Regulation Of Epidermal Growth Factor Receptor Signaling Research Articles

Cell Adhesion Molecules as Modulators of the Epidermal Growth Factor Receptor.

Cell adhesion molecules (CAMs) are cell surface glycoproteins mediating interactions of cells with other cells and the extracellular matrix. By mediating the adhesion and modulating activity of other plasma membrane proteins, CAMs are involved in regulating a multitude of cellular processes, including growth, proliferation, migration, and survival of cells. In this review, we present evidence showing that various CAMs interact with the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase inducing pro-proliferative and anti-apoptotic intracellular signaling in response to binding to several soluble ligands, including the epidermal growth factor. We discuss that CAMs are involved in regulating EGFR signaling by either potentiating or inhibiting the soluble ligand-dependent activation of EGFR. In addition, CAMs induce soluble ligand-independent forms of EGFR activity and regulate the levels of EGFR and its ligand-induced degradation. The CAM-dependent modulation of EGFR activity plays a key role in regulating the growth, proliferation, and survival of cells. Future research is needed to determine whether these processes can be targeted in both normal and cancerous cells by regulating interactions of EGFR with various CAMs.

Extracellular proximal interaction profiling by cell surface-targeted TurboID reveals LDLR as a partner of liganded EGFR.

Plasma membrane proteins play pivotal roles in receiving and transducing signals from other cells and from the environment and are vital for cellular functionality. Enzyme-based, proximity-dependent approaches, such as biotin identification (BioID), combined with mass spectrometry have begun to illuminate the landscape of proximal protein interactions within intracellular compartments. To extend the potential of these approaches to study the extracellular environment, we developed extracellular TurboID (ecTurboID), a method designed to profile the interactions between proteins on the surfaces of living cells over short timescales using the fast-acting biotin ligase TurboID. After optimizing our experimental and data analysis strategies to capture extracellular proximity interactions, we used ecTurboID to reveal the proximal interactomes of several plasma membrane proteins, including the epidermal growth factor receptor (EGFR). We found that EGF stimulation induced an association between EGFR and the low-density lipoprotein receptor (LDLR) and changed the interactome of LDLR by increasing its proximity with proteins that regulate EGFR signaling. The identification of this interaction between two well-studied and clinically relevant receptors illustrates the utility of our modified proximity labeling methodology for identifying dynamic extracellular associations between plasma membrane proteins.

The involvement of epidermal growth factor receptor/protein kinase Bsignaling in the tumor intrinsic PD-L1-induced malignant potential of oral squamous cell carcinoma.

Various antigen-presenting cells and tumor cells-expressing PD-L1 inhibits antitumor immune responses in the tumor microenvironment. Recently, numerous studies have shown that tumor cell intrinsic PD-L1 also plays important roles in tumor growth and progression. On the other hand, oral squamous cell carcinoma (OSCC) cells overexpress epidermal growth factor receptor (EGFR) and EGFR signal pathway exacerbates tumor progression. Therefore, this study assessed whether tumor-intrinsic PD-L1 facilitates malignant potential of OSCC cells through regulation of EGFR signaling. Two OSCC cell lines, SAS and HSC-3, were transfected with PD-L1 and EGFR-specific small interfering RNA (siRNA). Influences of PD-L1 knockdown on malignant potentials of OSCC cells were examined by Cell Counting kit-8 assay, transwell assay, sphere formation assay, flow cytometry, and Western blot. Effects of PD-L1 and EGFR knockdown on each expression were examined by quantitative real-time PCR (qRT-PCR), Western blot, and flow cytometry. Transfection of an PD-L1-siRNA into OSCC cells decreased the abilities of proliferation, stemness, and mobility of these cells significantly. PD-L1 knockdown also decreased EGFR expression through the promotion of proteasome- and lysosome-mediated degradation and following activation of the EGFR/protekin kinase B (AKT)signal pathway. Meanwhile, EGFR knockdown did not influence PD-L1 expression in SAS and HSC-3 cells, but treatment with a recombinant human EGF induced its expression. Treatment with erlotinib and cetuximab suppressed rhEGF-induced PD-L1 expression and localization in the cellular membrane of both OSCC cells. OSCC cells-expressing PD-L1 induced by EGF stimulation may promote malignancy intrinsically via the activation of the EGFR/AKT signaling cascade.

MFN2 suppresses clear cell renal cell carcinoma progression by modulating mitochondria-dependent dephosphorylation of EGFR.

Clear cell renal cell carcinoma (ccRCC) is the most lethal renal cancer. An overwhelming increase of patients experience tumor progression and unfavorable prognosis. However, the molecular events underlying ccRCC tumorigenesis and metastasis remain unclear. Therefore, uncovering the underlying mechanisms will pave the way for developing novel therapeutic targets for ccRCC. In this study, we sought to investigate the role of mitofusin-2 (MFN2) in supressing ccRCC tumorigenesis and metastasis. The expression pattern and clinical significance of MFN2 in ccRCC were analyzed by using the Cancer Genome Atlas datasets and samples from our independent ccRCC cohort. Both in vitro and in vivo experiments, including cell proliferation, xenograft mouse models and transgenic mouse model, were used to determine the role of MFN2 in regulating the malignant behaviors of ccRCC. RNA-sequencing, mass spectrum analysis, co-immunoprecipitation, bio-layer interferometry and immunofluorescence were employed to elucidate the molecular mechanisms for the tumor-supressing role of MFN2. we reported a tumor-suppressing pathway in ccRCC, characterized by mitochondria-dependent inactivation of epidermal growth factor receptor (EGFR) signaling. This process was mediated by the outer mitochondrial membrane (OMM) protein MFN2. MFN2 was down-regulated in ccRCC and associated with favorable prognosis of ccRCC patients. in vivo and in vitro assays demonstrated that MFN2 inhibited ccRCC tumor growth and metastasis by suppressing the EGFR signaling pathway. In a kidney-specific knockout mouse model, loss of MFN2 led to EGFR pathway activation and malignant lesions in kidney. Mechanistically, MFN2 preferably binded small GTPase Rab21 in its GTP-loading form, which was colocalized with endocytosed EGFR in ccRCC cells. Through this EGFR-Rab21-MFN2 interaction, endocytosed EGFR was docked to mitochondria and subsequently dephosphorylated by the OMM-residing tyrosine-protein phosphatase receptor type J (PTPRJ). Our findings uncover an important non-canonical mitochondria-dependent pathway regulating EGFR signaling by the Rab21-MFN2-PTPRJ axis, which contributes to the development of novel therapeutic strategies for ccRCC.

Inhibition of CDCP1 by 8-isopentenylnaringenin synergizes with EGFR inhibitors in lung cancer treatment.

CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance by regulating EGFR signaling pathways and is a potential target in lung cancer treatment. This study aims to identify a CDCP1 reducer that synergistically improves TKI treatment. Utilizing a high-throughput drug screening system, a phytoestrogen 8-isopentenylnaringenin (8PN) was identified. Upon 8PN treatment, CDCP1 protein levels and malignant features were reduced. 8PN exposure caused the accumulation of lung cancer cells in G0/G1 phase and increased the proportion of senescent cells. In EGFR TKI-resistant lung cancer cells, the combination of 8PN and TKI synergistically reduced cell malignance, inhibited downstream EGFR pathway signaling and exerted additive effects on cell death. Moreover, combination therapy effectively reduced tumor growth and enhanced tumor necrosis in tumor xenograft mice models. Mechanistically, 8PN increased interleukin (IL)6 and IL8 expression, induced neutrophil infiltration, and enhanced neutrophil-mediated cytotoxicity to attenuate lung cancer cell growth. In conclusion, 8PN enhances the anti-cancer efficacy of EGFR TKI on lung cancer, and triggers neutrophil-dependent necrosis, highlighting the potential to overcome TKI resistance in lung cancer patients who have EGFR mutation.

LRRK1 functions as a scaffold for PTP1B-mediated EGFR sorting into ILVs at the ER-endosome contact site.

Proper control of epidermal growth factor receptor (EGFR) signaling is important for maintaining cellular homeostasis. Given that EGFR signaling occurs at the plasma membrane and endosomes following internalization, endosomal trafficking of EGFR spatiotemporally regulates EGFR signaling. In this process, leucine-rich repeat kinase 1 (LRRK1) has multiple roles in kinase activity-dependent transport of EGFR-containing endosomes and kinase-independent sorting of EGFR into the intraluminal vesicles (ILVs) of multivesicular bodies. Active, phosphorylated EGFR inactivates the LRRK1 kinase activity by phosphorylating Y944. In this study, we demonstrate that LRRK1 facilitates EGFR dephosphorylation by PTP1B (also known as PTPN1), an endoplasmic reticulum (ER)-localized protein tyrosine phosphatase, at the ER-endosome contact site, after which EGFR is sorted into the ILVs of endosomes. LRRK1 is required for the PTP1B-EGFR interaction in response to EGF stimulation, resulting in the downregulation of EGFR signaling. Furthermore, PTP1B activates LRRK1 by dephosphorylating pY944 on the contact site, which promotes the transport of EGFR-containing endosomes to the perinuclear region. These findings provide evidence that the ER-endosome contact site functions as a hub for LRRK1-dependent signaling that regulates EGFR trafficking.

Regulation of EGFR signalling by palmitoylation and its role in tumorigenesis.

The epidermal growth factor receptor (EGFR) is an essential driver of oncogenic signalling, and EGFR inhibitors are some of the earliest examples of successful targeted therapies in multiple types of cancer. The tractability of EGFR as a therapeutic target is overshadowed by the inevitable drug resistance that develops. Overcoming resistance mechanisms requires a deeper understanding of EGFR regulation in cancer cells. In this review, we discuss our recent discovery that the palmitoyltransferase DHHC20 palmitoylates EGFR on the C-terminal domain and plays a critical role in signal regulation during oncogenesis. Inhibiting DHHC20 expression or mutating the palmitoylation site on EGFR alters the EGF-induced signalling kinetics from a transient signal to a sustained signal. The change in signalling is accompanied by a decrease in cell proliferation in multiple human cancer cell lines. Our in vivo studies demonstrate that ablating the gene Zdhhc20 by CRISPR/Cas9-mediated inhibition in a mouse model of oncogenic Kras-driven lung adenocarcinoma potently inhibits tumorigenesis. The negative effect on tumorigenesis is mediated by EGFR since the expression of a palmitoylation-resistant mutant form of EGFR also inhibits Kras-driven lung adenocarcinoma. Finally, reducing EGFR palmitoylation increases the sensitivity of multiple cancer cell lines to existing inhibitors of EGFR and downstream signalling effector pathways. We will discuss the implications of these effects and strategies for targeting these new vulnerabilities.

Neuropilin-1 Expression Associates with Poor Prognosis in HNSCC and Elicits EGFR Activation upon CDDP-Induced Cytotoxic Stress.

Simple SummaryNRP-1, a co-receptor of the EGFR, represented an interesting candidate to investigate in HNSCC, as Cetuximab, in combination with radio and chemotherapy, provided the first targeted therapy scheme approved by the FDA as a standard of care for patients with recurrent or metastatic HNSCC. High levels of NRP-1 expression significantly correlated with a shorter overall survival in both Oral Squamous Cell Carcinoma and Oropharyngeal Squamous Cell Carcinoma diagnosed patients, suggesting a prognostic role for this protein. In HNSCC cell lines in vitro experiments, NRP-1 sustained EGFR activation upon CDDP exposure, together with activation of downstream MAPK/AKT pathways. Furthermore, NRP-1 modulated the responsiveness to CDDP treatment.Head and neck squamous cell carcinoma (HNSCC) includes a group of aggressive malignancies characterized by the overexpression of the epidermal growth factor receptor (EGFR) in 90% of cases. Neuropilin-1 (NRP-1) acts as an EGFR co-receptor, enhancing, upon ligand stimulation, EGFR signaling in several cellular models. However, NRP-1 remains poorly characterized in HNSCC. By utilizing in vitro cellular models of HNSCC, we report that NRP-1 is involved in the regulation of EGFR signaling. In fact, NRP-1 can lead to cisplatin-induced EGFR phosphorylation, an escape mechanism activated by cancer cells upon cytotoxic stress. Furthermore, we evaluated Neuropilin-1 staining in tissue samples of an HNSCC case series (n = 218), unraveling a prognostic value for the Neuropilin-1 tissue expression. These data suggest a potential role for NRP-1 in HNSCC cancer progression, expanding the repertoire of signaling in which NRP-1 is involved and eliciting the need for further investigations on NRP-1 as a suitable target for HNSCC novel therapeutic approaches.

ZNRF1 Mediates Epidermal Growth Factor Receptor Ubiquitination to Control Receptor Lysosomal Trafficking and Degradation.

Activation of the epidermal growth factor receptor (EGFR) is crucial for development, tissue homeostasis, and immunity. Dysregulation of EGFR signaling is associated with numerous diseases. EGFR ubiquitination and endosomal trafficking are key events that regulate the termination of EGFR signaling, but their underlying mechanisms remain obscure. Here, we reveal that ZNRF1, an E3 ubiquitin ligase, controls ligand-induced EGFR signaling via mediating receptor ubiquitination. Deletion of ZNRF1 inhibits endosome-to-lysosome sorting of EGFR, resulting in delayed receptor degradation and prolonged downstream signaling. We further demonstrate that ZNRF1 and Casitas B-lineage lymphoma (CBL), another E3 ubiquitin ligase responsible for EGFR ubiquitination, mediate ubiquitination at distinct lysine residues on EGFR. Furthermore, loss of ZNRF1 results in increased susceptibility to herpes simplex virus 1 (HSV-1) infection due to enhanced EGFR-dependent viral entry. Our findings identify ZNRF1 as a novel regulator of EGFR signaling, which together with CBL controls ligand-induced EGFR ubiquitination and lysosomal trafficking.

P2RY14-EGFR Crosstalk is Perturbed by Loss of the  <i>NF1</i> Tumor Suppressor and Modulates Schwann Cell Precursor Self-Renewal And Neurofibroma Initiation

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by nerve tumors called neurofibromas. Expression profiling of human Schwann cells (SCs) and neurofibroma SC precursors (SCPs) identified enriched P2RY14 expression in neurofibroma SCPs. We show that genetic and pharmacological inhibition of P2RY14 in human and murine NF1 mutant SCPs reduces SCP self-renewal in vitro. We identified the mechanism of action being, P2RY14 forms a complex with the epidermal growth factor receptor (EGFR) to regulate cAMP and RAS signaling. In vivo, genetic deletion of P2RY14 in NF1 murine model delayed neurofibroma initiation, reduced cAMP and increased Caveolin 1 ( Cav1 ) expression. In a murine model where EGFR is overexpressed in SCs, genetic deletion of Cav1 increased neurofibroma initiation. Thus, P2RY14 regulates EGFR signaling, cAMP and Cav1 expression, facilitating SCP self-renewal and neurofibroma initiation. These gain and loss of function experiments suggest critical roles for P2RY14-EGFR crosstalk perturbed by NF1 tumor suppressor loss.

Abstract 2951: Knockdown of SCAMP3 suppresses the proliferation and emboli formation capacity of inflammatory breast cancer cells through EGFR signaling

Abstract Inflammatory Breast Cancer (IBC) is one of the most lethal forms of breast cancer (BC), where patients have a 43% increased risk of death compared to women with non-IBC advanced BC. Approximately 30% of triple-negative (TN) IBC cases overexpress the Epidermal Growth Factor Receptor (EGFR). EGFR amplification is associated with a poor overall survival rate compared with EGFR-negative IBC cases. We previously identified increased expression of the secretory carrier membrane protein 3 (SCAMP3) in EGFR overexpressing TN SUM149 IBC cells, tissues and in the IBC metastasis promoting structure, the tumor emboli. SCAMP3 is an endosome-associated protein known to regulate the degradation of EGFR. Thus, we aim to elucidate the role of SCAMP3 in the regulation of EGFR signaling and IBC progression. To investigate how SCAMP3 contributes to the IBC cellular response, we knocked out (KO) the expression of SCAMP3 in IBC SUM149 cells using the CRISPR/Cas9 technique. Wild type (WT) and KO IBC cells treated with EGF for 30 min in serum-starved conditions were used to assess cell viability after 24, 48, 72, and 96h, colony, and emboli formation capacity. Our results demonstrate a reduction in the proliferation, colony, and emboli formation capacity of SCAMP3 KO cells in comparison with the WT. Taken together our results show that SCAMP3 plays a promoting role in the progression of IBC through EGFR signaling modulation. Citation Format: Ivette J. Suarez-Arroyo, Jael Reyes-Chea, Michelle M. Martinez-Montemayor. Knockdown of SCAMP3 suppresses the proliferation and emboli formation capacity of inflammatory breast cancer cells through EGFR signaling [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 2951.

Cell Derived Matrix Fibulin-1 Associates With Epidermal Growth Factor Receptor to Inhibit Its Activation, Localization and Function in Lung Cancer Calu-1 Cells

Epidermal Growth Factor Receptor (EGFR) is a known promoter of tumor progression and is overexpressed in lung cancers. Growth factor receptors (including EGFR) are known to interact with extracellular matrix (ECM) proteins, which regulate their activation and function. Fibulin-1 (FBLN1) is a major component of the ECM in lung tissue, and its levels are known to be downregulated in non-small cell lung cancers (NSCLC). To test the possible role FBLN1 isoforms could have in regulating EGFR signaling and function in lung cancer, we performed siRNA mediated knockdown of FBLN1C and FBLN1D in NSCLC Calu-1 cells. Their loss significantly increased basal (with serum) and EGF (Epidermal Growth Factor) mediated EGFR activation without affecting net EGFR levels. Overexpression of FBLN1C and FBLN1D also inhibits EGFR activation confirming their regulatory crosstalk. Loss of FBLN1C and FBLN1D promotes EGFR-dependent cell migration, inhibited upon Erlotinib treatment. Mechanistically, both FBLN1 isoforms interact with EGFR, their association not dependent on its activation. Notably, cell-derived matrix (CDM) enriched FBLN1 binds EGFR. Calu-1 cells plated on CDM derived from FBLN1C and FBLN1D knockdown cells show a significant increase in EGF mediated EGFR activation. This promotes cell adhesion and spreading with active EGFR enriched at membrane ruffles. Both adhesion and spreading on CDMs is significantly reduced by Erlotinib treatment. Together, these findings show FBLN1C/1D, as part of the ECM, can bind and regulate EGFR activation and function in NSCLC Calu-1 cells. They further highlight the role tumor ECM composition could have in influencing EGFR dependent lung cancers.

MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation.

Mesenchymal stromal cells (hMSCs) display a pleiotropic function in bone regeneration. The signaling involved in osteoblast commitment is still not completely understood, and that determines the failure of current therapies being used. In our recent studies, we identified two miRNAs as regulators of hMSCs osteoblast differentiation driving hypoxia signaling and cytoskeletal reorganization. Other signalings involved in this process are epithelial to mesenchymal transition (EMT) and epidermal growth factor receptor (EGFR) signalings through the regulation of Yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression. In the current study, we investigated the role of miR-33a family as a (i) modulator of YAP/TAZ expression and (ii) a regulator of EGFR signaling during osteoblast commitments. Starting from the observation on hMSCs and primary osteoblast cell lines (Nh-Ost) in which EMT genes and miR-33a displayed a specific expression, we performed a gain and loss of function study with miR-33a-5p and 3p on hMSCs cells and Nh-Ost. After 24 h of transfections, we evaluated the modulation of EMT and osteoblast genes expression by qRT-PCR, Western blot, and Osteoimage assays. Through bioinformatic analysis, we identified YAP as the putative target of miR-33a-3p. Its role was investigated by gain and loss of function studies with miR-33a-3p on hMSCs; qRT-PCR and Western blot analyses were also carried out. Finally, the possible role of EGFR signaling in YAP/TAZ modulation by miR-33a-3p expression was evaluated. Human MSCs were treated with EGF-2 and EGFR inhibitor for different time points, and qRT-PCR and Western blot analyses were performed. The above-mentioned methods revealed a balance between miR-33a-5p and miR-33a-3p expression during hMSCs osteoblast differentiation. The human MSCs phenotype was maintained by miR-33a-5p, while the maintenance of the osteoblast phenotype in the Nh-Ost cell model was permitted by miR-33a-3p expression, which regulated YAP/TAZ through the modulation of EGFR signaling. The inhibition of EGFR blocked the effects of miR-33a-3p on YAP/TAZ modulation, favoring the maintenance of hMSCs in a committed phenotype. A new possible personalized therapeutic approach to bone regeneration was discussed, which might be mediated by customizing delivery of miR-33a in simultaneously targeting EGFR and YAP signaling with combined use of drugs.

Cytoskeleton protein 4.1R suppresses murine keratinocyte cell hyperproliferation via activating the Akt/ERK pathway in an EGFR-dependent manner

The correct functioning of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is required for normal skin development and homeostasis. Cellular hyperproliferation induced by dysregulation of EGFR is tightly associated with structural and functional defects of hair follicles, skin lesions, and tumorigenesis. However, a number of questions still remain regarding the mechanism of EGFR activation and signaling. Here, we report that 4.1R, a member of the membrane-cytoskeleton linker FERM family proteins, plays critical roles in EGFR activation and signaling in keratinocytes. We demonstrated that knockout of 4.1R augments the excessive proliferation potential of keratinocytes by immunohistochemical analysis using murine skin samples. 4.1R−/− keratinocytes display enhanced EGFR-mediated Akt/ERK signaling by upregulating EGFR expression and phosphorylation, which can be reversed by either EGFR or MEK phosphorylation inhibitors. Mechanistically, coimmunoprecipitation and immunofluorescent staining results confirmed that 4.1R can impair the activation of EGFR through direct binding to EGFR and reduce the downstream signaling. Taken together, a deficiency of 4.1R would therefore serve to sustain aberrant EGFR-mediated cellular signaling, leading to hyperproliferation. Our findings highlight the role of 4.1R in the regulation of EGFR signaling in keratinocytes and suggest that 4.1R acts as a novel regulator for EGFR activation.

Abstract 2541: Mastl regulates EGFR signaling to promote pancreatic cancer progression

Abstract Background: Anti-cancer treatments act primarily by damaging the DNA of cancer cells. Upon DNA damage, cells stop proliferation at cell cycle checkpoints, which provides them time for DNA repair. Hence, pharmacological inhibition of checkpoint kinases in combination with the DNA damaging anti-cancer therapies is now emerging as promising cancer treatment strategy. In this regard, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with a frightening resistance to chemotherapeutic and targeted approaches. Here, along with the KRAS-dependent signaling, receptor tyrosine kinases especially the EGF receptor (EGFR) signaling is strongly upregulated in PDAC. Importantly, Greatwall (Mastl; Microtubule-associated serine/threonine-protein kinase-like) promotes normal G2-mitosis transition and is highly upregulated in multiple cancer types, and have been demonstrated to associate with therapy resistance. However, potential role of Mastl in pancreatic cancer progression/therapy resistance remains unknown. Our central hypothesis is that MASTL expression is (a) involved in cancer progression and intrinsic drug resistance (b) constitutes a potential therapeutic target for PDAC (c) correlates with EGFR expression and regulation. Methods: We used immunoblotting, immunohistochemistry and TCGA database analysis to examine Mastl expression, its association with EGFR in pancreatic cancer progression, and patient survival. Genetic and pharmacological manipulations for Mastl expression were performed and effects on proliferation, apoptosis and cell cycle were determined. Results: A robust increase in Mastl expression characterized pancreatic cancer cells when compared with the non-transformed pancreatic cells. A similar upregulated expression of Mastl was found in samples originating from KPC mouse model of PDAC. Analysis of the human pancreatic cancer samples and the TCGA database strongly supported this outcome and suggested a positive association of the Mastl expression with cancer progression and patient mortality. Interestingly, genetic inhibition of Mastl expression in pancreatic cancer cells not only inhibited the ability of these cells to proliferate and invade through the matrix but also inhibited activation of the ErbB-family of tyrosine kinase receptors. Further studies revealed that overexpressing K-ras/mutant p53 pathway in HPNE cells (untransformed pancreatic cells) modulates Mastl expression, and thus suggested a causal correlation of Mastl with K-Ras/EGFR signaling in pancreatic cancer cells. Conclusion: Our results suggest a cross talk of Mastl with K-ras/EGFR signaling pathway in pancreatic cancer progression. We hypothesize that combinatorial therapy targeting Mastl along with Gemcitabine would overcome the drug resistance in the PDAC. Thus, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors. Citation Format: Iram Fatima, Shailender S. Chauhan, Srijayaprakash Babu Uppada, Geoffrey A. Talmon, Amar B. Singh, Surinder K. Batra, Punita Dhawan. Mastl regulates EGFR signaling to promote pancreatic cancer progression [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 2541.

The Dlg Module and Clathrin-Mediated Endocytosis Regulate EGFR Signaling and Cyst Cell-Germline Coordination in the Drosophila Testis.

SummaryTissue homeostasis and repair relies on proper communication of stem cells and their differentiating daughters with the local tissue microenvironment. In the Drosophila male germline adult stem cell lineage, germ cells proliferate and progressively differentiate enclosed in supportive somatic cyst cells, forming a small organoid, the functional unit of differentiation. Here we show that cell polarity and vesicle trafficking influence signal transduction in cyst cells, with profound effects on the germ cells they enclose. Our data suggest that the cortical components Dlg, Scrib, Lgl and the clathrin-mediated endocytic (CME) machinery downregulate epidermal growth factor receptor (EGFR) signaling. Knockdown of dlg, scrib, lgl, or CME components in cyst cells resulted in germ cell death, similar to increased signal transduction via the EGFR, while lowering EGFR or downstream signaling components rescued the defects. This work provides insights into how cell polarity and endocytosis cooperate to regulate signal transduction and sculpt developing tissues.

Fyn is recruited to specialized clathrin coated pits and regulates EGF receptor signaling

The Epidermal Growth Factor (EGF) Receptor (EGFR) is a key receptor tyrosine kinase that controls many facets of cell physiology, including proliferation, survival and migration. EGF stimulation elicits EGFR phosphorylation and/or membrane recruitment of Grb2, Gab1 and phosphatidylinositol‐3‐kinase (PI3K), resulting in production of phosphatidylinositol‐3,4,5‐trisphosphate (PIP3), leading to Akt activation. Concomitantly upon ligand binding, EGFR is simultaneously recruited to clathrin‐coated pits (CCPs), eventually leading to receptor endocytosis. We previously uncovered that perturbation of clathrin, but not of receptor endocytosis, impairs EGF‐stimulated activation of Akt signaling (Garay et al. 2015. Mol Biol Cell.26:3504–19). Moreover, we found that some key EGFR signaling intermediates such as phosphorylated Gab1 are enriched with some CCPs. We thus proposed that in addition to their function as endocytic portals, some CCPs have a direct role in controlling EGFR signaling. How CCPs and clathrin directly control the spatiotemporal properties of EGFR signaling at the plasma membrane prior to receptor endocytosis had remained poorly understood. We uncovered that perturbations of TOM1L1 (a clathrin‐binding regulator of Src‐family kinases) and the TOM1L1‐binding Src‐family kinase Fyn impaired EGF‐stimulated Akt phosphorylation, similarly to the effects of clathrin perturbations. We found that TOM1L1 and Fyn are enriched within a subset of CCPs that also contain EGFR, and that perturbation of TOM1L1 prevented Fyn recruitment to CCPs. Importantly, TOM1L1‐ or Fyn‐positive CCPs exhibit unique properties, such as distinct lifetimes, compared to other CCPs. Thus, our results indicate that a subset of CCPs is specialized by enrichment with key signaling regulators to control EGFR signaling, a phenomenon required for subsequent Akt activation. Given the importance of EGFR in driving growth and survival of certain types of cancer, obtaining a better understanding of how CCPs organize key EGFR signals in space and time within the PM may lead to the development of novel anti‐cancer treatments.Support or Funding InformationFunding for this research was provided by an Operating Grant (no. 125854), a Project Grant and a New Investigator Salary Award from the Canadian Institutes of Health Research, as well as an Early Researcher Award (Ontario Ministry of Research, Innovation and Science) to C.N.A., and an Ontario Graduate Scholarship to S.L.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dephosphorylation of Epidermal Growth Factor Receptor by Protein Tyrosine Phosphatase 1B

Phosphorylation and dephosphorylation are fundamental regulatory mechanisms in a myriad of proteins. Protein tyrosine phosphatases (PTPs) and their integration with protein tyrosine kinases contribute to an important role in the maintenance of normal cell function. More specifically, the reversible phosphorylation of tyrosine residues in receptor tyrosine kinases provides regulation of cellular signaling. PTP1B, Protein Tyrosine Phosphatase 1B, is one of the most well studied PTPs and has been shown to dephosphorylate epidermal growth factor receptor (EGFR). Its purpose is to downregulate EGFR signaling through dephosphorylation of tyrosine residues. In doing so, PTP1B displays both tumor suppressing and tumor promoting effects dependent on cellular conditions. The detailed molecular mechanism of PTP1B in its dephosphorylation of EGFR has not yet been determined. We hypothesize that regulation of EGFR signaling by wild type PTP1B, established by a specific order of dephosphorylation, is important in maintaining in normal cellular function. We examined EGFR dephosphorylation by PTP1B and observed that there is a specific order of dephosphorylation. Furthermore, K197E PTP1B, found in thyroid cancer, had dramatic decrease in rate of EGFR dephosphorylation as well as change in order of dephosphorylation compared to the wild type PTP1B. Our studies presented here provide evidence of PTP1B's ability to fine tune EGFR signaling pathway, and we plan to deduce interactions between EGFR and PTP1B by continuing this study with individual EGFR peptides. Moreover, this research offer insights in cancer therapeutics since dysregulation of EGFR signaling due to PTP1B mutation would affect normal cellular signaling leading to uncontrollable proliferation and differentiation. Tyrosine Residues Dephosphorylation rate WT PTP1B (sec−1) Dephosphorylation rate K197E PTP1B (sec−1) Fold Difference Y992 0.88 ± 0.16 0.042 ± 0.008 21.0 Y1068 0.67 ± 0.046 0.054 ± 0.011 12.4 Y1148 0.60 ± 0.24 0.225 ± 0.0043 2.67 Y1173 2.12 ± 0.63 0.040 ± 0.0083 53 Western Blots This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EGFR and Prion protein promote signaling via FOXO3a-KLF5 resulting in clinical resistance to platinum agents in colorectal cancer.

Epidermal growth factor receptor (EGFR) supports colorectal cancer progression via oncogenic signaling. Anti‐EGFR therapy is being investigated as a clinical option for colorectal cancer, and an observed interaction between EGFR and Prion protein has been detected in neuronal cells. We hypothesized that PrPC expression levels may regulate EGFR signaling and that detailed understanding of this signaling pathway may enable identification of resistance mechanisms and new actionable targets in colorectal cancer. We performed molecular pathway analysis following knockdown of PrPC or inhibition of EGFR signaling via gefitinib to identify changes in expression of key signaling proteins that determine cellular sensitivity or resistance to cisplatin. Expression of these proteins was examined in matched primary and metastatic patient samples and was correlated for resistance to therapy and progression of disease. Utilizing three colorectal cancer cell lines, we observed a correlation between high expression of PrPC and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrPC contributed to signaling via colocalization with EGFR, which could be overcome by targeting p38 mitogen‐activated protein kinases (p38 MAPK). We revealed that the level of Krüppel‐like factor 5 (KLF5), a target downstream of p38 MAPK, was predictive for cell line and patient response to platinum agents. Further, high KLF5 expression was observed in BRAF‐mutant colorectal cancer. Our study indicates that the EGFR to KLF5 pathway is predictive of patient progression on platinum‐based therapy.

Endogenous Membrane Receptor Labeling by Reactive Cytokines and Growth Factors to Chase Their Dynamics in Live Cells

Summary Cytokine and growth factor receptors localized on cell membranes play key roles in many biological events. Because the dynamic behaviors of receptors are deeply involved in cytokine- and growth-factor-dependent signaling pathways, reliable techniques for real-time imaging of receptors in live cells are strongly desired. Here, we describe a method of covalently labeling and imaging membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with 4-dimethylaminopyridine (DMAP), an organocatalyst that facilitates acyl transfer. Construction of the DMAP cytokines and growth factors relies on non-covalent coordination chemistry between an oligo-histidine tag and dimeric Ni(II)-nitrilotriacetate in an in situ manner. This supramolecular approach is relatively simple and flexible and can be applied to labeling not only transiently but also endogenously expressed receptors. Moreover, given the benefit of this traceless labeling of the receptors, our technique allows real-time imaging of the ligand-induced dynamics of endogenously expressed receptors in live cells.