respectively. PDZK1 wild type, but not mutant overexpression enhanced the inhibitory effect of erlotinib on EGFR signaling and TNBC malignancy in vitro and in vivo. Conclusion: PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for TNBC to reverse the tolerance of TNBC cells to erlotinib. Citation Format: Junfang Zheng, Yuanzhen Ma, Zhiyu Fang, Yijun Qi. PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 549.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6956.

Abstract 1866: Discovery of mutation-independent EGFR degrading bispecific antibodies that suppress tumor growth in preclinical tumor models

Abstract Extracellular targeted protein degradation (eTPD) has emerged as a promising new drug modality focused on targeted elimination of extracellular and transmembrane proteins. In contrast to intracellular protein degraders, such as proteolysis targeting chimeras (PROTACs) and molecular glues which require ubiquitin-proteosome cellular degradation pathways, extracellular protein degraders can additionally harness endosomal-lysosomal protein degradation. Two recently published examples of extracellular protein degraders include AbTACs (antibody-based PROTAC) which co-engage a protein of interest (POI) and transmembrane E3 ligases, and KineTACs (cytokine receptor-targeting chimeras) which utilize endogenous cytokine receptors to degrade extracellular POIs. These bispecific antibody degrader platforms not only have advantageous pharmacological and drug-like manufacturing properties, but can also be engineered for tissue-specificity and to address multiple complementary targets, with the goal of increased efficacy and decreased toxicity. To that end, we have greatly expanded the extracellular degrader repertoire beyond AbTACs and KineTACs, with a novel bispecific antibody degrader platform called TrainTACs (tissue receptor antigen internalization targeting chimeras). To demonstrate the potential of this novel extracellular degrading platform, we developed degraders for the canonical receptor tyrosine kinase epidermal growth factor receptor (EGFR). EGFR is an oncogenic driver, that has been clinically validated in lung, colorectal and head and neck cancers, but patient benefit has been limited by treatment-related acquired resistance mutations and on-target/off-tumor dose-limiting toxicities. The potential of our TrainTACs, AbTACs and KineTACs to overcome the limitations of current therapeutic modalities was assessed. Gene expression profiles from tumor and normal tissues were evaluated to identify potential degraders co-expressed with EGFR in tumors. More than 70 unique bispecific antibody constructs spanning 20 receptors were generated and screened in tumor cell-based assays to evaluate EGFR antagonism, internalization, and degradation. TrainTACs, AbTACs and KineTACs degraded EGFR in multiple tumor cell lines covering a range of EGFR mutations. Degradation of EGFR led to deep inhibition of EGFR signaling, robust inhibitory effects on tumor spheroids, and in xenograft mouse tumor models. In conclusion, eTPD represents a promising new drug modality, and TrainTACs, AbTACs and KineTACs have expanded the toolbox of extracellular targeted protein degraders that can be utilized in a target-, tissue- and disease-specific manner. Citation Format: Jonathan Sitrin, Lisa Marshall, Hai Tran, Kenneth Ng, Kimberly Hoi, Josef Gramespacher, Zhong Huang, Andy Goodrich, Filomena Housley, May Dayao, Man-Tzu Wang, Katarina Pance, Aleysha Chen, Kevin Carlin, Lichao Zhang, James Lee, Rami Hannoush, Ken Flanagan, Maia Vinogradova, Isaac Rondon, Shyra Gardai. Discovery of mutation-independent EGFR degrading bispecific antibodies that suppress tumor growth in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1866.

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Targeted dual degradation of HER2 and EGFR obliterates oncogenic signaling, overcomes therapy resistance, and inhibits metastatic lesions in HER2-positive breast cancer models

