Effect Of Epidermal Growth Factor Receptor Activation Research Articles

MATRIX METALLOPROTEINASE (MMP-9) INDUCED INCREASE IN INTESTINAL EPITHELIAL TIGHT JUNCTION BARRIER IS MEDIATED BY EGFR ACTIVATION

Abstract BACKGROUND The defective intestinal tight junction (TJ) barrier has a key pathogenic factor in Crohn’s disease (CD) and other inflammatory conditions of the gut. Clinical studies in CD patients have shown that a persistent increase in intestinal permeability is predictive of poor clinical outcome. Matrix metalloproteinase-9 (MMP-9) has been shown to be markedly increased in intestinal tissues of patients with CD and it has been postulated that MMP-9 plays an important role on the pathogenesis of intestinal inflammation in CD. Our recent studies suggested that MMP-9 at clinically relevant concentrations causes an increase in intestinal epithelial TJ permeability in-vitro and in-vivo by inducing intracellular signaling pathways via activating distinct basolateral membrane receptors. However, the specific protein receptors and the intracellular signaling cascades involved have not been fully identified. One of the potential protein receptors that might be a target of MMP-9 on the basolateral membrane of the intestinal epithelial cells is the epidermal growth factor receptor (EGFR), which has been shown to have opposing roles in the pathophysiology of CD. AIMS Therefore, our aims in this study were to examine the effect of MMP-9 on EGFR activation and delineate the role of EGRF in MMP-9 induced increase in intestinal epithelial TJ permeability by using Caco-2 monolayers as an in-vitro model system. RESULTS MMP-9 (400 ng/ml) caused a time-dependent increase in EGFR activation as assessed by EGFR phosphorylation, and by in-vitro kinase activity measurement of EGFR in filter-grown Caco-2 monolayers. Inhibition of EGFR by pharmacologic inhibitor, PD 153035, prevented the MMP-9 induced increase in Caco-2 TJ permeability measured by trans-epithelial resistance (TER) and mucosal-to-serosal flux of a paracellular marker dextran 10kd. MMP-9 caused a decrease in the TJ protein occludin expression. Inhibition of EGFR by PD 153035 prevented the MMP-9 induced degradation of occludin expression. In addition, the effect of EGFR activation by a known activator, NSC 228155 25, in the absence of MMP-9, caused an increase in Caco-2 TJ permeability and a decrease in occludin expression. CONCLUSION Our data showed that MMP-9 induced increase in intestinal epithelial TJ permeability is mediated in part by the activation of EGFR and the subsequent degradation of occludin expression. Identifying the specific membrane receptors that mediate the MMP-9 induced increase in intestinal TJ permeability could be important in developing future therapeutic strategies in IBD.

Epidermal growth factor receptor and STAT3 signal through KRAS have mutually opposite effects on CTEN

Background: C-terminal tensin-like (CTEN) is a protein located at focal adhesions and has been reported to be an oncogene in the colon, breast, lung, and gastric cancer. In this study, we investigated whether two other proposed mechanisms, i.e., epidermal growth factor receptor (EGFR) and Signal transducer and activator of transcription 3 (STAT3) signaling were involved in regulating CTEN expression. Methodology: Initially, we manipulated EGFR signaling by (i) stimulation with epidermal growth factor (EGF) and (ii) inhibition by the PD153035 in the colorectal cancer cell lines SW620 and C32. In C32, EGF stimulation resulted in the upregulation of KRAS and CTEN, whereas exposure to PD153035 resulted in the downregulation of both KRAS and CTEN. EGFR activation and inhibition were reflected by, respectively, increased and decreased cell motility although the effect of EGFR activation was lost by CTEN knockdown. In SW620, which harbors a KRAS mutation, modulating EGFR activity in this way did not affect either KRAS or CTEN. STAT3 signaling has also been reported to positively regulate CTEN. We tested this in SW620 by directly knocking down STAT3 and exposing cells to interleukin-6 (an activator of STAT3). STAT3 knockdown resulted in increased CTEN, whereas STAT3 activation resulted in the downregulation of CTEN. Results: Testing for KRAS expression showed that STAT3 was negatively regulating KRAS, and this was reflected in the CTEN expression. Functional analysis, however, showed that the inhibition of STAT3 resulted in a reduction of cell motility in a K RAS and CTEN-independent manner. Conclusion: We conclude that both EGFR signals through KRAS to modulate CTEN (and consequently integrin- linked kinase/focal adhesion kinase) and stimulates cell motility. STAT3, however, negatively regulates KRAS and consequently CTEN although its net effect is to stimulate motility through an alternative mechanism.

EGFR-Signaling and Autophagy: How they Fit in the Cancer Landscape

Key significance of an overachieving Epidermal Growth Factor Receptor (EGFR)-signaling in cancer aggressiveness and poor prognosis is well recognized. In accordance, EGFR is either amplified or mutated in majority of the cancers of epithelial origin, and therefore has been recognized as a principal target for anticancer therapy. However, despite initial clinical efficacy of the anti-EGFR therapy in cancer treatment, long-term attempt to mute the cancer boosting effects of EGFR-dependent signaling meets resistance in cancer cells. Notably, effects of EGFR activation are pleotropic. Also, under conditions of anti-EGFR therapy in cancer cells, feedback activation of the pro-survival signaling by activation of other growth factor receptors can occur. However, a critical role of autophagy in the resistance against anti-EGFR therapy is fast emerging. Interestingly, EGFR regulates autophagy in a context-dependent manner. Furthermore, EGFR deregulated tumors demonstrate differential dependence upon autophagy for their survival and growth. Also, inhibiting EGFR-signaling promotes autophagy. These intriguing considerations are complicated further by findings that EGFR regulates autophagy in kinase-dependent or independent manner. Thus, for effective clinical cancer treatment using anti-EGFR regimen, it is critical that we understand molecular details of the nexus between the EGFR-signaling and autophagy.

Epidermal growth factor receptor (EGFR) and Stat3 signal through Kras and have mutually opposite effects on Cten

BackgroundCten is a protein located at focal adhesions and has beenreported to be an oncogene in colon, breast, lung andgastric cancer [1-3]. We have previously shown thanCten is a target of K-ras in colorectal and pancreatic can-cer [4]. In this study, we investigated whether two otherproposed mechanisms i.e. EGFR and Stat3 signaling wereinvolved in regulating Cten expression.Materials and methodsEGF was used to stimulate the EGFR while the PD153035to inhibit the EGFR. On the other hand, IL-6 used to sti-mulate the STAT3 and siRNA for STAT3 was used forknockdown. The effect was confirmed using Western blotand QPCR and cell motility was assessed by transwellchambers.ResultsInitially we manipulated EGFR signaling by (i) stimulationwith EGF and (ii) inhibition by the PD153035 in the color-ectal cancer cell lines SW620 and C32. In C32, EGF stimu-lation resulted in up-regulation of Kras and Cten whilstexposure to PD153035 resulted in down-regulation of bothKras and Cten. EGFR activation and inhibition wasreflected by, respectively, increased and decreased cellmotility although the effect of EGFR activation was lost byCten Knockdown. In SW620, which harbours a KRASmutation, modulating EGFR activity in this way had noeffect on either Kras or Cten. Stat3 signaling has also beenreported to positively regulate Cten. We tested this inSW620 by directly knocking down Stat3 and exposing cellsto interleukin-6 (an activator of Stat3). Stat3 knockdownresulted in increased Cten whilst Stat3 activation resultedin downregulation of Cten. Testing for Kras expressionshowed that Stat3 was negatively regulating Kras and thiswas reflected in the Cten expression. Functional analysishowever showed that inhibition of Stat3 resulted in areduction of cell motility in a Kras and Cten-independentmanner.ConclusionsWe conclude that both EGFR signals through Krasto modulate Cten (and consequently ILK/FAK) and stimu-lates cell motility. Stat3 however negatively regulates Krasand consequently Cten although its net effect is tostimulate motility through an alternative mechanism.

Abstract 5542: Activation of epidermal growth factor receptor potentiates effects of radiation in A549 lung cancer cells

Abstract Introduction : For patients with advanced non-small cell lung cancer (NSCLC), chemotherapy alone or in combination with radiation (IR) is considered the standard treatment. However, after the documentation of the over-expression of epidermal growth factor receptor (EGFR) in 50% to 80% of NSCLCs and transforming growth factor-alpha in 81% to 93% of NSCLCs, EGFR has emerged as an important target molecule in NSCLC therapy. The cellular effects of EGFR activation include increased proliferation, promotion of cell motility, adhesion, invasion, angiogenesis and enhanced cell survival. However, activation of EGFR in some tumor cell lines excessively expressing EGFR resulted in cellular apoptosis and growth inhibition. This study attempts to define the role of EGFR kinase activity in sensitizing the responsiveness of EGFR-overexpressed NSCLC cells to IR. Methods: Cell survival, proliferation, cell cycle distribution, reporter activity of EGR-1 and protein expression was studied by colony forming assays, cell counting, flow cytometry, 14Cchloramphenicol acetyl transferase (CAT) reporter assay and Western analysis respectively in A549 and H460 cells. Results: Interestingly, we observed that EGFR activation mediated sensitization of IR effects in A549 cells over-expressing EGFR. Tyrosine kinase inhibitor (TKI), AG1478 blocked EGF effect on sensitization of IR in A549 cells but not in H460 cells. To understand the molecular mechanism underlying the response of A549 cells to IR and EGFR, we investigated the effects of EGFR signaling and IR on expression of early growth response (EGR-1), a transcription factor that sensitizes IR therapy, in both IR-sensitive and insensitive lung cancer cell lines. However, we observed that EGR-1 is not involved in EGFR signaling-mediated IR sensitization since inhibition of Erk (EGFR induces expression of Egr-1 through activation of Erk1/2), did not block EGFR-mediated sensitization of IR effect. Further, we observed that JNK kinase activity mediated EGF-dependent IR effects. Interestingly, acute induction of serum EGF expression level was observed in the patients who responded to IR therapy. Conclusions: Results from this study show that EGFR activation and IR synergistically activate JNK signaling that leads to cellular apoptosis that has important implications for anti-EGFR therapy or IR therapy. Although, preclinical studies have shown that EGFR inhibition can render an additive or synergistic effect with radiotherapy in NSCLCs, clinical benefits are modest. This study suggests that tyrosine kinase activity of EGFR might be required for IR sensitivity. Thus, the inhibition of EGFR by TKIs may lead to reduced responsiveness of NSCLCs to IR thereby compromising the treatment efficacy. Understanding the role of EGFR activity in sensitization of NSCLC cells to IR is therefore extremely important for both radiotherapy and the EGFR TKI-based chemotherapy. 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 5542.

Abstract 261: Epidermal growth factor receptor (EGFR)-mediated upregulation of matrix metalloproteinase-1(MMP-1) in glioblastoma cell lines involves multiple signaling pathways

Abstract Glioblastoma Multiforme (GBM) is a devastating disease with an extremely poor prognosis primarily due to its invasive and infiltrative nature. Matrix metalloproteinases (MMPs) are important enzymes that regulate tumor microenvironment, alter cellular behavior and increase invasion and metastasis in wide variety of cancers. Previous studies from our laboratory have shown that MMP-1 is over expressed in GBM tissue and can influence glioma cell invasion in vitro. Since the epidermal growth factor receptor (EGFR) is amplified in human GBM, we wanted to determine the effect of EGFR activation by epidermal growth factor (EGF) on MMP-1 expression and invasion in human glioma cell lines and if so, determine the signaling mechanisms responsible for this induction. We stimulated glioma cell lines (T98 and U87) with EGF in the presence and absence of the EGFR inhibitor, AG1478. Inhibitors to multiple signaling pathways activated by EGFR induction were also used. All inhibitors tested were added to cells for 2 hrs prior to addition of EGF. In addition, transfections using AKT and STAT3 siRNA were conducted. Western Blotting was performed to examine the levels of MMP-1, EGFR, AKT, STAT3 and ERK. We observed that with EGF treatment, in addition to EGFR activation, AKT was phosphorylated, MMP-1 was induced and that in the presence of AG1478, AKT and EGFR phosphorylation was inhibited in a dose dependent manner. Importantly, MMP-1 up-regulation by EGF was abrogated in cells pretreated with AG1478. EGFR has been shown to up-regulate MAPK, PI3K and STAT3 pathways in glioma and other malignancies. Involvement of the MAPK pathway in the EGFR- mediated induction of MMP-1 was examined using the MAPK inhibitor, U0126. Only a modest decrease in MMP-1 levels was observed following treatment with EGF and U0126, suggesting potential inputs from other signaling pathways. Although there was a modest inhibition of AKT protein levels in cells treated with AKT siRNA, the levels of MMP-1 in these cells were significantly reduced. Current experiments focus on the role of AKT/PI3K and STAT3 signaling pathways in MMP-1 mediated glioma cell invasion. 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 261.

EGF Receptor Activation Decreases Retroviral Gene Transfer through Protein Kinase C-δ

Although much progress has been made in the design of retrovirus vectors, the interactions of recombinant retrovirus with host cells remain largely elusive. The inability of recombinant retrovirus to transduce non-dividing cells prompted several studies to determine optimal cocktails of growth factors and/or extracellular matrix molecules to promote gene transfer to slowly diving cells and stem cells. In contrast to previous reports that growth factors increased gene transfer, we found that treatment of human epidermal keratinocytes and several cell lines with epidermal growth factor receptor (EGFR) ligands EGF, transforming growth factor-α, or heparin-binding-EGF decreased gene transfer. Conversely, treatment with an EGFR function-blocking antibody or inhibition of EGFR tyrosine phosphorylation enhanced gene transfer in a dose-dependent manner. In addition, blocking protein kinase C (PKC)-δ but not PKC-ζ, with chemical inhibitors or small interfering RNA reversed the effects of EGF and restored gene transfer, indicating that the effect of EGFR activation is mediated through PKC-δ. Lastly, cell cycle analysis showed that the effect of EGFR activation on retroviral gene transfer was independent of the cell cycle status of target cells. Our results implicate EGFR and PKC-δ in retroviral infection and may have implications for retrovirus gene transfer or design of antiretroviral therapies.

PTEN mutation cooperates with EGFR activation in human glioblastoma cells to increase VEGF mRNA levels by transactivating an element in the proximal promoter

Purpose: Glioblastomas often express high levels of vascular endothelial growth factor (VEGF), even under normoxic conditions. Genetic alterations that commonly occur in these tumors, including PTEN mutations and epidermal growth factor receptor (EGFR) overexpression, may contribute to this increased VEGF expression. Our previous work showed that, compared to parental U87MG human glioblastoma cells, VEGF mRNA levels are decreased in U87/T691, a derivative line in which EGFR signaling is inhibited by introduction of a truncated p185Neu protein (Cancer Research 60: 5879-5886, 2000). In that study, we also showed that the effect of EGFR activation on VEGF was mediated at the level of transcription via a PI3K dependent pathway. The purpose of the current study was to assess the role of PTEN, a negative regulator of PI3K, and its interaction with EGFR activation in regulating VEGF expression. Materials and Methods: Protein lysates were obtained from U87MG and U87/T691 cells for use in Western blotting for phosphorylated Akt. U87MG and U87/T691 cells were infected with adenovirus expressing either wildtype PTEN or phosphatase dead PTEN, then RNA was harvested for Northern blotting for VEGF. Nuclear extracts were also obtained from these cells for use in gel shift assays. Luciferase assays were performed with lysates of U87MG cells transfected with luciferase reporter constructs containing fragments of the VEGF promoter. Results: The level of phosphorylated Akt, a marker for PI3K activation, was lower in U87/T691 cells compared to U87MG cells, an expected result because of inhibition of the EGFR in the former. Treatment of U87/T691 cells with the PI3K inhibitor LY294002 further decreased the level of phosphorylated Akt and VEGF mRNA. Therefore, in spite of EGFR inhibition, U87/T691 cells contain residual PI3K activity that regulates VEGF expression. To determine whether the PTEN status of these cells influences VEGF mRNA levels, wildtype PTEN was introduced into U87MG and U87/T691 cells using adenovirus, This resulted in a decrease in both phosphorylated Akt and VEGF mRNA levels. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing either wildtype PTEN or dominant negative PI3K decreased activity of a luciferase reporter containing −88/+54 bp of the VEGF promoter (+1 is the transcription start site). LY294002 also blocked induction of activity of this reporter by EGF stimulation. Gel shift assays were also performed using nuclear lysates of U87MG cells infected with either wildtype or phosphatase dead PTEN adenovirus. In these assays, the radioactively labeled probe used was an oligonucleotide spanning −88 to −65. These assays showed that introduction of wildtype PTEN decreased binding of a protein complex to this region in the promoter. Conclusion: In human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally upregulating the proximal VEGF promoter (−88/+54) via a PI3K-dependent pathway. Furthermore, PI3K activation leads to increased binding of a protein complex to a segment of the promoter located between −88 and −65.