EGFR Tyrosine Phosphorylation Research Articles

Increased endogenous PKG I activity attenuates EGF-induced proliferation and migration of epithelial ovarian cancer via the MAPK/ERK pathway

The type I cGMP-dependent protein kinase (PKG I) is recognized as a tumor suppressor, but its role in EGFR regulated epithelial ovarian cancer (EOC) progression remains unclear. We evaluated the in vivo and in vitro effects of activated PKG I in EGF-induced EOC cell proliferation, migration, and invasion. The expressions of EGFR and PKG I were elevated, but the activated PKG I was decreased in EOC tissues of patients and cells lines. The addition of 8-Br-cGMP, a specific PKG I activator, attenuated the EGF-induced EOC cell proliferation, migration, and invasion in vitro. Similarly, activated PKG I also attenuated EOC progression in vivo using an EOC xenograft nude mouse model. The activated PKG I interacted with EGFR, causing increased threonine (693) phosphorylation and decreased tyrosine (1068) phosphorylation of EGFR, which resulted in disrupted EGFR-SOS1-Grb2 combination. Subsequently, the cytoplasmic phosphorylation of downstream proteins (c-Raf, MEK1/2, and ERK1/2) were declined, impeding the phosphorylated ERK1/2’s nucleus translocation, and this reduction of phosphorylated tyrosine (1068) EGFR and ERK1/2 were also abolished by Rp-8-Br-cGMPS. Our results suggest that the activation of PKG I attenuates EGF-induced EOC progression, and the 8-Br-cGMP-PKG I-EGFR/MEK/ERK axis might be a potential target for EOC therapy.

Cisplatin causes covalent inhibition of protein-tyrosine phosphatase 1B (PTP1B) through reaction with its active site cysteine: Molecular, cellular and in vivo mice studies

Protein tyrosine phosphatase 1B (PTP1B) is a critical regulator of different signalling cascades such as the EGFR pathway. The biological importance of PTP1B is further evidenced by knockout mice studies and the identification of recurrent mutations/deletions in PTP1B linked to metabolic and oncogenic alterations. Cisplatin is among the most widely used anticancer drug. The biological effects of cisplatin are thought to arise primarily from DNA damaging events involving cisplatin-DNA adducts. However, increasing evidence indicate that the biological properties of cisplatin could also rely on the perturbation of other processes such as cell signalling through direct interaction with certain cysteine residues in proteins. Here, we provide molecular, cellular and in vivo evidence suggesting that PTP1B is a target of cisplatin. Mechanistic studies indicate that cisplatin inhibited PTP1B in an irreversible manner and binds covalently to the catalytic cysteine residue of the enzyme. Accordingly, experiments conducted in cells and mice exposed to cisplatin showed inhibition of endogenous PTP1B and concomitant increase in tyrosine phosphorylation of EGFR. These findings are consistent with previous studies showing tyrosine phosphorylation-dependent activation of the EGFR pathway by cisplatin and with recent studies suggesting PTP1B inhibition by cisplatin and other platinum complexes. Importantly, our work provides novel mechanistic evidence that PTP1B is a protein target of cisplatin and is inhibited by this drug at molecular, cellular and in vivo levels. In addition, our work may contribute to the understanding of the pathways undergoing modulation upon cisplatin administration beyond of the established genotoxic effect of cisplatin.

The Transactivation of the Erbb Family of Receptor Tyrosine Kinases Is Regulated by Neurotensin Receptors in Cancer

Neurotensin (NTS)-like peptides are autocrine growth factors for lung cancer. NTS is present in and secreted from lung cancer cells and binds to G protein-coupled receptors causing signal transduction and proliferation. The growth of non-small cell lung cancer (NSCLC) cells is stimulated by NTS and inhibited by SR48692, a small molecule NTSR1 antagonist. Adding NTS to NSCLC cells increases the tyrosine phosphorylation of ErbB receptor tyrosine kinases EGFR, HER2, and HER3 by transactivation. The NTSR1 regulation of EGFR, HER2, and HER3 transactivation is blocked by SR48692, specific tyrosine kinase inhibitors, and certain monoclonal antibodies. Additional agents which impair the transactivation process include PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), and N-acetyl-cysteine (antioxidant). The results indicate growth stimulation caused by the adding NTS to NSCLC cells may be mediated by transactivation of ErbB RTKs.

The G Protein-Coupled Receptor PAC1 Regulates Transactivation of the Receptor Tyrosine Kinase HER3.

Peptide G protein-coupled receptors (GPCRs) for pituitary adenylate cyclase activating polypeptide (PACAP) regulate the growth of non-small cell lung cancer (NSCLC) cells. PACAP binds with high affinity to PAC1, which causes transactivation of receptor tyrosine kinases (RTK) for the EGFR and HER2 but its effect on HER3 is unknown. Using 3 NSCLC cell lines (NCI-H358, NCI-H441, and Calu-3), proteins for EGFR, HER2, HER3, and PAC1 were detected. The increase in EGFR tyrosine phosphorylation caused by PACAP was blocked by the EGFR tyrosine kinase inhibitor (TKI) gefitinib, or PACAP(6-38), a PAC1 antagonist. The increase in HER2 tyrosine phosphorylation caused by PACAP was inhibited by trastuzumab, a monoclonal antibody (mAb) for HER2, or PACAP(6-38). The increase in HER3 tyrosine phosphorylation caused by PACAP was inhibited by HER3 mAb3481 or PACAP(6-38). Immunoprecipitation experiments indicated the PACAP addition to Calu-3 cells resulted in the formation of EGFR/HER3 and HER2/HER3 heterodimers. Addition of the HER3 agonist neuregulin (NRG)-1 increased HER3 tyrosine phosphorylation in non-small-cell lung cancer (NSCLC) cells. PACAP or NRG-1 increased the proliferation of NSCLC cells, whereas PACAP(6-38), gefitinib, trastuzumab, or mAb3481 inhibited proliferation. The results indicate that PAC1 regulates the proliferation of NSCLC cells as a result of transactivation of the EGFR, HER2, and HER3.

Abstract 2075: GPCRs for pituitary adenylate cyclase activating polypeptide regulate transactivation of HER3 in non-small cell lung cancer cells

Abstract HER3, which lacks intrinsic kinase activity, plays an important role in cancer proliferation by formation of heterodimers with the EGFR or HER2 (Kiavue et al., Oncogene, 2019). Peptide GPCRs for pituitary adenylate cyclase activating polypeptide (PACAP) stimulate growth by transactivation of the EGFR and HER2 (Moody et al., JPET, 2012 and Peptides, 2019), but its effect on HER3 is unknown. The ability of PACAP to cause tyrosine phosphorylation of the EGFR, HER2 and HER3 was investigated. Using a panel of 17 non-small cell lung cancer (NSCLC) cell lines, mRNA for EGFR, HER2, HER3 was detected in 94%, 88%, and 100% of the cell lines, respectively. Addition of 100 nM PACAP but not vasoactive intestinal peptide to A549 cells increased EGFR, HER2 and HER3 tyrosine phosphorylation by 420, 240 and 190%, respectively. The increase is EGFR tyrosine phosphorylation was blocked by gefitinib or PACAP(6-38), a PAC1 antagonist. The increase in HER2 tyrosine phosphorylation caused by PACAP was inhibited by Herceptin or PACAP(6-38). The increase in HER3 tyrosine phosphorylation caused by PACAP was inhibited by the HER3 blocking antibody mAb3481 or PACAP(6-38). Immunoprecipitation experiments indicated the PACAP addition to A549 cells resulted in the formation of EGFR/HER2 and HER2/HER3 heterodimers, whereas addition of EGF resulted in EGFR/HER2 heterodimers and addition of the HER3 agonist neuregulin-1 resulted in HER2/HER3 heterodimers. Addition of N-acetyl-cysteine (antioxidant), Tiron (superoxide scavenger) or diphenylene iodonium (Nox/Duox inhibitor) impaired the ability of PACAP to cause EGFR, HER2 or HER3 transactivation. The results indicate that reactive oxygen species are essential for PACAP to cause EGFR, HER2 or HER3 transactivation in non small cell lung cancer cells. Citation Format: Terry W. Moody, Lingaku Lee, Robert T. Jensen. GPCRs for pituitary adenylate cyclase activating polypeptide regulate transactivation of HER3 in non-small cell lung cancer cells [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 2075.

Trefoil factor 3 in perinatal pancreas is increased by gestational low protein diet and associated with accelerated β-cell maturation

ABSTRACTThe endocrine pancreas expands markedly in the first postnatal days and the insulin producing β-cells initiate a functional maturation preceded by a morphological change of the islets of Langerhans. Trefoil factor 3 (TFF3) is a secreted peptide expressed in intestinal epithelia, where it promotes migration, but its role in the pancreas is not characterized. The aim of this study was to examine the expression and function of TFF3 in perinatal rat pancreas, ex vivo cultured fetal rat pancreas and in the rat β-cell line INS-1E.Control or gestational low-protein diet perinatal rat pancreas was harvested at embryonic day 20 (E20), day of birth (P0) and postnatal day 2 (P2). TFF3 mRNA was upregulated 4.5-fold at P0 vs. E20 and downregulated again at P2. In protein-undernourished pups induction of TFF3 at P0 was further increased to 9.7-fold and was increased at P2. TFF3 caused tyrosine phosphorylation of EGFR in INS-1E β-cells, and purified recombinant TFF3 increased both attachment and spreading of INS-1E β-cells. In ex vivo cultures of collagenase digested fetal rat pancreas, a model of perinatal β-cell maturation, TFF3 increased cellular spreading as well as insulin mRNA levels. TFF3 also increased the expression of Pref1/Dlk1 that shares similarities in expression and regulation with TFF3.These results suggest that TFF3 may promote adhesion and spreading of cells to accelerate β-cell maturation. This study indicates a functional role for TFF3 in pancreatic β-cell maturation in the perinatal period, which is altered by low protein diet during gestation.

P-216 - Thioredoxin 1 plays a key role in S-nitrosylation of cell death receptors and apoptosis in Sorafenib-treated hepatocarcinoma cells

Sorafenib is the first line molecular targeted therapy for patients in advanced hepatocarcinoma stage. The aim of the study was the identification of the relationship between redox and antitumoral properties of Sorafenib in liver cancer cells. Sorafenib induces mitochondrial depolarization and superoxide anion production. However, this effect was counteracted by relevant drug-related scavenging activity, determined using fluorescent and EPR approaches, which were associated with a reduced Nrf2-dependent Thioredoxin 1 (Trx1) signaling. The downregulation of Trx1 was associated with NOS3-denitrosylation, low nitric oxide generation and reduction of caspase-8 activity in HepG2. The reduction of S-nitrosylation of Cys199 and Cys304 residues in CD95 was related to low caspase-8 activation in Sorafenib-treated HepG2 cells. Different overexpression and interfering in vitro designs showed that Trx1 regulated S-transnitrosylation between NOS3 and CD95, caspase-8 activation, EGFR tyrosine phosphorylation and cell proliferation. The two in vivo models, thioacetamide-induced HCC and the subcutaneous HepG2-implantation in nude mice, showed that the antitumoral properties of Sorafenib were related to reduced Trx1 expression, CD95 S-nitrosylation and caspase-8 activity in tumors.

Sialylation of EGFR by the ST6Gal-I sialyltransferase promotes EGFR activation and resistance to gefitinib-mediated cell death

BackgroundThe ST6Gal-I sialyltransferase is upregulated in numerous cancers, and high expression of this enzyme correlates with poor patient prognosis in various malignancies, including ovarian cancer. Through its sialylation of a select cohort of cell surface receptors, ST6Gal-I modulates cell signaling to promote tumor cell survival. The goal of the present study was to investigate the influence of ST6Gal-I on another important receptor that controls cancer cell behavior, EGFR. Additionally, the effect of ST6Gal-I on cancer cells treated with the common EGFR inhibitor, gefitinib, was evaluated.ResultsUsing the OV4 ovarian cancer cell line, which lacks endogenous ST6Gal-I expression, a kinomics assay revealed that cells with forced overexpression of ST6Gal-I exhibited increased global tyrosine kinase activity, a finding confirmed by immunoblotting whole cell lysates with an anti-phosphotyrosine antibody. Interestingly, the kinomics assay suggested that one of the most highly activated tyrosine kinases in ST6Gal-I-overexpressing OV4 cells was EGFR. Based on these findings, additional analyses were performed to investigate the effect of ST6Gal-I on EGFR activation. To this end, we utilized, in addition to OV4 cells, the SKOV3 ovarian cancer cell line, engineered with both ST6Gal-I overexpression and knockdown, as well as the BxPC3 pancreatic cancer cell line with knockdown of ST6Gal-I. In all three cell lines, we determined that EGFR is a substrate of ST6Gal-I, and that the sialylation status of EGFR directly correlates with ST6Gal-I expression. Cells with differential ST6Gal-I expression were subsequently evaluated for EGFR tyrosine phosphorylation. Cells with high ST6Gal-I expression were found to have elevated levels of basal and EGF-induced EGFR activation. Conversely, knockdown of ST6Gal-I greatly attenuated EGFR activation, both basally and post EGF treatment. Finally, to illustrate the functional importance of ST6Gal-I in regulating EGFR-dependent survival, cells were treated with gefitinib, an EGFR inhibitor widely used for cancer therapy. These studies showed that ST6Gal-I promotes resistance to gefitinib-mediated apoptosis, as measured by caspase activity assays.ConclusionResults herein indicate that ST6Gal-I promotes EGFR activation and protects against gefitinib-mediated cell death. Establishing the tumor-associated ST6Gal-I sialyltransferase as a regulator of EGFR provides novel insight into the role of glycosylation in growth factor signaling and chemoresistance.

Development of a human epidermal growth factor derivative with EGFR-blocking and depleted biological activities: A comparative in vitro study using EGFR-positive breast cancer cells

Epidermal growth factor (EGF) is a local growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. EGF and EGFR are involved in many aspects of the development of carcinomas. Because EGFR has been found to be over-expressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. In this study we designed a mutated form of hEGF (mEGF) with a deletion of four amino acids residues (Gln43, Tyr44, Arg45 and Asp46) in order to show importance of Leu spatial location for EGFR binding/activation. Expression vector pET32a+ and E. Coli, strain Rosetta-gami B (DE3) were used to enhance solubility of the recombinant protein with yielding approximately 10mg/l of cell culture. The purified cleaved hEGF as well as non-cleaved fusion protein were biologically active, which was confirmed by their equal ability to stimulate proliferation of MCF7 cells. The mEGF showed specificity and high affinity for EGFR binding, however binding affinity of mEGF for EGFR was reduced about 11.5 fold compared with that of hEGF. The mEGF effect on the MCF7 cell proliferation had a relatively different outcome; mEGF simulated differential cell growth in a dose dependent manner. On the other hand, in MDA-MB468 cells, hEGF and mEGF induced growth inhibition, which was much more severe for hEGF than that of mEGF. Also, hEGF strongly induced the phosphorylation of EGF receptor in MDA-MB468 cells while mEGF induced poor EGFR phosphorylation. The same observations were also made for migration of cancer cells, especially induction of MDA-MB468 migration by mEGF was significantly lower than that of hEGF, suggesting a connection between tyrosine phosphorylation of EGFR and cell migration. Docking analysis revealed that the binding affinity and the buried surface area of mEGF to EGFR complex are lower than those of hEGF/EGFR. Although theoretical studies confirmed reduction in mEGF-EGFR binding affinity, the data of the present study indicate that mEGF is a potential EGFR blocker but may highlight it as excellent delivery agent of protein/non-protein toxins as well as for α-, β-, γ-emitting radio-immunotherapy.

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ETA receptor (R) and ETBR on cancer cells. High levels of tumor ET-1 and ETAR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ETAR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ETAR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca2+ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.

P-Benzoquinone initiates non-invasive urothelial cancer through aberrant tyrosine phosphorylation of EGFR, MAP kinase activation and cell cycle deregulation: Prevention by vitamin C.

According to WHO classification system, non-invasive urothelial carcinoma represents urothelial carcinoma in situ (CIS) and dysplasia. Dysplastic urothelium often progresses to CIS that further advances to urothelial carcinoma (UC). The strongest risk factor for UC is cigarette smoking. However, the pathogenesis of cigarette smoke (CS)-induced UC is poorly understood. Earlier we had shown that p-benzoquinone (p-BQ), a major toxic quinone derived from p-benzosemiquinone of CS in vivo, is a causative factor for various CS-induced diseases. Here, using a guinea pig model we showed that prolonged treatment with p-BQ led to non-invasive UC, specifically carcinoma in situ (CIS) of the renal pelvis and dysplasia in the ureter and bladder. The mechanisms of carcinogenesis were p-BQ-induced oxidative damage and apoptosis that were later suppressed and followed by activation of epidermal growth factor receptor, aberrant phosphorylation of intracellular tyrosine residues, activation of MAP kinase pathway and persistent growth signaling. This was accompanied by deregulation of cell cycle as shown by marked decrease in the expression of p21waf1/cip1 and cyclin D1 proteins as well as hyperphosphorylation of pRb. UC has been characterised by histopathology and immunohistochemistry showing aberrant CK20, increased Ki-67, and marked p53 nuclear immunopositivity with uniformly negative labelling of CD44. Oral supplementation of vitamin C (30mg/kg body weight/day) prevented CIS of the renal pelvis and dysplasia in the ureter and bladder. Since majority of non-invasive UC progresses to invasive cancer with increased risk of mortality, our preclinical study might help to devise effective strategies for early intervention of the disease.

Inhibition of SREBP increases gefitinib sensitivity in non-small cell lung cancer cells.

The clinical success of EGFR inhibitors in patients with lung cancer is limited by the inevitable development of treatment resistance. Here, we show that inhibition of SREBP increase gefitinib sensitivity in vitro and in vivo. Interference of SREBP1 binding partner MARVELD1 potentiate the therapeutic effect of gefitinib as well. Mechanistically, SREBP inhibition decreases the cell membrane fluidity, results in a decreased tyrosine phosphorylation of EGFR. Therefore, targeting lipid metabolism combined with EGFR-TKIs is potentially a novel therapeutic strategies for cancer treatment.

Abstract 1809: Endothelin causes transactivation of the EGFR and Her-2/Neu in non-small cell lung cancer cells

Abstract Endothelin-1 (ET-1) is a 23 amino acid peptide which is present in many lung cancer cell lines (Ahmed et al., Am. J. Respir. Cell Mol. Biol. 2000; 22: 422). Expression of ET-1 or the ETA receptor is associated with poor prognosis of non-small cell lung cancer (NSCLC) patients (Boldrini et al., Eur. J. Cancer 2005; 41: 2828). ET-1 stimulates whereas the ETA antagonist ZD4054 inhibits the proliferation of cancer cells (Bagnato et al., Br. J. Pharmacol. 2011: 163; 220). Here the effects of ET-1 on the transactivation of the EGFR and Her-2/Neu were investigated in NSCLC cells. Twelve of 13 NSCLC cell lines tested had both ETA and ETB mRNA. Addition of ET-1 to adenocarcinoma cell line NCI-H838, which had both ETA and ETB mRNA, elevated cytosolic Ca2+, increased tyrosine phosphorylation of the ERK, EGFR and Her-2/Neu and increased colony number. The ability of ET-1 to increase cytosolic Ca2+, tyrosine phosphorylation of ERK, EGFR and Her-2/Neu and colony number was inhibited by ZD4054 or BQ123 (ETA antagonists). The transactivation of EGFR and Her-2/Neu by ET-1 was inhibited by lapatinib (EGFR and Her-2/Neu TKI), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or N-acetylcysteine (anti-oxidant). ET-1 stimulated whereas lapatinib and ZD4054 inhibited the clonal growth of NSCLC cells. The results indicate that ET-1 may regulate the proliferation of NSCLC cells in an EGFR and Her-2/Neu-dependent manner. Citation Format: Terry W. Moody, Irene Ramos Alvarez, Samuel Mantey, Robert T. Jensen. Endothelin causes transactivation of the EGFR and Her-2/Neu in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1809.

Bone metastatic prostate cancer and resistance to tyrosine kinase inhibitors: an intimate relationship between loss of miR-203 and up-regulation of EGFR signaling

A tumor suppressor role for miR-203 in RAS-dependent prostate cancer metastasis has been described recently by our group. We have explored the regulatory mechanisms by which miR-203 is being regulated through EGFR signaling. We investigated the molecular mechanism of metastasis and identified novel roles of genes that interact with miR-203 downstream of activated Ras. We showed an amplifying regulatory loop involving the direct interaction of miR-203 with the EGFR ligands, AREG, EREG, and TGFA 3’UTR. Using clinical specimens and database analysis, our data suggested that decreasing miR-203 and increasing EGFR ligands, AREG, EREG, and TGFA expressions are correlated with prostate cancer progression. Since tyrosine kinase inhibitors (TKIs) have been shown to inhibit tyrosine phosphorylation of EGFR in a dose-dependent manner, we examined a role for miR-203 in TKIs-induced apoptosis in RAS-activated prostate cancer. We investigated the mechanisms by which miR-203 overexpression contributes to TKIs-resistant RAS-activated prostate cancer cells apoptosis. We have shown indications for candidate miR-203 targets that are either influenced by anti-apoptotic proteins (e.g. API5, BIRC2, and TRIAP1) or positively influenced by a novel NF-κB-inducible oncogenic molecule, TNFAIP8. These observations suggest that the latter category may be synergistically affected by the regulatory loop of miR-203 depletion and anti-apoptotic proteins overexpression. Our results provided evidence showing a role of miR-203 in regulating the expression of EGFR signaling genes in response to TKIs-resistance during prostate cancer progression.

Abstract 2125: G-protein coupled receptors for cholecystokinin regulate transactivation of EGF receptors in lung cancer cells

Abstract Cholecystokinin (CCK) receptors (CCK-AR and CCK-BR) are G-protein coupled receptors (GPCR) which are present on lung cancer cells (Yoder and Moody, Peptides 1987; 8: 103). CCK stimulates the proliferation of NCI-H345 cells, whereas the CCK-B receptor antagonist CI-988 inhibits proliferation (Moody et al., JPET 2001; 299: 1154). Peptide receptors for some gastrointestinal hormones/neurotransmitters mediate lung cancer growth by causing EGFR transactivation, but it is unknown if this occurs with CCK-BR. CCK-BR mRNA was detected in NCI-H345 and H727 lung cancer cells. Addition of CCK-8 or gastrin I (100 nM) to NCI-H345 or H727 cells, which have wild type EGFR, increased EGFR Tyr1068 phosphorylation after 5 min. The ability of CCK-8 to cause EGFR or ERK tyrosine phosphorylation was blocked by CI-988 (CCK-BR antagonist), gefitinib (EGFR tyrosine kinase inhibitor), PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), tiron (superoxide scavenger) and U-73122 (phospholipase C inhibitor) but not H89 (protein kinase A inhibitor). U-73122 or CI-988 inhibited the ability of CCK to cause phosphatidylinositol turnover and elevate cytosolic Ca2+. CI-988 or gefitinib inhibited the growth of NCI-H345 or H727 cells. CI-988 and gefitinib synergistically inhibited the growth of lung cancer cells. The results indicate that the CCK-BR regulates lung cancer proliferation in an EGFR-dependent mechanism through activation of Src, MMP and reactive oxygen species. Citation Format: Terry W. Moody, Suk Hee Lee, Paola Moreno Perez, Robert T Jensen. G-protein coupled receptors for cholecystokinin regulate transactivation of EGF receptors in lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2125. doi:10.1158/1538-7445.AM2014-2125

Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.

Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway.

In rabbit proximal tubular cells, ANG II type 2-receptor (AT2)-induced arachidonic acid release is PLA2 coupled and dependent of G protein βγ (Gβγ) subunits. Moreover, ANG II activates ERK1/2 and transactivates EGFR via a c-Src-dependent mechanism. Arachidonic acid has been shown to mimic this effect, at least in part, by an undetermined mechanism. In this study, we determined the effects of ANG II on fibronectin expression in cultured rabbit proximal tubule cells and elucidated the signaling pathways associated with such expression. We found that ANG II and transfection of Gβγ subunits directly increased fibronectin protein expression, and this increase was inhibited by overexpression of β-adrenergic receptor kinase (βARK)-ct or DN-Src. Moreover, ANG II-induced fibronectin protein expression was significantly abrogated by the AT2 receptor antagonist PD123319. In addition, inhibition of cystolic PLA2 diminished ANG II-induced fibronectin expression. Endogenous arachidonic acid mimicked ANG II-induced fibronectin expression. We also found that overexpression of Gβγ subunits induced c-Src, ERK1/2, and EGFR tyrosine phosphorylation, which can be inhibited by overexpression of βARK-ct or DN-Src. Gβγ also induced c-Src SH2 domain association with the EGFR. Supporting these findings, in rabbit proximal tubular epithelium, immunoblot analysis indicated that βγ expression was significant. Interestingly, arachidonic acid- and eicosatetraenoic acid-induced responses were preserved in the presence of βARK-ct. This is the first report demonstrating the regulation of EGFR, ERK1/2, c-Src, and fibronectin by Gβγ subunits in renal epithelial cells. Moreover, this work demonstrates a role for Gβγ heterotrimeric proteins in ANG II, but not arachidonic acid, signaling in renal epithelial cells.

Role of genistein on EGFR phosphorylation in acute liver damage induce by administration of carbon tetrachloride in rat (653.17)

Our aim was to study the effect of genistein on EGFR phosphorylation on liver rats with acute damage induces by administration of carbon tetrachloride (CCl4). For this study, we used male Wistar rats which were randomly divided into four groups: control, genistein 5mg/kg oral (2 weeks), acute damage CCl4 4mg/kg i.p. (4 weeks) and acute damage+genistein. Rats were sacrificed and liver were collected for determinate EGFR expression and tyrosine phosphorylation by western blot and immunoprecipitation. EGFR expression was elevated in groups with acute liver damage and acute damage+genistein. On the other hand when we analyzed EGFR phosphorylation we founded a reduction on Tyr845 EGFR phosphorylation in group with liver damage, nevertheless the genistein increased this phosphorylation 5 fold compared with CCl4 group, on Tyr1045 the phosphorylation was to decrease in acute damage and acute damage+genistein group. On Tyr992 and Tyr1068 EGFR phosphorylation genistein and acute damage not showed modification. In conclusion, the genistein can be involved in process like proliferation, apoptosis and EGFR degradation in the liver during acute damage, because it modifies specific tyrosine phosphorylation in EGFR.

Abstract 1046: AS1411 activity is regulated by epidermal growth factor receptor signaling pathway.

Abstract The anticancer AS1411 agent is a G-rich phosphodiester oligodeoxynucleotide, which forms a stable quadruplex structure and binds specifically to nucleolin as an aptamer. It efficiently inhibits proliferation and induces cell death in many types of cancer cells, but has little effect on normal cells. We have also shown that AS1411 is taken up by macropinocytosis and stimulates further macropinocytosis by a nucleolin-dependent mechanism in several cancer cells. AS1411 activity correlates with stimulated macropinocytosis, suggesting this hyperstimulation of macropinocytosis may explain the unusual cancer cell death caused by AS1411. Macropinocytosis is a ligand-independent endocytic pathway that is normally activated by growth factor receptor stimulation. On the other hand, nucleolin has been reported that can enhance growth factor signaling by binding directly to some growth factor receptors. Therefore in this study, we determine the participation of growth factor signaling pathway in AS1411-induced macropinocytosis and cell death. Pre-incubation of DU145 cells with a specific EGFR inhibitor, but not PDGFR or JAK2 inhibitors, significantly inhibited the stimulation of AS1411-mediated macropinocytosis, suggesting that AS1411 activates EGFR signaling pathways. This was confirmed by observing that DU145 cells treated with AS1411 induced tyrosine phosphorylation of EGFR and activation of its downstream effectors, including p38, PI3K and Rac1. Furthermore, AS1411 seem to induce the interaction between EGFR and nucleolin in accord with EGFR activation. We also found that the activation of EGFR signaling pathway by AS1411 might regulate its cancer cellular response. Simulation of macropinocytosis by AS1411 was significantly inhibited in DU145 cells pre-treated with pharmacological inhibitors of Rac1 or PI3K, but not by Src inhibitor. Moreover the ability of AS1411 to inhibit cell proliferation and induce cell death was reduced by an EGFR-neutralizing antibody (cetuximab) or by downregulating the expression of Rac1 using siRNAs. These results indicate that AS1411 mediates the activation of EGFR signaling pathway to induce cancer cell death, and suggest that AS1411 might redirect EGFR signaling pathway from a cell survival signal to a cell death signal. Citation Format: Elsa M. Reyes-Reyes, Francesca R. Salipur, Paula J. Bates. AS1411 activity is regulated by epidermal growth factor receptor signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2013-1046

Photodynamic therapy activated signaling from epidermal growth factor receptor and STAT3

Patients with serosal (pleural or peritoneal) spread of malignancy have few definitive treatment options and consequently have a very poor prognosis. We have previously shown that photodynamic therapy (PDT) can be an effective treatment for these patients, but that the therapeutic index is relatively narrow. Here, we test the hypothesis that EGFR and STAT3 activation increase survival following PDT, and that inhibiting these pathways leads to increased PDT-mediated direct cellular cytotoxicity by examining BPD-PDT in OvCa and NSCLC cells. We found that BPD-mediated PDT stimulated EGFR tyrosine phosphorylation and nuclear translocation, and that EGFR inhibition by erlotinib resulted in reduction of PDT-mediated EGFR activation and nuclear translocation. Nuclear translocation and PDT-mediated activation of EGFR were also observed in response to BPD-mediated PDT in multiple cell lines, including OvCa, NSCLC and head and neck cancer cells, and was observed to occur in response to porfimer sodium-mediated PDT. In addition, we found that PDT stimulates nuclear translocation of STAT3 and STAT3/EGFR association and that inhibiting STAT3 signaling prior to PDT leads to increased PDT cytotoxicity. Finally, we found that inhibition of EGFR signaling leads to increased PDT cytotoxicity through a mechanism that involves increased apoptotic cell death. Taken together, these results demonstrate that PDT stimulates the nuclear accumulation of both EGFR and STAT3 and that targeting these survival pathways is a potentially promising strategy that could be adapted for clinical trials of PDT for patients with serosal spread of malignancy.