AimsHuman epidermal growth factor receptor 2 (HER2) is an oncogenic receptor tyrosine kinase amplified in approximately 20% of breast cancer (BC). HER2-targeted therapies are the linchpin of treating HER2-positive BC. However, drug resistance is common, and the main resistance mechanism is unknown. We tested the hypothesis that drug resistance results mainly from inadequate or lack of inhibition of HER2 and its family member epidermal growth factor receptor (EGFR). MethodsWe used clinically relevant cell and tumor models to assess the impact of targeted degradation of HER2 and EGFR on trastuzumab resistance. Trastuzumab is the most common clinically used HER2 inhibitor. Targeted degradation of HER2 and EGFR was achieved using recombinant human protein PEPDG278D, which binds to the extracellular domains of the receptors. siRNA knockdown was used to assess the relative importance of EGFR and HER2 in trastuzumab resistance. ResultsBoth HER2 and EGFR are overexpressed in all trastuzumab-resistant HER2-positive BC cell and tumor models and that all trastuzumab-resistant models are highly vulnerable to targeted degradation of HER2 and EGFR. Degradation of HER2 and EGFR induced by PEPDG278D causes extensive inhibition of oncogenic signaling in trastuzumab-resistant HER2-positive BC cells. This is accompanied by strong growth inhibition of cultured cells, orthotopic patient-derived xenografts, and metastatic lesions in the brain and lung of trastuzumab-resistant HER2-positive BC. siRNA knockdown indicates that eliminating both HER2 and EGFR is necessary to maximize therapeutic outcome. ConclusionsThis study unravels the therapeutic vulnerability of trastuzumab-resistant HER2-positive BC and shows that an agent that targets the degradation of both HER2 and EGFR is highly effective in overcoming drug resistance in this disease. The findings provide new insights and innovations for advancing treatment of drug-resistant HER2-positive breast cancer that remains an unmet problem.

SERPINE2 promotes liver cancer metastasis by inhibiting c-Cbl-mediated EGFR ubiquitination and degradation.

Liver cancer is a malignancy with high morbidity and mortality rates. Serpin family E member 2 (SERPINE2) has been reported to play a key role in the metastasis of many tumors. In this study, we aimed to investigate the potential mechanism of SERPINE2 in liver cancer metastasis. The Cancer Genome Atlas database (TCGA), including DNA methylation and transcriptome sequencing data, was utilized to identify the crucial oncogene associated with DNA methylation and cancer progression in liver cancer. Data from the TCGA and RNA sequencing for 94 pairs of liver cancer tissues were used to explore the correlation between SERPINE2 expression and clinical parameters of patients. DNA methylation sequencing was used to detect the DNA methylation levels in liver cancer tissues and cells. RNA sequencing, cytokine assays, immunoprecipitation (IP) and mass spectrometry (MS) assays, protein stability assays, and ubiquitination assays were performed to explore the regulatory mechanism of SERPINE2 in liver cancer metastasis. Patient-derived xenografts and tumor organoid models were established to determine the role of SERPINE2 in the treatment of liver cancer using sorafenib. Based on the public database screening, SERPINE2 was identified as a tumor promoter regulated by DNA methylation. SERPINE2 expression was significantly higher in liver cancer tissues and was associated with the dismal prognosis in patients with liver cancer. SERPINE2 promoted liver cancer metastasis by enhancing cell pseudopodia formation, cell adhesion, cancer-associated fibroblast activation, extracellular matrix remodeling, and angiogenesis. IP/MS assays confirmed that SERPINE2 activated epidermal growth factor receptor (EGFR) and its downstream signaling pathways by interacting with EGFR. Mechanistically, SERPINE2 inhibited EGFR ubiquitination and maintained its protein stability by competing with the E3 ubiquitin ligase, c-Cbl. Additionally, EGFR was activated in liver cancer cells after sorafenib treatment, and SERPINE2 knockdown-induced EGFR downregulation significantly enhanced the therapeutic efficacy of sorafenib against liver cancer. Furthermore, we found that SERPINE2 knockdown also had a sensitizing effect on lenvatinib treatment. SERPINE2 promoted liver cancer metastasis by preventing EGFR degradation via c-Cbl-mediated ubiquitination, suggesting that inhibition of the SERPINE2-EGFR axis may be a potential target for liver cancer treatment.

Key roles of autophagosome/endosome maturation mediated by Syntaxin17 in methamphetamine-induced neuronal damage in mice

BackgroundAutophagic defects are involved in Methamphetamine (Meth)-induced neurotoxicity. Syntaxin 17 (Stx17), a member of the SNARE protein family, participating in several stages of autophagy, including autophagosome-late endosome/lysosome fusion. However, the role of Stx17 and potential mechanisms in autophagic defects induced by Meth remain poorly understood.MethodsTo address the mechanism of Meth-induced cognitive impairment, the adenovirus (AV) and adeno-associated virus (AAV) were injected into the hippocampus for stereotaxis to overexpress Stx17 in vivo to examine the cognitive ability via morris water maze and novel object recognition. In molecular level, the synaptic injury and autophagic defects were evaluated. To address the Meth induced neuronal damage, the epidermal growth factor receptor (EGFR) degradation assay was performed to evaluate the degradability of the “cargos” mediated by Meth, and mechanistically, the maturation of the vesicles, including autophagosomes and endosomes, were validated by the Co-IP and the GTP-agarose affinity isolation assays.ResultsOverexpression of Stx17 in the hippocampus markedly rescued the Meth-induced cognitive impairment and synaptic loss. For endosomes, Meth exposure upregulated Rab5 expression and its guanine-nucleotide exchange factor (GEF) (immature endosome), with a commensurate decreased active form of Rab7 (Rab7-GTP) and impeded the binding of Rab7 to CCZ1 (mature endosome); for autophagosomes, Meth treatment elicited a dramatic reduction in the overlap between Stx17 and autophagosomes but increased the colocalization of ATG5 and autophagosomes (immature autophagosomes). After Stx17 overexpression, the Rab7-GTP levels in purified late endosomes were substantially increased in parallel with the elevated mature autophagosomes, facilitating cargo (Aβ42, p-tau, and EGFR) degradation in the vesicles, which finally ameliorated Meth-induced synaptic loss and memory deficits in mice.ConclusionStx17 decrease mediated by Meth contributes to vesicle fusion defects which may ascribe to the immature autophagosomes and endosomes, leading to autophagic dysfunction and finalizes neuronal damage and cognitive impairments. Therefore, targeting Stx17 may be a novel therapeutic strategy for Meth-induced neuronal injury.

Endosomal Arl4A attenuates EGFR degradation by binding to the ESCRT-II component VPS36

Ligand-induced epidermal growth factor receptor (EGFR) endocytosis followed by endosomal EGFR signaling and lysosomal degradation plays important roles in controlling multiple biological processes. ADP-ribosylation factor (Arf)-like protein 4 A (Arl4A) functions at the plasma membrane to mediate cytoskeletal remodeling and cell migration, whereas its localization at endosomal compartments remains functionally unknown. Here, we report that Arl4A attenuates EGFR degradation by binding to the endosomal sorting complex required for transport (ESCRT)-II component VPS36. Arl4A plays a role in prolonging the duration of EGFR ubiquitinylation and deterring endocytosed EGFR transport from endosomes to lysosomes under EGF stimulation. Mechanistically, the Arl4A-VPS36 direct interaction stabilizes VPS36 and ESCRT-III association, affecting subsequent recruitment of deubiquitinating-enzyme USP8 by CHMP2A. Impaired Arl4A-VPS36 interaction enhances EGFR degradation and clearance of EGFR ubiquitinylation. Together, we discover that Arl4A negatively regulates EGFR degradation by binding to VPS36 and attenuating ESCRT-mediated late endosomal EGFR sorting.

Cbl and Cbl-b independently regulate EGFR through distinct receptor interaction modes.

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant manner by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking, and signaling.

ESCRT-III Component CHMP4C Attenuates Cardiac Hypertrophy by Targeting the Endo-Lysosomal Degradation of EGFR.

Cardiac hypertrophy and subsequent heart failure impose a considerable burden on public health worldwide. Impaired protein degradation, especially endo-lysosome-mediated degradation of membrane proteins, is associated with cardiac hypertrophy progression. CHMP4C (charged multivesicular body protein 4C), a critical constituent of multivesicular bodies, is involved in cellular trafficking and signaling. However, the specific role of CHMP4C in the progression of cardiac hypertrophy remains largely unknown. Mouse models with CHMP4C knockout or cardiadc-specific overexpression were subjected to transverse aortic constriction surgery for 4 weeks. Cardiac morphology and function were assessed through histological staining and echocardiography. Confocal imaging and coimmunoprecipitation assays were performed to identify the direct target of CHMP4C. An EGFR (epidermal growth factor receptor) inhibitor was administrated to determine whether effects of CHMP4C on cardiac hypertrophy were EGFR dependent. CHMP4C was significantly upregulated in both pressure-overloaded mice and spontaneously hypertensive rats. Compared with wild-type mice, CHMP4C deficiency exacerbated transverse aortic constriction-induced cardiac hypertrophy, whereas CHMP4C overexpression in cardiomyocytes attenuated cardiac dysfunction. Mechanistically, the effect of CHMP4C on cardiac hypertrophy relied on the EGFR signaling pathway. Fluorescent staining and coimmunoprecipitation assays confirmed that CHMP4C interacts directly with EGFR and promotes lysosome-mediated degradation of activated EGFR, thus attenuating cardiac hypertrophy. Notably, an EGFR inhibitor canertinib counteracted the exacerbation of cardiac hypertrophy induced by CHMP4C knockdown in vitro and in vivo. CHMP4C represses cardiac hypertrophy by modulating lysosomal degradation of EGFR and is a potential therapeutic candidate for cardiac hypertrophy.

D-mannose induces TFE3-dependent lysosomal degradation of EGFR and inhibits the progression of NSCLC.

In non-small cell lung cancer (NSCLC), the overexpression or abnormal activation of epidermal growth factor receptor (EGFR) is associated with tumor progression and drug resistance. EGFR tyrosine kinase inhibitors (TKIs) are currently the first-line treatment of NSCLC. However, patients inevitably acquired EGFR TKIs resistance mutations, which led to disease progression, so it is urgent to find new treatment. Here, we report that D-mannose up-regulates lysosomal activity by enhancing TFE3-mediated lysosomal biogenesis, thereby increasing the degradation of EGFR and significantly down-regulating its protein level. Therefore, D-mannose significantly inhibited the proliferation, migration and invasion of wild-type EGFR (WT-EGFR) and EGFR mutant cells (E746-A750 deletion, L858R and T790M mutations) in vitro. Oral administration of D-mannose strongly inhibited tumor growth in mice, showing similar effects with osimertinib. Taken together, these data suggest that D-mannose may represent a new strategy for clinical treatment of NSCLC.

An EGCG Derivative in Combination with Nimotuzumab for the Treatment of Wild-Type EGFR NSCLC.

Inhibiting the tyrosine kinase activity of epidermal growth factor receptor (EGFR) using small-molecule tyrosine kinase inhibitors (TKIs) or monoclonal antibodies is often ineffective in treating cancers harboring wild-type EGFR. Given the fact that EGFR possesses a kinase-independent pro-survival function, more effective inhibition of EGFR-mediated signals is therefore necessary. In this study, we investigated the effects of using a combination of low-dose nimotuzumab and theasinensin A to evaluate whether the inhibitory effect of nimotuzumab on NCI-H441 cancer cells was enhanced. Here, theasinensin A, a novel epigallocatechin-3-gallate (EGCG) derivative, was identified and its potent anticancer activity against wild-type EGFR NSCLC was demonstrated in vitro; the anticancer activity was induced through degradation of EGFR. Mechanistic studies further revealed that theasinensin A bound directly to the EGFR extracellular domain, which decreased interaction with its ligand EGF in combination with nimotuzumab. Theasinensin A significantly promoted EGFR degradation and repressed downstream survival pathways in combination with nimotuzumab. Meanwhile, treatment with theasinensin A and nimotuzumab prevented xenograft growth, whereas the single agents had limited effect. Thus, the combination therapy of theasinensin A with nimotuzumab is a powerful candidate for treatment of wild-type EGFR cancers.

Downregulation of AP1S1 causes the lysosomal degradation of EGFR in non-small cell lung cancer.

Matrix stiffness has been shown to play a critical role in cancer progression by influencing various cellular processes, including epidermal growth factor (EGF) signaling. However, the underlying molecular mechanisms are not fully understood. Here, we investigated the role of adaptor-related protein complex 1 subunit sigma 1 (AP1S1), a component of adaptor protein complex-1, in the regulation of EGF receptor (EGFR) intracellular trafficking during cancer cell progression. We found that AP1S1 expression was upregulated under stiff matrix conditions, resulting in the regulation of EGFR trafficking in non-small cell lung adenocarcinoma cells. Knockout of AP1S1 caused the lysosomal degradation of EGFR, leading to suppressed EGF-induced anaplastic lymphoma receptor tyrosine kinase phosphorylation. In addition, the downregulation of AP1S1 increased the sensitivity of H1975 cancer cells, which are resistant to tyrosine kinase inhibitors, to erlotinib. Collectively, our results suggest that AP1S1 could regulate EGFR recycling under stiff matrix conditions, and AP1S1 inhibition could be a novel strategy for treating cancer cells resistant to EGFR-targeted anticancer drugs.

EGF, TGF-α and Amphiregulin Differently Regulate Endometrium-Derived Mesenchymal Stromal/Stem Cells.

The prototypical receptor tyrosine kinase epidermal growth factor receptor (EGFR) is regulated by a set of its ligands, which determines the specificity of signaling and intracellular fate of the receptor. The EGFR signaling system is well characterized in immortalized cell lines such as HeLa derived from tumor tissues, but much less is known about EGFR function in untransformed multipotent stromal/stem cells (MSCs). We compared the effect of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and amphiregulin (AREG) on physiological responses in endometrial MSCs (enMSC) and HeLa cells. In addition, using Western blotting and confocal microscopy, we studied the internalization and degradation of EGFR stimulated by the three ligands in these cell lines. We demonstrated that unlike HeLa, EGF and TGF-α, but not AREG, stimulated enMSC proliferation and prevented decidual differentiation in an EGFR-dependent manner. In HeLa cells, EGF targeted EGFR for degradation, while TGF-α stimulated its recycling. Surprisingly, in enMSC, both ligands caused EGFR degradation. In both cell lines, AREG-EGFR internalization was not registered. In HeLa cells, EGFR was degraded within 2 h, restoring its level in 24 h, while in enMSC, degradation took more than 4-8 h, and the low EGFR level persisted for several days. This indicates that EGFR homeostasis in MSCs may differ significantly from that in immortalized cell lines.

Identification of EGF Receptor and Thrombospondin-1 as Endogenous Targets of ER-Associated Degradation Enhancer EDEM1 in HeLa Cells.

Secretory and membrane proteins are vital for cell activities, including intra- and intercellular communication. Therefore, protein quality control in the endoplasmic reticulum (ER) is an essential and crucial process for eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) targets misfolded proteins during the protein maturation process in the ER and leads to their disposal. This process maintains the ER productive function and prevents misfolded protein stress (i.e., ER stress). The ERAD-stimulating factor ER degradation-enhancing α mannosidase-like 1 protein (EDEM1) acts on misfolded proteins to accelerate ERAD, thereby maintaining the productivity of the ER. However, the detail mechanism underlying the function of EDEM1 in ERAD is not completely understood due to a lack of established physiological substrate proteins. In this study, we attempted to identify substrate proteins for EDEM1 using siRNA. The matrix component thrombospondin-1 (TSP1) and epidermal growth factor receptor (EGFR) were identified as candidate targets of EDEM1. Their protein maturation status and cellular localization were markedly affected by knockdown of EDEM1. We also showed that EDEM1 physically associates with EGFR and enhances EGFR degradation via ERAD. Our data highlight the physiological role of EDEM1 in maintaining specific target proteins and provide a potential approach to the regulation of expression of clinically important proteins.

Influence of EGF and pro-NGF on EGFR/SORTILIN interaction and clinical impact in head and neck squamous cell carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) remains a cancer with a poor prognosis, with a 5-year survival rate of less than 50%. Although epidermal growth factor receptor (EGFR) is almost always overexpressed, targeted anti-EGFR therapies have modest efficacy and are mainly used in palliative care. Growth factors such as Nerve Growth Factor (NGF) and its precursor proNGF have been shown in our laboratory to play a role in tumor growth and aggressiveness. Interestingly, an interaction between Sortilin, a proNGF receptor, and EGFR has been observed. This interaction appears to interfere with the pro-oncogenic signaling of EGF and modulate the membrane expression of EGFR. The aim of this study was to characterize this interaction biologically, to assess its impact on clinical prognosis and to analyze its role in the cellular trafficking of EGFR. Using immunohistochemical staining on tumor sections from patients treated at our university center and PLA (Proximity Ligation Assay) labeling, we showed that Sortilin expression is significantly associated with reduced 5-year survival. However, when Sortilin was associated with EGFR, this association was not found. Using the Cal-27 and Cal-33 cancer cell lines, we observed that proNGF reduces the effects of EGF on cell growth by inducing the internalization of its receptor. These results therefore suggest a regulatory role for Sortilin in the degradation or renewal of EGFR on the membrane. It would be interesting in future work to show the intracellular fate of EGFR and the role of (pro)neurotrophins in these mechanisms.

EGFR-T790M Mutation–Derived Interactome Rerouted EGFR Translocation Contributing to Gefitinib Resistance in Non-Small Cell Lung Cancer

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer.