DeepEGFR a graph neural network for bioactivity classification of EGFR inhibitors

Inhibition of Brain Epidermal Growth Factor Receptor Activation: A Novel Target in Neurodegenerative Diseases and Brain Injuries.

Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury

Background: Platinum agents are a standard of care for treatment many cancers. Clinical data has shown that patients (pts) with CRC with mutant (MT) KRAS do not respond to EGFR inhibitors (Amado et al 2009). Furthermore, results from phase 3 clinical trials with erlotinib, cetuximab, or panitumumab have shown that pts whose tumors express MT KRAS have shorter progression-free survival and overall survival when an EGFR inhibitor is added to a platinum-containing regimen vs chemotherapy alone (Eberhard et al 2005, Bokemeyer et al 2011, Douillard et al 2010). However, KRAS status is not predictive of outcomes in pts receiving Ox- or irinotecan-containing regimens without an EGFR inhibitor (Richman et al 2009). This negative interaction has not been observed in pts whose tumors express either wild type (WT) KRAS receiving an EGFR inhibitor and chemotherapy or whose tumors express MT KRAS receiving an EGFR inhibitor with irinotecan (Peeters et al 2010, Van Cutsem et al 2009). Our goal was to gain a molecular mechanistic understanding of the negative interaction between EGFR inhibitors and Ox in KRAS MT CRC cells. Methods: To investigate the negative interaction between Ox and EGFR inhibitors, isogenic MT and WT KRAS-expressing HCT116 CRC cells were treated with Ox, SN-38 (the active metabolite of irinotecan), panitumumab, or gefitinib as single agents or in combination for 72 hrs. Viability was measured using an ATPlite assay. To determine the cellular distribution of EGFR, cells were serum-starved, labeled with a fluorescent anti-EGFR mAb, and visualized by a confocal microscopy. To analyze the effects of single agent or combination treatment on downstream phosphorylation of the PI3K or MAPK pathways, cells were treated for 24 hrs and phospho-proteins were detected by Western blotting. To investigate whether negative interaction between Ox and EGFR inhibitors could be reversed, cells were treated with Ox and gefitinib in combination with inhibitors to MEK, PI3K or Src for 72 hrs. Results: Treatment with gefitinib, but not panitumumab, reversed the anti-proliferative effects of Ox in the MT KRAS-expressing CRC cells vs Ox alone (p<0.0005). Neither treatment reversed the anti-proliferative effects of Ox observed in the WT KRAS-expressing cells or SN-38 in either the MT or WT KRAS-expressing cells. In the MT KRAS-expressing cells, EGFR was predominantly expressed intracellularly; in contrast, distinct cell surface staining was observed in the W T-KRAS expressing cells. This difference correlated with the inability of panitumumab to reverse the anti-proliferative effects of Ox in the CRC cells. Combination treatment of MT KRAS CRC cells with Ox and gefitinib increased pAKT which was not observed with Ox alone. Treatment of the mutant KRAS-expressing cells with a MEK, PI3K or Src inhibitor reversed the negative interaction between Ox and gefitinib (p<0.0005). Conclusion: We developed a preclinical model to further understand the negative interaction between MT KRAS-expressing cancer cells, platinum agents, and EGFR inhibitors. Feedback though AKT may contribute to the increased resistance to Ox in KRAS MT cells. Inhibitors of MEK, PI3K, or Src reversed the negative interaction between the EGFR inhibitors and Ox in a mutant-KRAS genetic background. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A150.

Epidermal Growth Factor Receptor Is a Critical Mediator of Ultraviolet B Irradiation-Induced Signal Transduction in Immortalized Human Keratinocyte HaCaT Cells

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 >> pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 >> pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

Sustained Activation of EGFR Triggers Renal Fibrogenesis after Acute Kidney Injury

Ready, Set, Go: The EGF Receptor at the Pancreatic Cancer Starting Line

FGFR4 increases EGFR oncogenic signaling in lung adenocarcinoma, and their combined inhibition is highly effective

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

Editor's evaluation: pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells

Author response: pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells

Decision letter: pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells

Pancreatic cancer is a lethal disease with little understanding of the etiology and no effective therapies. While aberrant epidermal growth factor receptor (EGFR), Src and Stat3 are detected in pancreatic cancer and implicated in the disease, their exact roles in the support of the disease phenotype remain poorly defined. Moreover, the potential for a cross-talk between EGFR, Src and Stat3 poses a challenge to any therapy that targets only one of these entities. In this study, we sought to define the EGFR, Src and Stat3 signaling integration and to investigate the contributory roles of the three entities to the pancreatic cancer phenotype. We found in Panc-1 and Colo-357 lines that phospho-Y845EGFR, pY1068EGFR and pY1086EGFR levels are responsive to c-Src inhibition, in contrast to pY1173EGFR that is solely EGFR kinase-dependent. Treatment of Panc-1 and Colo-357 cells with the EGFR inhibitor, Iressa (ZD 1839) or the Src inhibitor, Dasatinib (Das) suppressed the constitutively-active Stat3 activity, suggesting that both EGFR and Src activities promote aberrant Stat3 activation in pancreatic cancer cells. However, the early suppression of aberrantly-active Stat3 by the EGFR and Src inhibition in Panc-1 cells is countered by a Janus kinase (Jaks)-dependent re-activation, suggesting that Jaks activity could represent a compensatory mechanism for Stat3 induction. The abrogation of c-Src activity by either Das treatment or the over-expression of a kinase-dead c-Src mutant suppressed the levels of phospho-FAK, phospho-p130Cas, phospho-cortactin, phospho-p120catenin, and phospho-paxillin, suggesting that Src activity induces the mediators of motility and migration in pancreatic cancer cells. By contrast, neither EGFR nor Src inhibition modulated the baseline enhanced phospho-ErkMAPK or phospho-Akt levels in Panc-1 and Colo-357 cells, consistent with the observation that the inhibition of EGFR or Src alone only weakly suppressed cell growth and survival of pancreatic cancer cells. By contrast, the concurrent inhibition of Stat3 and EGFR or Src induced greater viability loss and apoptosis, and diminished the migration/invasion of pancreatic cancer cells in vitro. Of therapeutic significance, the concurrent inhibition of Stat3 and EGFR or Src, as compared to mono-targeting modality, induced a stronger human pancreatic tumor growth inhibition in xenografts models. We infer that the large tumor growth inhibition in vivo is due to the simultaneous suppression of the abnormal functions of Stat3 and EGFR or Src. These studies strongly suggest that the concurrent targeting of Stat3 and EGFR or Src could be a beneficial therapeutic approach for pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2528.

<div>Abstract<p><b>Purpose:</b> Skin toxicity is the main side effect of epidermal growth factor receptor (EGFR) inhibitors, often leading to dose reduction or discontinuation. We hypothesized that phosphatase inhibition in the skin keratinocytes may prevent receptor dephosphorylation caused by EGFR inhibitors and be used as a new potential strategy for the prevention or treatment of this side effect.</p><p><b>Experimental Design:</b> Menadione (Vitamin K3) was used as the prototype compound to test our hypothesis. HaCat human skin keratinocyte cells and A431 human squamous carcinoma cells were used. EGFR inhibition was measured by Western blotting and immunofluorescence. Phosphatase inhibition and reactive oxygen species (ROS) generation were measured by standard ELISA and fluorescence assays.</p><p><b>Results:</b> Menadione caused significant and reversible EGFR activation in a dose-dependent manner starting at nontoxic concentrations. EGFR activation by menadione was associated with reversible protein tyrosine phosphatase inhibition, which seemed to be mediated by ROS generation as exposure to antioxidants prevented both menadione-induced ROS generation and phosphatase inhibition. Short-term coincubation of cells with nontoxic concentrations of menadione and the EGFR inhibitors erlotinib or cetuximab prevented EGFR dephosphorylation. Seventy-two–hour coincubation of cells with the highest nontoxic concentration of menadione and erlotinib provided for a fourfold cell growth inhibitory protection in HaCat human keratinocyte cells.</p><p><b>Conclusions:</b> Menadione at nontoxic concentrations causes EGFR activation and prevents EGFR dephosphorylation by erlotinib and cetuximab. This effect seems to be mediated by ROS generation and secondary phosphatase inhibition. Mild oxidative stress in skin keratinocytes by topical menadione may protect the skin from the toxicity secondary to EGFR inhibitors without causing cytotoxicity. <i>Clin Cancer Res; 17(21); 6766–77. ©2011 AACR</i>.</p></div>