Epidermal Growth Factor Receptor Recycling Research Articles

TMED2 promotes glioma tumorigenesis by being involved in EGFR recycling transport

Aberrant epidermal growth factor receptor (EGFR) signaling is the core signaling commonly activated in glioma. The transmembrane emp24 protein transport domain protein 2 (TMED2) interacts with cargo proteins involved in protein sorting and transport between endoplasmic reticulum (ER) and Golgi apparatus. In this study, we found the correlation between TMED2 with glioma progression and EGFR signaling through database analysis. Moreover, we demonstrated that TMED2 is essential for glioma cell proliferation, migration, and invasion at the cellular levels, as well as tumor formation in mouse models, underscoring its significance in the pathobiology of gliomas. Mechanistically, TMED2 was found to enhance EGFR-AKT signaling by facilitating EGFR recycling, thereby providing the initial evidence of TMED2's involvement in the membrane protein recycling process. In summary, our findings shed light on the roles and underlying mechanisms of TMED2 in the regulation of glioma tumorigenesis and EGFR signaling, suggesting that targeting TMED2 could emerge as a promising therapeutic strategy for gliomas and other tumors associated with aberrant EGFR signaling.

Leucine Zipper Downregulated in Cancer-1 Interacts with Clathrin Adaptors to Control Epidermal Growth Factor Receptor (EGFR) Internalization and Gefitinib Response in EGFR-Mutated Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). Clathrin-mediated internalization (CMI) sustains EGFR signaling. AXL is associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutated (EGFRM) NSCLC. We investigated the effects of Leucine zipper downregulated in cancer-1 (LDOC1) on EGFR CMI and NSCLC treatment. Coimmunoprecipitation, double immunofluorescence staining, confocal microscopy analysis, cell surface labelling assays, and immunohistochemistry studies were conducted. We revealed that LDOC1 interacts with clathrin adaptors through binding motifs. LDOC1 depletion promotes internalization and plasma membrane recycling of EGFR in EGFRM NSCLC PC9 and HCC827 cells. Membranous and cytoplasmic EGFR decreased and increased, respectively, in LDOC1 (-) NSCLC tumors. LDOC1 depletion enhanced and sustained activation of EGFR, AXL, and HER2 and enhanced activation of HER3 in PC9 and HCC827 cells. Sensitivity to first-generation EGFR-TKIs (gefitinib and erlotinib) was significantly reduced in LDOC1-depleted PC9 and HCC827 cells. Moreover, LDOC1 downregulation was significantly associated (p < 0.001) with poor overall survival in patients with EGFRM NSCLC receiving gefitinib (n = 100). In conclusion, LDOC1 may regulate the efficacy of first-generation EGFR-TKIs by participating in the CMI of EGFR. Accordingly, LDOC1 may function as a prognostic biomarker for EGFRM NSCLC.

Rab11 suppresses head and neck carcinoma by regulating EGFR and EpCAM exosome secretion

ObjectivesRab11(Rab11a and Rab11b) localizes primarily along recycling endosomes in cells and is involved in various intracellular trafficking processes, including membrane receptor recycling and secretion of exosomes or small extracellular vesicles (EVs). Although Rab11 is closely associated with the progression and metastasis of various cancer types, little is known about Rab11’ role in head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the roles of Rab11a and Rab11b in HNSCC. MethodsThe clinical significance of Rab11 expression in HNSCC was investigated using a public database and tissue microarray analysis. Stable cell lines with loss and gain of Rab11a or Rab11b were originally established to investigate their roles in the proliferative, migratory, and invasive capabilities of HNSCC cells. ResultsDatabase analysis revealed a significant association between Rab11b mRNA expression and a favorable patient survival rate in HNSCC. Tissue microarray analysis revealed that Rab11b expression was the highest in normal tissues and gradually decreased across the stages of HNSCC progression. Overexpression of Rab11a or Rab11b resulted in a decrease in epidermal growth factor receptor (EGFR), Epithelial cell adhesion molecule (EpCAM) exosome secretion, and the migratory and invasive potential of HNSCC cells. The knockdown of Rab11a or Rab11b increased EpCAM/CD9 exosome secretion in addition to the migratory and invasive potential of HNSCC cells. ConclusionsRab11 suppresses HNSCC by regulating EGFR recycling and EpCAM exosome secretion in HNSCC cells. Our results indicate that Rab11b is a superior prognostic indicator of HNSCC and holds promise for developing novel therapeutic strategies.

CASC4/GOLM2 drives high grade serous carcinoma anoikis resistance through the recycling of EGFR.

Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.

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.

Abstract 2551: CASC4 drives anoikis resistance in high grade serous ovarian cancer through recycling of EGFR

Abstract Introduction: Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian cancer (HGSOC) subtype accounts for almost 60% of ovarian cancers and is the deadliest. HGSOC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. Survival of tumor cells in the peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). CRISPR/Cas9 and transcriptomic screens identified the Golgi protein CASC4 (GOLM2, H63) as a “driver” of anoikis resistance. As CASC4 is highly uncharacterized in literature, we sought to determine how CASC4 confers anoikis resistance to HGSOC cells. Methods: Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse clinical outcomes, such as worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we used HGSOC cell lines PEO1, CaOV3, and OVCAR3 with shRNA-mediated CASC4 knockdowns (CASC4 KD), cultured in forced suspension. Results: Culturing cells in suspension, to recapitulate the peritoneal fluid environment in vitro, showed that CASC4 KD hampers cell proliferation and colony formation ability, and increases apoptosis. Additionally, a Reverse Phase Protein Assay (RPPA) showed that CASC4 KD results in significant changes in proteins known to be localized to the cell surface or secreted, including decreased protein levels of the receptor tyrosine kinase (RTK) Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways. A paralog of CASC4, GOLM1, is known to drive hepatocellular carcinoma through various mechanisms, such as the recycling of internalized RTKs, including EGFR. Indeed, our experiments showed that CASC4 KD in ovarian cancer cells leads to decreased EGFR levels and decreased EGFR recycling. Conclusion: Knocking down CASC4 results in decreased survivability in suspension, decreased EGFR levels at the plasma membrane, and decreased recycling of EGFR. Further elucidating mechanisms of CASC4-dependent anoikis resistance could lead to the development of novel therapeutic approaches, such as inhibitory peptides, and may assist in developing newer biomarkers for predicting ovarian cancer malignancy. Citation Format: Jaidev Bapat, Lindsay W. Brubaker, Zachary L. Watson, Tomomi M. Yamamoto, Benjamin G. Bitler. CASC4 drives anoikis resistance in high grade serous ovarian cancer through recycling of EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2551.

Opposite changes in the expression of clathrin and caveolin-1 in normal and cancerous human prostate tissue: putative clathrin-mediated recycling of EGFR

Endocytosis, an important macromolecule uptake process in cells, is known to be dysregulated in cancer. Clathrin and caveolin-1 proteins play a major role in receptor-mediated endocytosis. We have used a quantitative, unbiased and semi-automated method to measure in situ protein expression of clathrin and caveolin-1 in cancerous and paired normal (cancer adjacent, non-cancerous) human prostate tissue. There was a significant (p < 0.0001) increase in the expression of clathrin in prostate cancer samples (N = 29, n = 91) compared to normal tissue (N = 29, n = 67) (N = number of patients, n = number of cores in tissue arrays). Conversely, there was a significant (p < 0.0001) decrease in expression of caveolin-1 in prostate cancer tissue compared to normal prostate tissue. The opposite change in expression of the two proteins was highly correlated to increasing cancer aggressiveness. There was also a concurrent increase in the expression of epidermal growth factor receptor (EGFR), a key receptor in carcinogenesis, with clathrin in prostate cancer tissue, indicating recycling of EGFR through clathrin-mediated endocytosis (CME). These results indicate that in prostate cancer, caveolin-1-mediated endocytosis (CavME) may be acting as a brake and increase in CME may facilitate tumorigenicity and aggressiveness of prostate cancer through recycling of EGFR. Changes in the expression of these proteins can also potentially be used as a biomarker for prostate cancer to aid in diagnosis and prognosis and clinical decision-making.

VPS35 promotes cell proliferation via EGFR recycling and enhances EGFR inhibitors response in gastric cancer.

Vacuolar protein sorting-associated protein 35 (VPS35) is a core component of the retromer complex which mediates intracellular protein transport. It is well known that dysfunctional VPS35 functions in the accumulation of pathogenic proteins. In our previous study, VPS35 was found to be a potential gene related to poor prognosis in gastric cancer. However, the biological functions of VPS35 in gastric cancer remain unclear. Cell viability assays were performed to examine whether VPS35 affected cell proliferation. Immunoprecipitation and biotin assays showed that VPS35 bound to epidermal growth factor receptor (EGFR) in the cytoplasm and recycled it to the cell surface. Patient-derived xenografts and organoids were used to evaluate the effect of VPS35 on the response of gastric cancer to EGFR inhibitors. VPS35 expression levels were upregulated in tumour tissues and correlated with local tumour invasion and poor survival in patients with gastric cancer. VPS35 promoted cell proliferation and increased tumour growth. Mechanistically, VPS35 selectively bound to endocytosed EGFR in early endosomes and recycled it back to the cell surface, leading to the downstream activation of the ERK1/2 pathway. We also found that high VPS35 expression levels increased the sensitivity of the xenograft and organoid models to EGFR inhibitors. VPS35 promotes cell proliferation by recycling EGFR to the cell surface, amplifying the network of receptor trafficking. VPS35 expression levels are positively correlated with gastric cancer sensitivity to EGFR inhibitors, which offers a potential method to stratify patients for EGFR inhibitor utilisation. National Natural Science Foundation of China.

ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.

Abstract 3678: The role of SCAMP3 in EGFR trafficking and breast cancer cell response

Abstract Breast cancer (BC) affects approximately 1 in 8 women and is the second leading cause of cancer among women in the United States. BC tumors frequently overexpress Epidermal Growth Factor Receptor (EGFR) which contributes to BC progression and poor clinical outcomes. Studies associate the endocytosis related protein, Secretory Carrier Membrane Protein 3 (SCAMP3), with the regulation of EGFR recycling and degradation. Recently, we published that SCAMP3 is overexpressed in BC cells and tumors. Others have identified SCAMP3 as an indicator of poor prognosis in hepatocellular carcinoma. Therefore, we hypothesize that SCAMP3 promotes the trafficking of EGFR, induces EGFR signaling and BC progression. To evaluate whether SCAMP3 has a direct interaction with EGFR, we treated SUM-149 (EGFR+) and MDA-MB-468 (EGFR+++) BC cells with 100ng/mL EGF at different time points, then we evaluated the localization of both proteins by immunofluorescence. Results showed that SCAMP3 co-localizes with EGFR at the cytoplasm at early stimulation times and the perinuclear area after 30 mins in both cell lines. To evaluate if SCAMP3 has a role in BC progression, we knocked down SCAMP3 in SUM-149 cells using the CRISPR/Cas9 technique, and we assessed the effects in cell viability, colony, and sphere formation capacity and EGFR signaling. Knockdown (KO) and wild type (WT) cells were treated with the ligand at the same concentration described above for 30 min. Our results showed that SCAMP3 knockdown decreased cell viability, colony number and the area of formed spheres. The activation of ERK1/2 was also reduced in SCAMP3 KO cells. Taken together, SCAMP3 is involved in the internalization of EGFR, cell viability, proliferation, and tumor sphere formation. We conclude that SCAMP3 has an essential role in EGFR trafficking and BC progression through EGFR downstream signaling modulation. Citation Format: Ariana C. Acevedo Diaz, Jael Reyes- Chea, Michelle M. Martinez-Montemayor, Ivette J. Suárez-Arroyo. The role of SCAMP3 in EGFR trafficking and breast cancer cell response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3678.

TRIB3-EGFR interaction promotes lung cancer progression and defines a therapeutic target

High expression or aberrant activation of epidermal growth factor receptor (EGFR) is related to tumor progression and therapy resistance across cancer types, including non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) are first-line therapy for NSCLC. However, patients eventually deteriorate after inevitable acquisition of EGFR TKI-resistant mutations, highlighting the need for therapeutics with alternative mechanisms of action. Here, we report that the elevated tribbles pseudokinase 3 (TRIB3) is positively associated with EGFR stability and NSCLC progression. TRIB3 interacts with EGFR and recruits PKCα to induce a Thr654 phosphorylation and WWP1-induced Lys689 ubiquitination in the EGFR juxtamembrane region, which enhances EGFR recycling, stability, downstream activity, and NSCLC stemness. Disturbing the TRIB3-EGFR interaction with a stapled peptide attenuates NSCLC progression by accelerating EGFR degradation and sensitizes NSCLC cells to chemotherapeutic agents. These findings indicate that targeting EGFR degradation is a previously unappreciated therapeutic option in EGFR-related NSCLC.

Abstract 2644: Histone deacetylase 6 plays a critical role in sulfiredoxin-mediated activation of the epidermal growth factor receptor signaling in human colorectal cancer

Abstract Mammalian cells develop various transmembrane receptors including the family of receptor tyrosine kinases (RTKs) to respond to extracellular stimuli. Epidermal growth factor receptor (EGFR) is the first identified member of the RTKs and it plays a pivotal role in colorectal cancer by stimulating cell proliferation and survival. Binding of extracellular ligands, mainly EGF, leads to the dimerization of EGFR that triggers the activation of downstream mitogen activated protein kinases. In addition to the amount of ligand binding, the strength and duration of EGFR signaling are also affected by the posttranslational modifications of the receptor itself, such as phosphorylation, ubiquitination, acetylation, etc. In the previous study, we demonstrated that Sulfiredoxin (Srx) enhances the EGFR signaling in colorectal cancer cells through the inhibition of receptor acetylation at lysine residue 1061. However, the molecular mechanism by which Srx regulates the level of EGFR acetylation is still unclear. In this study, we investigated (1) the mechanism of EGFR acetylation and its regulation by Srx; (2) the functional consequence of EGFR acetylation on EGF-induced receptor dimerization, recycling, trafficking and degradation. Human colon cancer cell lines were cultured and maintained in standard conditions. Western blotting, immunoprecipitation and Mass spectrometry were used to identify cysteine oxidation of HDAC6 and characterize enzymes that are involved in the acetylation of EGFR. Stable cells that overexpress or loss of target genes, such as Srx, HDAC6, EGFR and/or mutants, were established using the 3rd generation lentiviral ShRNA infection or the CRISPR-Cas9 system. The activity of histone deacetylases was determined using commercial kit in the presence/absence of pan- or isoform-specific chemical inhibitors of HDACs. Upon EGF stimulation, the dimerization, recycling, trafficking and degradation of EGFR were demonstrated through a combination of immunoblotting, flow-cytometry and immunofluorescent imaging. We demonstrated that HDAC6 plays a central role in the regulation of EGFR acetylation through its interaction with EGFR. Depletion of Srx in human colon cancer cells increases the levels of intracellular reactive oxygen species, leading to inhibition of HDAC6 activity through regulating the redox state of cysteines 407/417. Depletion of HDAC6 not only leads to increased level of EGFR acetylation but also suppresses the binding between EGFR and its adaptor proteins. EGF-induced receptor dimerization and recycling are altered in HDAC6 knockout cells, result in inhibition of EGFR downstream signaling. Taken together, this study indicates that Srx enhances the strength and duration of EGF signaling through the ROS-HDAC6-EGFR axis in human colon cancer cells. Citation Format: Qi Ying, Hong Jiang, B. Mark Evers, Qiou Wei. Histone deacetylase 6 plays a critical role in sulfiredoxin-mediated activation of the epidermal growth factor receptor signaling in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2644.

Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

SummaryAdaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling.

The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

Mutations in the gene encoding for leucine-rich repeat kinase 2 (LRRK2) are a common cause of hereditary Parkinson's disease. LRRK2 regulates various intracellular vesicular trafficking pathways, including endolysosomal degradative events such as epidermal growth factor receptor (EGFR) degradation. Recent studies have revealed that a subset of RAB proteins involved in secretory and endocytic recycling are LRRK2 kinase substrates in vivo. However, the effects of LRRK2-mediated phosphorylation of these substrates on membrane trafficking remain unknown. Here, using an array of immunofluorescence and pulldown assays, we report that expression of active or phosphodeficient RAB8A variants rescues the G2019S LRRK2–mediated effects on endolysosomal membrane trafficking. Similarly, up-regulation of the RAB11–Rabin8–RAB8A cascade, which activates RAB8A, also reverted these trafficking deficits. Loss of RAB8A mimicked the effects of G2019S LRRK2 on endolysosomal trafficking and decreased RAB7A activity. Expression of pathogenic G2019S LRRK2 or loss of RAB8A interfered with EGFR degradation by causing its accumulation in a RAB4-positive endocytic compartment, which was accompanied by a deficit in EGFR recycling and was rescued upon expression of active RAB7A. Dominant-negative RAB7A expression resulted in similar deficits in EGF degradation, accumulation in a RAB4 compartment, and deficits in EGFR recycling, which were all rescued upon expression of active RAB8A. Taken together, these findings suggest that, by impairing RAB8A function, pathogenic G2019S LRRK2 deregulates endolysosomal transport and endocytic recycling events.

Decorin antagonizes corneal fibroblast migration via caveolae-mediated endocytosis of epidermal growth factor receptor

Decorin (Dcn), a small leucine-rich proteoglycan, is involved in the regulation of corneal wound healing. Epidermal growth factor receptor (EGFR) plays a critical role in corneal fibroblasts proliferation, migration and extracellular matrix (ECM) modulation upon injury or infection. The present study aimed to investigate the mechanistic role of Dcn in EGFR internalization to the regulation of corneal stromal fibroblasts (CSFs) migration, a key step in the corneal wound healing. Human corneal stromal fibroblasts (hCSF) cultures were generated from donor corneas. At 70% confluence, cells were switched to serum-free conditions for 48 h and then treated with decorin (250 nM) in the presence or absence of EGF (100 ng/ml) for various time points (10–60 min). Cell lysates were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry, and western blots to analyze EGFR phosphorylation. The scratch-wound assay was performed to evaluate the effects of decorin on EGF-mediated hCSF migration. Dcn caused a rapid EGFR phosphorylation within 10 min of exposure in RTK blot defining its role as a biological ligand for EGFR in hCSFs. Prolonged exposure to Dcn caused complete disappearance of EGFR and inhibition of the hCSF migration in the scratch wound assay suggesting Dcn binding to EGFR causes EGFR down-regulation. Immunostaining studies indicated that Dcn-treatment to hCSFs internalizes Dcn-EGFR complex, which does not require tyrosine kinase activity when treated with the AG1478 inhibitor and co-localizes the complex to the perinuclear region. Next, we found that Dcn-EGFR complex does not follow canonical early endosome internalization as revealed by the EEA1 antibody instead binds to the CD63 antibody directed for degradation by the late endosome. We also found that Dcn regulates the EGFR recycling by preventing its binding to Rab11, a specific antibody for recycling endosome. Further, hCSFs-pretreated with pharmacological inhibitors, methyl-β-cyclodextrin and chlorpromazine and supplemented with Dcn suggested EGFR trafficking via the caveolae-mediated pathway. These results suggest that Dcn acts as a biological ligand for EGFR and modulates hCSF migration via EGFR down-regulation, thus playing a vital role in corneal wound healing.

BCAP31 drives TNBC development by modulating ligand-independent EGFR trafficking and spontaneous EGFR phosphorylation.

Identification of novel targets for triple-negative breast cancer (TNBC) is an urgent task as targeted therapies have increased the lifespans of Oestrogen Receptor +/ Progesterone Receptor + and HER2+ cancer patients.Methods: genes involved in protein processing in the endoplasmic reticulum, which have been reported to be key players in cancer, were used in loss-of-function screening to evaluate the oncogenic roles of these genes to identify candidate target genes in TNBC. In vitro and in vivo function assays as well as clinical prognostic analysis were used to study the oncogenic role of the gene. Molecular and cell based assays were further employed to investigate the mechanisms.Results: B Cell Receptor Associated Protein 31 (BCAP31), the expression of which is correlated with early recurrence and poor survival among patients, was identified an oncogene in our assay. In vitro studies further suggested that BCAP31 acts as a key oncogene by promoting TNBC development. We also showed that BCAP31 interacts with epidermal growth factor receptor (EGFR) and serves as an inhibitor of ligand-independent EGFR recycling, sustaining EGFR autophosphorylation and activation of downstream signalling.Conclusion: These findings reveal the functional role of BCAP31, an ER-related protein, in EGFR dysregulation and TNBC development.

Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network.

SummaryThe proto-oncogenic epidermal growth factor receptor (EGFR) is a tyrosine kinase whose sensitivity to growth factors and signal duration determines cellular behavior. We resolve how EGFR's response to epidermal growth factor (EGF) originates from dynamically established recursive interactions with spatially organized protein tyrosine phosphatases (PTPs). Reciprocal genetic PTP perturbations enabled identification of receptor-like PTPRG/J at the plasma membrane and ER-associated PTPN2 as the major EGFR dephosphorylating activities. Imaging spatial-temporal PTP reactivity revealed that vesicular trafficking establishes a spatially distributed negative feedback with PTPN2 that determines signal duration. On the other hand, single-cell dose-response analysis uncovered a reactive oxygen species-mediated toggle switch between autocatalytically activated monomeric EGFR and the tumor suppressor PTPRG that governs EGFR's sensitivity to EGF. Vesicular recycling of monomeric EGFR unifies the interactions with these PTPs on distinct membrane systems, dynamically generating a network architecture that can sense and respond to time-varying growth factor signals.

Intersectin-s interaction with DENND2B facilitates recycling of epidermal growth factor receptor.

Epidermal growth factor (EGF) activates the EGF receptor (EGFR) and stimulates its internalization and trafficking to lysosomes for degradation. However, a percentage of EGFR undergoes ligand-independent endocytosis and is rapidly recycled back to the plasma membrane. Importantly, alterations in EGFR recycling are a common hallmark of cancer, and yet, our understanding of the machineries controlling the fate of endocytosed EGFR is incomplete. Intersectin-s is a multi-domain adaptor protein that is required for internalization of EGFR Here, we discover that intersectin-s binds DENND2B, a guanine nucleotide exchange factor for the exocytic GTPase Rab13, and this interaction promotes recycling of ligand-free EGFR to the cell surface. Intriguingly, upon EGF treatment, DENND2B is phosphorylated by protein kinase D and dissociates from intersectin-s, allowing for receptor targeting to degradation. Our study thus reveals a novel mechanism controlling the fate of internalized EGFR with important implications for cancer.

Activation of Endosome-Associated Inert EGF Receptor Following Internalization.

Our current understanding of endosomal signaling is incomplete given our inability to generate endosomal signals in isolation from plasma membrane signals. We present a novel method to specifically activate epidermal growth factor (EGF) receptor (EGFR) following its internalization into endosomes. This method will allow us to address questions such as whether endosomal signaling is sufficient to activate signal transduction pathways and produce biological responses. In this method, the cells are treated with EGF in the presence of AG1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks recycling of EGFR. The treatment results in the internalization of nonactivated EGF-EGFR complex into endosomes. Next, the removal of AG1478 and monensin causes endosome-associated EGFR activation. Phosphorylated surface EGFR levels are undetectable during the treatment. This is a unique and novel approach to study endosomal signaling and its physiological relevance. The protocol is transferable to study endosomal signaling of other receptor tyrosine kinases.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature