Epidermal Growth Factor Receptor Downstream Signaling Research Articles

Down-regulation of epidermal growth factor receptor by curcumin-induced UBE1L in human bronchial epithelial cells

UBE1L, ubiquitin-activating enzyme E1-like, is the activating enzyme of ISG15ylation (ISG15, interferon stimulated gene 15). Loss of UBE1L and activation of epidermal growth factor receptor (EGFR) signaling are common events in lung carcinogenesis. Curcumin, a well-studied chemopreventive agent, is known to down-regulate EGFR. The present study demonstrated that curcumin decreased EGFR expression in human bronchial epithelial (HBE) Beas-2B cells and lung cancer A549 cells. For the first time, UBE1L was found to be induced by curcumin in HBE cells. Interestingly, overexpression of UBE1L reduced EGFR at posttranslational level in HBE cells. UBE1L triggered EGFR membrane internalization and promoted complex formation between ISG15 and EGFR. Curcumin decreased EGFR downstream signaling pAKT and nuclear factor κB (NF-κB). Overexpression or knockdown of UBE1L also resulted in down-regulation or up-regulation of phosphoinositide 3-kinase/AKT/NF-κB correspondently. In human samples, there was an inverse relationship between UBE1L and EGFR/AKT/NF-κB in non-small cell lung cancer tissues and adjacent tissues. These results uncover a novel chemopreventive mechanism of curcumin in inducing UBE1L and down-regulating EGFR signaling in HBE cells.

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.

Abstract C37: Reduced annexin A6 expression enhances EGFR degradation and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors

Abstract The role of annexin A6 (AnxA6), a calcium-dependent phospholipid binding protein, in breast carcinogenesis remains poorly understood. Some reports suggest that AnxA6 inhibits the activation and down-stream signaling of epidermal growth factor receptor (EGFR). On the contrary, other studies have shown that AnxA6 not only stabilizes activated receptors on the cell surface but also promotes the expression of differentiation markers in chondrocytes. Together with our recent study showing that AnxA6 expression is reduced in breast cancer, we investigated whether AnxA6 expression status influences breast cancer outcome in patients and whether this may be attributed to its modulation of EGFR expression. We show that low AnxA6 expression correlates with reduced distant metastasis-free and overall survival, but is associated with a higher probability for recurrence-free survivalof patients with basal-like breast cancer. To determine whether these effects are mediated via EGFR, a predictive marker of basal-like breast cancer, we examined the effects of altered expression of AnxA6 in AnxA6-high invasive triple negative breast cancer cells. Interestingly, depletion of AnxA6 in invasive BT-549 cells was accompanied by decreased expression of EGFR protein and mRNA. We also show that in invasive breast cancer cells, AnxA6 expression is required for sustained membrane localization of activated EGFR and persistent downstream signaling. Consequently, AnxA6-depleted cells exhibited decreased cell motility, and increased sensitivity to the tyrosine kinase inhibitors (TKIs), lapatinib and PD153035. On the contrary, over-expression of AnxA6 in AnxA6-low HCC1806 cells did not affect their motility but as expected, inhibited their growth in 3D matrigel cultures. These data suggest that AnxA6-modulation of EGFR expression and activity underlies at least in part, the changes in proliferation, metastatic potentials and sensitivity to EGFR-targeted TKIs of invasive breast cancer cells. Our data also suggest that AnxA6 expression status may predict the survival and likelihood to respond to EGFR-targeted therapies of patients with basal-like breast cancer. Citation Format: Rainelli B. Koumangoye, Josiah Ochieng, Amos M. Sakwe. Reduced annexin A6 expression enhances EGFR degradation and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C37.

Abstract A052: Inhibition of triple-negative and Her-2 resistant breast cancer proliferation and migration by Annexin A2 antibodies

Abstract Introduction: The triple negative breast cancer (TNBC) phenotype, which lacks the presence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (ErbB-2 or Her-2), is highly aggressive and resistant to chemotherapy. In fact, some of the patients under hormone deprivation and/or Herceptin therapy acquire resistance. Therefore, there is an urgent need to identify new diagnostic as well as therapeutic markers for treatment of such patients. Herceptin, an immunotherapy directed against the Her-2 receptor, inhibits cancer growth and progression in Her-2 positive breast cancer by blocking the downstream survival pathways. In these cells however, the expression of epidermal growth factor receptor (EGFR) is significantly low. Interestingly, EGFR expression is significantly increased in triple negative and Her-2 resistant breast cancer thereby contributing to cancer growth and progression. Present studies were designed to investigate whether or not Annexin A2 (AnxA2), a calcium dependent phospholipid binding protein, regulated EGFR downstream signaling pathway and if the functions of EGFR can be inhibited by the AnxA2 antibody in TNBC and Herceptin resistant cancer cell lines. Methods: AnxA2 function at cell surface of the triple negative breast cancer cell line MDA-MB-231 and Her-2 resistant breast cancer cell line JIMT-1 was blocked incubating with AnxA2 antibody (2μg/ml) after 12h of serum starvation. The cells were treated with/without EGF (50ng/ml) for 20 min after 2h of antibody treatment. The cell lysate was analyzed for pEGFR and EGFR mediated downstream signaling by Western blotting. Cell migration was analyzed by scratch wound healing assay. Results: The results of the present study indicate that AnxA2 interacts with EGFR at the cell surface and plays an important role in the regulation of EGFR mediated downstream signaling. Treatment of MDA-MB-231 and JIMT-1 cells with AnxA2 antibody causes significant decrease in EGF-induced phosphorylation of EGFR at Y845 and Y1068 sites. Our results also demonstrate that blocking cell surface AnxA2 functions causes the downregulation of proteins such as pAKT, and pERK1/2 which are regulated by EGFR resulting in lower cell survival, proliferation, and migration. Conclusions: Our data indicate that association of AnxA2 with EGFR in the membrane domain plays a positive regulatory role in keeping EGFR signaling events in an activated state in both triple negative and Her-2 resistant breast cancer cells, thus making AnxA2 an important therapeutic target. Citation Format: Pankaj Chaudhary, Jamboor K. Vishwanatha. Inhibition of triple-negative and Her-2 resistant breast cancer proliferation and migration by Annexin A2 antibodies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A052.

The Autophagy Inhibitor Chloroquine Overcomes the Innate Resistance of Wild-Type EGFR Non-Small-Cell Lung Cancer Cells to Erlotinib

The epidermal growth factor receptor (EGFR) inhibitor erlotinib is much less effective in non-small-cell lung cancer (NSCLC) tumors with wild-type EGFR, than in tumors with activating EGFR mutations. Autophagy is a tightly regulated lysosomal self-digestion process, which may alternatively promote cell survival or type II cell death. This study assessed the role of autophagy in erlotinib-mediated cytotoxicity. We used wild-type EGFR erlotinib-sensitive and erlotinib-resistant NSCLC cell lines to determine whether inhibiting autophagy by a therapeutic agent potentiated the antitumor activity of erlotinib in vitro and in vivo. Erlotinib at a clinically relevant concentration (2 μM) induced autophagy in NSCLC cells with wild-type EGFR, and the degree of induction was greater in cells that were resistant than sensitive, suggesting that autophagy is cytoprotective. This was confirmed by knockdown of the autophagy-related gene Atg-5, and by using the autophagy inhibitor chloroquine (CQ), both of which increased the cytotoxicity of erlotinib. The synergistic activity of CQ was not because of the potentiation of erlotinib's effects on autophagy, cell-cycle arrest, and inhibition of both EGFR or downstream signaling of EGFR. Rather, CQ markedly activated apoptosis in the cells. The ability of CQ to potentiate the antitumor activity of erlotinib was also seen in mice bearing NSCLC tumor xenografts. The ability to adapt to anti-EGFR therapy by triggering autophagy may be a key determinant for resistance to erlotinib in wild-type EGFR NSCLC. Inhibition of autophagy by CQ represents a novel strategy to broaden the spectrum of erlotinib efficacy in wild-type EGFR NSCLC tumors.

Nitric oxide donating anilinopyrimidines: Synthesis and biological evaluation as EGFR inhibitors

To search for potent nitric oxide (NO) donating epidermal growth factor receptor (EGFR) inhibitors, a series of phenylsulfonylfuroxan-based anilinopyrimidines 10a–h were synthesized and biologically evaluated. Compounds 10f–h exhibited potent inhibitory activity against EGFR L858R/T790M and were as potent as WZ4002 in inhibition of H1975 cells harboring EGFR L858R/T790M. Additionally, 10h produced high levels of NO in H1975 cells but not in normal human cells, and its antiproliferative activity was diminished by hemoglobin, an NO scavenger. Furthermore, 10h inhibited EGFR activation and downstream signaling in H1975 cells. These results suggest that the strong antiproliferative activity of 10h could be attributed to the synergic effects of high levels of NO production and inhibition of EGFR and downstream signaling in the cancer cells.

Novel Hybrids of (Phenylsulfonyl)furoxan and Anilinopyrimidine as Potent and Selective Epidermal Growth Factor Receptor Inhibitors for Intervention of Non-Small-Cell Lung Cancer

A series of hybrids (12a-k) from (phenylsulfonyl)furoxan and anilinopyrimidine were synthesized and biologically evaluated as epidermal growth factor receptor (EGFR) inhibitors for intervention of non-small-cell lung cancer (NSCLC). Compound 12k exhibited strong and selective EGFR L858R/T790M inhibitory activity (IC50 = 0.047 μM) and displayed antiproliferative effects on EGFR mutation NSCLC cell lines HCC827 (del E746_A750) and H1975 (L858R/T790M) with IC50 values of 0.007 and 0.029 μM, respectively. Additionally, 12k released high levels of NO in H1975 cells but not in normal human cells, and its activity was diminished by pretreatment with a NO scavenger. Furthermore, 12k induced apoptosis of H1975 and HCC827 cells more strongly than WZ4002 (1), inhibited EGFR downstream signaling in H1975 cells, and suppressed the nuclear factor-κB activation in H1975 cells, while 1 had no significant effects under the same conditions. Finally, 12k substantially inhibited tumor growth in an H1975 xenograft mouse model. Overall, 12k might be a promising candidate for the treatment of NSCLC.

Abstract 2255: Distinct biological effect of inhibiting the metabolic pathway using active compounds derived from Chinese medicinal herbs in non-small cell lung cancer cells with different EGFR mutational profiles.

Abstract Lung cancer is the leading cause of cancer death worldwide. Epidermal Growth Factor Receptor (EGFR) mutation(s) is/are common in non-small cell lung cancer (NSCLC) in Eastern Oriental population, resulting in constitutive activation of EGFR downstream anti-apoptotic signaling and lung tumor formation. Molecular targeted therapy using tyrosine kinase inhibitor (TKI) targeting EGFR shows promising initial response, however, drug resistance and relapse are universal, making the treatment outcome undesirable. Drug resistance could be due to additional EGFR mutation or activation of alternative survival pathways. Therefore, it is important to identify new inhibitors and new molecular targets to tackle TKI-resistance. Chinese Medicinal Herbs (CMHs) has been used to treat variety systemic diseases for many years in China, including diabetes, neurodegenerative disease and cancer. The multi-targeting nature and potential alternation effects on metabolic pathways of CMHs could be a new era for novel cancer drug discovery. Hence, we have reviewed the literature and selected ten single purified compounds derived from CMHs which exhibited the highest potential of cancer suppression effect in NSCLC. We have recruited three EGFR-dependent NSCLC cell lines for drug screening using cytotoxicity assay. A549 is used as EGFR wild-type control. Two TKI-resistant NSCLC cell lines were used, H1975 harbors double mutation (EGFRL858R+T790M) and H1650 harbors EGFRexon 19 deletion with activation of alternative cell survival pathway. H2228 is a NSCLC cell line which harbours EML4-ALK fusion gene and was used as EGFR-independent cell line control. MTT assay revealed that six out of ten candidate agents showed significant cancer-inhibiting effects in H1650, H1975 cells. Three compounds exhibited IC50 value at micro-molar levels while another three compounds exhibited IC50 at as low as nano-molar levels. One compound exhibited specificity on EGFR-dependent NSCLC cell lines, which showed 10-fold more selective than the EGFR-independent H2228 cells. Cell cycle analysis and western blot showed that one effective compound altered the metabolic pathway of glucose metabolism and induced cell cycle arrest at G1 phase in NSCLC with EGFR mutation. However, the anti-proliferative effects were distinct in NSCLC cell lines with different EGFR mutation patterns. Our compound significantly induced cell cycle arrest in four NSCLC in EGFR mutant cell lines but the inhibiting effect was not significant in EGFR wild-type cell line. Immunobloting assay revealed that glycolytic enzymes and cell cycle regulatory gene expression were altered after 72 hr compound treatment in the responsive cells. Further investigation is required to elucidate the underlying reason of drug selectivity and the treatment mechanism of these compounds. Citation Format: Xing Xing Fan, Maria Pik Wong, Zhi Wei Cao, Jian Lin Wu, Hua Zhou, Zhi Hong Jiang, Liang Liu, Elaine Lai-Han Leung. Distinct biological effect of inhibiting the metabolic pathway using active compounds derived from Chinese medicinal herbs in non-small cell lung cancer cells with different EGFR mutational profiles. [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 2255. doi:10.1158/1538-7445.AM2013-2255

Abstract 530: Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells.

Abstract Approximately half of all basal type breast cancers show evidence of autocrine activation of the epidermal growth factor receptor (EGFR) by amphiregulin (AREG). In addition, AREG expression is associated with aggressive and chemoresistant forms of breast cancer. Studies in our laboratory show that when AREG is the activating ligand, EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To investigate the effect of AREG-mediated activation of EGFR on the phenotype of AREG-expressing cancers, AREG expression was knocked-down in SUM149 cells, a highly tumorigenic and metastatic cell line isolated from a basal subtype inflammatory breast cancer, which expresses high levels of AREG and overexpresses EGFR. Microarray analysis of AREG knockdown cells revealed a number of relevant pathways affected by AREG-EGFR signaling. These pathways include Notch signaling, MAPK activation, IL-1 signaling and multiple components of the Wnt signaling pathway. In particular, knockdown of AREG or inhibition of EGFR signaling in AREG stimulated cells resulted in the upregulation of DKK-1, an antagonist of canonical Wnt signaling, and the expression of several WNT target genes. Flow cytometric analysis of these cells demonstrated that AREG knockdown resulted in a significant reduction in CD44+ cells while simultaneously increasing the non-stem cell fraction of CD44-/CD24+ cells. Because CD44hi/CD24lo is characteristic of undifferentiated cells that have the potential to initiate tumor formation, the tumorigenicity of these cells was investigated by transplanting parental and AREG knock-down SUM149 cells into the mammary fat pads of NOD-SCID mice. The tumorigenic activity of the AREG knock-down derivatives was significantly reduced compared to the parental SUM149 cells. In particular, the AREG knockdown clone that expressed the lowest level of AREG expression and the highest level of DKK-1 expression failed to form tumors in NOD-SCID mice. To investigate the link between AREG-mediated activation of EGFR, receptor trafficking, and downstream signaling pathways that are responsible for changes in expression of WNT pathway genes, we are investigating differences in EGFR phosphorylation and downstream signaling stimulated by AREG versus EGF. In conjunction with our previous findings that AREG-mediated EGFR signaling results in activation of NF-κβ and the upregulation of IL1α/β, our current results suggest a link between these pathways, WNT signaling, and tumorigenicity in SUM-149 cells. Citation Format: Christiana S. Kappler, Stephen P. Ethier. Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells. [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 530. doi:10.1158/1538-7445.AM2013-530

Mutation analysis of the EGFR gene and downstream signalling pathway in histologic samples of malignant pleural mesothelioma

Background:As epidermal growth factor receptor (EGFR) is involved in the pathogenesis of malignant pleural mesotheliomas (MPMs), the anti-EGFR drugs may be effective in treating MPM patients. Mutations of the EGFR gene or its downstream effectors may cause constitutive activation leading to cell proliferation, and the inhibition of apoptosis and metastases. Consequently, molecular profiling is essential for select patients with MPM who may respond to anti-EGFR therapies.Methods:After manual macrodissection, genomic DNA was extracted from 77 histological samples of MPM: 59 epithelioid, 10 biphasic, and 8 sarcomatoid. Epidermal growth factor receptor gene mutations were sought by means of real-time polymerase chain reaction (PCR) and direct sequencing, KRAS gene mutations by mutant-enriched PCR, and PIK3CA and BRAF gene mutations by direct sequencing.Results:Gene mutations were identified in nine cases (12%): five KRAS, three BRAF, and one PI3KCA mutation; no EGFR gene mutations were detected. There was no difference in disease-specific survival between the patients with or without gene mutations (P=0.552).Conclusions:Mutations in EGFR downstream pathways are not rare in MPM. Although none of those found in this study seemed to be prognostically significant, they may support a more specific selection of patients for future trials.

Low electric fields induce ligand-independent activation of EGF receptor and ERK via electrochemical elevation of H+ and ROS concentrations

Physiological electric fields are involved in many biological processes and known to elicit their effects during long exposures ranging from a few hours to days. Following exposure to electric fields of physiological amplitude, epidermal growth factor receptor (EGFR) was demonstrated to be redistributed and upregulated with further intracellular signaling such as the MAPK signaling cascade. In our study we demonstrated EGFR activation and signaling induced by short train of pulsed low electric field (LEF) (10V/cm, pulse-width 180μs, 500Hz, 2min) in serum-free medium, following 24-hour starvation, and in the absence of exogenous EGF ligand, suggesting a ligand-independent pathway for EGFR activation. This ligandless activation was further confirmed by using neutralizing antibodies (LA1) that block the EGFR ligand-binding site. EGFR activation was found to be EGFR kinase dependent, yet with no dimerization following exposure to LEF. ERK activation was found to be mainly a result of EGFR downstream signaling though it partially occurred via EGFR-independent way. We demonstrate that reactive oxygen species and especially decrease in pH generated during exposure to LEF are involved in EGFR ligandless activation. We propose a possible mechanism for the LEF-induced EGFR ligand-independent activation and show activation of other receptor tyrosine kinases following exposure to LEF.

Inhibitory effects of omega-3 fatty acids on injury-induced epidermal growth factor receptor transactivation contribute to delayed wound healing

Epidermal growth factor receptor (EGFR)-mediated signaling is required for optimal intestinal wound healing. Since n-3 polyunsaturated fatty acids (PUFA), specifically docosahexaenoic acid (DHA), alter EGFR signaling and suppress downstream activation of key signaling pathways, we hypothesized that DHA would be detrimental to the process of intestinal wound healing. Using a mouse immortalized colonocyte model, DHA uniquely reduced EGFR ligand-induced receptor activation, whereas DHA and its metabolic precursor eicosapentaenoic acid (EPA) reduced wound-induced EGFR transactivation compared with control (no fatty acid or linoleic acid). Under wounding conditions, the suppression of EGFR activation was associated with a reduction in downstream activation of cytoskeletal remodeling proteins (PLCγ1, Rac1, and Cdc42). Subsequently, DHA and EPA reduced cell migration in response to wounding. Mice were fed a corn oil-, DHA-, or EPA-enriched diet prior to intestinal wounding (2.5% dextran sodium sulfate for 5 days followed by termination after 0, 3, or 6 days of recovery). Mortality was increased in EPA-fed mice and colonic histological injury scores were increased in EPA- and DHA-fed mice compared with corn oil-fed (control) mice. Although kinetics of colonic EGFR activation and downstream signaling (PLCγ1, Rac1, and Cdc42) were delayed by both n-3 PUFA, colonic repair was increased in EPA- relative to DHA-fed mice. These results indicate that, during the early response to intestinal wounding, DHA and EPA uniquely delay the activation of key wound-healing processes in the colon. This effect is mediated, at least in part, via suppression of EGFR-mediated signaling and downstream cytoskeletal remodeling.

ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17

The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H2O2 participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y2 receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y2R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y2R activation as a proximal step in EGFR transactivation and downstream signaling.

Role of Amphiregulin in Suppressed Liver Regeneration after Small-For-Size Liver Transplantation in Mice

Introduction: Liver regeneration is inhibited in small-for-size liver grafts, but the underlying mechanisms remain unclear. The aim of this study was to investigate alterations in mitotic cytokines/growth factors after partial liver transplantation. Methods: Livers of male C57BL/6 mice were explanted after reduction to half-size grafts (HSG) and ˜third-size grafts (TSG). Full-size grafts (FSG), HSG and TSG were stored at 0-1oC for 3 h before implantation. Livers were harvested 48 h after liver transplantation for evaluation of liver regeneration. Results: 5-Bromodeoxyuridine (BrdU) labeling increased from 0.04% to ˜23% in HSG but did not increase in FSG and TSG at 48 h. Mitotic index increased from ˜0.1% to ˜7.5% in HSG but did not increase in FSG and TSG. Proliferating cell nuclear antigen (PCNA) expression was barely detectable by immunoblotting in livers from sham-operated mice and FSG but increased markedly in HSG. PCNA expression remained low in TSG. These data demonstrate suppressed hepatocyte proliferation in TSG. mRNAs of mitotic cytokines TNFα and IL-6 and hepatic growth factor (HGF) increased in HSG and increased further in TSG. Epidermal growth factor receptor (EGFR) ligands EGF and heparin-binding EGF did not increase significantly in either HGF or TSG. TGFα, which is also an EFGR ligand, increased equally in HSG and TSG. Therefore, these cytokines/growth factors are unlikely responsible for inhibited regeneration of TSG. In contrast, amphiregulin (AR), another EGFR ligand, increased in HSG after transplantation but did not change in FSG and TSG. Compared to HSG, TSG exhibited decreased AR protein and mRNA, indicating suppressed AR synthesis. An AR neutralizing antibody (100 μg/mouse, iv) blunted liver regeneration in HSG. AR supplementation (5 μg/mouse, iv immediately after transplantation) did not attenuate liver injury (ALT release, necrosis, and apoptosis) but stimulated liver regeneration, prevented hyperbilirubinemia and improved survival from 10% to 75% after transplantation of TSG. Phospho-EGFR increased in HSG but not in TSG. AR supplementation stimulated EGFR activation in TSG. The EGFR inhibitor PD153035 (30 mg/kg, ip) blunted liver regeneration in HSG and abrogated AR-stimulated liver regeneration in TSG. Phosphorylation of Akt, c-Jun-N-terminal kinase-1/2, and extracellular signal-regulated kinase-1/2, molecules that mediate EGFR down-stream signaling and expression of cyclin D1 and cyclin E, increased overtly in HSG but only slightly in TSG. AR supplementation enhanced the activation of these kinases and expression of cyclins in TSG. Conclusion: Suppression of regeneration of small-for-size liver grafts is likely due, at least in part, to decreased formation of AR and activation of EGFR signaling pathways. Supplementation of AR could be a promising therapy to stimulate regeneration of partial liver grafts (NIDDK).

Oxytocin Promotes Long-Term Potentiation by Enhancing Epidermal Growth Factor Receptor-Mediated Local Translation of Protein Kinase Mζ

In addition to triggering the birthing process and milk release, the hypothalamic neuropeptide oxytocin (OXT) plays an important role in the regulation of complex social cognition and behavior. Previous work has shown that OXT can regulate hippocampal synaptic plasticity and improve hippocampus-dependent cognitive functions in the female mice, but the underlying mechanisms remain largely unclear. Here, we demonstrate that OXT promotes the maintenance of long-term potentiation (LTP) induced by one train of tetanic stimulation (TS) in the CA1 region of hippocampal slices from both nulliparous female and male rats through a previously unknown mechanism involving OXT receptor (OXTR)-dependent and epidermal growth factor receptor (EGFR)-mediated local translation of an atypical protein kinase C isoform, protein kinase Mζ (PKMζ), in dendrites. Using pharmacological and biochemical approaches, we show that both the conventional OXTR-associated signaling pathway (G(q/11)-coupled phospholipase C) and the transactivated EGFR downstream signaling pathways (phosphatidylinositol 3 kinase and extracellular signal-regulated kinase 1/2) are involved in the regulation of OXT. In addition, OXT stimulates local dendritic PKMζ mRNA translation via activation of a mammalian target of rapamycin-regulated mechanism. Furthermore, blockade of OXTR results in a modest decrease in the ability to maintain late-phase LTP induced by three trains of TS. These results reveal a novel OXTR-to-EGFR communication to regulate the new synthesis of PKMζ, which functions to promote the maintenance of LTP at hippocampal CA1 synapses.

Reciprocal Regulation of Annexin A2 and EGFR with Her-2 in Her-2 Negative and Herceptin-Resistant Breast Cancer

Alternative survival pathways are commonly seen to be upregulated upon inhibition of receptor tyrosine kinases (RTK), including Her-2. It is established that treatment with Herceptin leads to selective overexpression and activation of epidermal growth factor receptor (EGFR) and Src which further contributes to oncogenesis in Herceptin resistant and triple negative breast cancer (TNBC) patients. Here, we show a co-regulated upregulation in the expression of Annexin A2 (AnxA2), a known substrate of Src and one of the regulators of EGFR receptor endocytosis, in Herceptin resistant and Her-2 negative breast cancer. Immunohistochemical expression analysis revealed a reciprocal regulation between Her-2 and AnxA2 in breast cancer clinical samples as well as in cell lines as confirmed by protein and RNA analysis. The siRNA and Herceptin mediated downregulation/inhibition of Her-2 in Her-2 amplified cells induced AnxA2 expression and membrane translocation. In this study we report a possible involvement of AnxA2 in maintaining constitutively activated EGFR downstream signaling intermediates and hence in cell proliferation, migration and viability. This effect was consistent in Herceptin resistant JIMT-1 cells as well as in Her-2 negative breast cancer. The siRNA mediated AnxA2 downregulation leads to increased apoptosis, decreased cell viability and migration. Our studies further indicate the role of AnxA2 in EGFR-Src membrane bound signaling complex and ligand induced activation of downstream signaling pathways. Targeting this AnxA2 dependent positive regulation of EGFR signaling cascade may be of therapeutic value in Her-2 negative breast cancer.

Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation

BackgroundEpidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear.Methodology/Principal FindingsHere we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization.Conclusions/SignificanceCortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation.

Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR.

CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells

IntroductionOur previous study has demonstrated Cyclosporin A (CsA) promotes the proliferation of human trophoblast cells. Therefore, we further investigate the intracellular signaling pathway involved in the CsA-induced proliferation of human trophoblast cells.MethodsEnzyme-linked immunosorbent assay (ELISA) was performed to evaluate the regulation of CsA on CXCL12 secretion in human trophoblast cells. Immunofluorescence analysis and western blotting analysis were used to investigate the role of CXCL12/CXCR4 axis in the CsA-induced epidermal growth factor receptor (EGFR) phosphorylation in human trophoblast cells. 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay was performed to analyze the involvement of EGFR and its downstream extracellular signal-regulated protein kinase (ERK) signaling pathway in the CsA-induced proliferation of human trophoblast cells.ResultsLow concentration of CsA promoted the secretion of CXCL12, and recombinant human CXCL12 promoted the phosphorylation of EGFR in primary human trophoblast cells and choriocarcinoma cell line JEG-3. The inhibition of CXCL12 or CXCR4 by either neutralizing antibodies or small interfering RNA (siRNA) could completely block the CsA-induced EGFR phosphorylation. The CsA-induced proliferation of human trophoblast cells was effectively abrogated by the EGFR inhibitor AG1478 as well as the ERK inhibitor U0126, but not by the PI3K/PKB inhibitor LY294002. CsA promoted the activation of ERK in JEG-3 cells, which was markedly abrogated in the presence of CXCL12 siRNA, or CXCR4 siRNA, or AG1478.ConclusionsCsA may promote EGFR activation via CXCL12/CXCR4 axis, and EGFR downstream ERK signaling pathway may be involved in the CsA-induced proliferation of human trophoblast cells.

Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11–18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11–18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11–18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.