Induced Epidermal Growth Factor Receptor Research Articles

17-β-estradiol and phytoestrogens elicit NO production and vasodilatation through PI3K, PKA and EGF receptors pathways, evidencing functional selectivity

Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-β-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2′,7′-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.

Citrus sudachi Peel Extract Suppresses Cell Proliferation and Promotes the Differentiation of Keratinocytes through Inhibition of the EGFR-ERK Signaling Pathway.

Citrus sudachi is a well-known fruit in Tokushima Prefecture, Japan, and its peels are rich in phytochemicals, including phenolic compounds. Although it is expected that the extract of the C. sudachi peel elicits various beneficial physiological activities, the effect on the skin has not been investigated. In this study, we report that the aqueous extract from the peel of C. sudachi suppresses cell proliferation of the immortalized human keratinocyte cell line, HaCaT, and primary normal human epidermal keratinocytes. The extract of C. sudachi peel suppressed epidermal growth factor (EGF)-induced EGF receptor activation and tumor necrosis factor (TNF)-α-induced extracellular regulated kinase (ERK) 1/2 activation, which suggests that the extract exerts its inhibitory effect through inhibition of both the EGF receptor (EGFR) and its downstream molecules. Additionally, the extract of C. sudachi peel potentiated calcium-induced keratinocyte differentiation. These results suggest that the extract of C. sudachi peel may have beneficial effects against skin diseases that are characterized by hyperproliferation of epidermal keratinocytes, such as those seen in psoriasis and in cutaneous squamous cell carcinoma.

Piperonylic acid stimulates keratinocyte growth and survival by activating epidermal growth factor receptor (EGFR)

Epidermal growth factor (EGF) stimulates cell growth, proliferation, and survival. The biological benefits of EGF have been utilized in medical uses for improving wound healing as well as in today’s skin cosmetics. EGF has been found in urine, saliva, milk, and plasma, but its efficient isolation remains a difficult task. With technical advances, recombinant protein purification technique has been used for EGF production. However, the recombinant EGF is still expensive and keeping it with stable activity is difficult to be used widely. Thus, a molecule that can mimic the EGF activity would be a useful alternative of EGF. Herein, we have discovered that a natural small molecule piperonylic acid shows EGF-like activity in HaCaT keratinocytes. Piperonylic acid induced EGF receptor (EGFR) activation and resulted in serial activation of the downstream modulators. The activated signaling pathway eventually up-regulated gene expression of egr-1, c-fos, c-jun, and c-myc, which are involved in cell growth and survival. Moreover, piperonylic acid showed promoting role in keratinocyte growth and survival from UVB-induced cellular damages. This study has revealed the EGF-like activity of piperonylic acid and proposed that the piperonylic acid could be a promising component for skin wound healing agents or cosmetic ingredient.

Abstract A65: A novel regulatory mechanism involving Ras-mediated activation of the zinc-finger transcription factor, SAF-1/MAZ induces EGFR/HER1 expression in breast cancer cells

Abstract Tumor microenvironment (TME) plays a critical role in tumor growth, invasion and metastasis. In TME, epidermal growth factor receptor (EGFR) family members, including HER1, HER2, HER3 and HER4, are involved in determining aggressive growth of breast cancer due to their ability to transduce the growth promoting functions of growth factors. This activity is potentiated by the over-expression of these receptor molecules in cancer cells. To reduce the activity of EGFR molecules, various inhibitors have been developed. EGFR/HER1 tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, show antitumor activity but these drugs have not meet their primary goal of improved survival in the overall patient population. A similar TKI, lapatinib, has shown some limited success in breast cancer. While these therapeutic options focus on reducing the functional activity of EGFR protein, the fundamental and major problem of over-expression of EGFR in cancer cells has not been addressed. In two-thirds of aggressive breast cancer patients, transcriptional induction of EGFR causes high EGFR/HER1 level. Therefore, the goal of this study is to identify a molecular mechanism by which EGFR is over-expressed in breast cancer cells. To understand how transcriptional induction might occur, we have explored a novel biosynthetic pathway for EGFR over-expression. Our findings reveal that EGFR promoter is significantly more active in MDA-MB-231 cells in comparison to MDA-MB-468 cells. When transcription factor SAF-1 was ectopically expressed in these cells, EGFR promoter activity was further increased in MDA-MB-231 cells. Since MDA-MB-231 cells contain a highly active form of Ras, the data suggested a possible Ras-mediated activation of SAF-1 which in turn induces EGFR expression. Consistent with these findings, inhibition of K- and H-Ras, by using both siRNA and CRISPR/Cas9-mediated knock-out systems, reduced the expression of EGFR in MDA-MB-231 cells. The effect was more profound when K-Ras was targeted for inhibition. Since SAF-1/MAZ is seen to be activated by Ras, our data implicates K-Ras - SAF-1/MAZ - EGFR axis in breast cancer cell growth. Our findings may provide new targets for breast cancer therapy. Supported by grants from College of Veterinary Medicine Faculty Research Award Grant and MU Center for Botanical Interaction Studies Pilot Project Grant. Citation Format: Alpana Ray, Brett Havis, Bimal Ray. A novel regulatory mechanism involving Ras-mediated activation of the zinc-finger transcription factor, SAF-1/MAZ induces EGFR/HER1 expression in breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A65.

Cyclic GMP Dependent Protein Kinase (PKG) as a mediator of EGFR-Induced Apoptosis in Breast Cancer

Background The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR mediated apoptosis in response to EGF ligand. Previous studies within our laboratory have shown that when the EGFR is activated and retained at the cell membrane, MDA-MB-468 cells undergo cell growth. Conversely, once the activated EGFR is internalized within endosomes, the cells undergo apoptosis [1]. This contrasting response at subcellular locations is defined as spatial regulation. The goal of this project is to identify the kinases responsible for the induction of apoptosis from within the endosomes. Research has shown that cGMP dependent protein kinase (PKG) activation within this cell line induces apoptosis in a dose dependent manner [2]. We hypothesize that PKG plays an intermediate role in the EGFR induction of apoptosis.

Abstract 2201: EGFR regulates the expression and activity of ARID3B through phosphoinositide 3-kinase

Abstract ARID3B is a transcription factor that is upregulated in ovarian cancer and its expression correlates with relapse following chemotherapy. However, the mechanism that leads to ARID3B's elevated expression in ovarian cancer has not been elucidated. EGFR is commonly overexpressed in ovarian cancer, as is phosphoinositide 3-kinase (PI3K), lending us to believe that this pathway may be regulating ARID3B. We demonstrated that epidermal growth factor receptor (EGFR) signaling induces expression of ARID3B through the ETS family transcription factor PEA3. However, the exact mechanism that controls EGFR induction of ARID3B has not been elucidated, and the impact of EGFR on ARID3B activity is unknown. To find the optimal time point for EGF induction of ARID3B mRNA, ovarian cancer cells were serum-starved and then treated with EGF for different amounts of time. We then examined the expression of ARID3B mRNA induced by this treatment in each case. We found that ARID3B was most strongly induced between 2 and 4 hours after treatment with EGF. We then performed dose curve experiments for the PI3K inhibitors, wortmannin and LY294002. We performed qRT-PCR to examine ARID3B mRNA expression under the different concentrations of inhibitors. Western blots for the presence of phosphorylated Akt under each concentration demonstrated the effectiveness of the inhibitors. Next, ovarian cancer cells were serum-starved and then treated with EGF or EGF in combination with PI3K inhibitors. Both immunoprecipitation and chromatin immunoprecipitation (ChIP) were performed on these samples. Western blot for phospho-serine was conducted on immunoprecipitated samples to test for serine phosphorylation of ARID3B in response to growth factor signaling. We found that ARID3B is serine phosphorylated. Next, we performed ChIP to assess the binding of ARID3B to select target genes. We found on the genes we tested, EGF treatment decreased DNA promoter binding. Binding was greatest in the serum-starved samples and the samples treated with the PI3K inhibitors. Very little binding was seen in the EGF treated cells. These results were consistent for a number of ARID3B target genes. In future studies, we will determine which serine residues are phosphorylated by doing a mass spectrometric analysis. Then, site directed mutagenesis will be conducted to test the requirement of serine phosphorylation to target gene regulation. By understanding how ARID3B functions as a transcription factor, we will gain critical insight into ovarian cancer relapse and progression. Citation Format: William M. Hallas, Karen Cowden Dahl. EGFR regulates the expression and activity of ARID3B through phosphoinositide 3-kinase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2201. doi:10.1158/1538-7445.AM2015-2201

Abstract 4993: β-arrestin-2 regulates CXCR7-mediated EGFR transactivation and tumor cell proliferation in prostate cancer cells

Abstract We have previously shown that the chemokine receptor CXCR7 trans-activates the epidermal growth factor receptor (EGFR) and is implicated in prostate tumor growth. However, the molecular mechanisms of CXCR7 mediated EGFR-trans-activation are not known. In the present work we show that CXCR7 induced EGFR transactivation is independent of EGF-like ligands such as amphiregulin and heparin-bound EGF. Importantly, treatment with pertussis toxin, a known G-protein inhibitor, had no effect on EGFR phosphorylation, indicating that G-protein signaling was not involved in CXCR7 mediated EGFR transactivation. Since CXCR7 is known to signal through β-arrestin-2 in its ligand dependent manner, we evaluated the role of β-arrestin-2 in CXCR7 induced EGFR trans-activation in prostate cancer cells. We used shRNA to stably down regulate β-arrestin-2 in LNCaP cells to evaluate the role of β-arrestin-2 in CXCR7 mediated induction of EGFR and downstream mitogenic signaling such as ERK1/2 and the phosphoinositide 3-kinase/Akt pathway. Interestingly, down regulation of β-arrestin-2 significantly induced phosphorylation of EGFR at Y1110 and Y1068 along with the phosphorylation/activation of ERK and AKT and an induction of proliferation/colony formation in prostate tumor cells. Moreover, shRNA mediated down regulation of β-arrestin-2 resulted in a decrease of p21 which is in accordance with the observed increased proliferation rate. The results of our study suggest that β-arrestin-2 is crucial negative regulator of CXCR7/EGFR signaling and underscore the importance of β-arrestin-2 in regulation of CXCR7/EGFR mediated tumor cell proliferation. Citation Format: Georgios Kallifatidis, Daniel Munoz, Rajendra K. Singh, Bal L. Lokeshwar. β-arrestin-2 regulates CXCR7-mediated EGFR transactivation and tumor cell proliferation in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4993. doi:10.1158/1538-7445.AM2015-4993

COX-2 and EGFR: Partners in Crime Split by Aspirin

COX-2 and EGFR: Partners in Crime Split by Aspirin

Abstract NG01: Blockade of EGFR and HER3 enhances PI3K/Akt antitumor activity in triple negative breast cancer

Abstract NG01: Blockade of EGFR and HER3 enhances PI3K/Akt antitumor activity in triple negative breast cancer

Inhibition of the αvβ6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-β (TGF-β) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-β induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-β pathway in the lung is unknown. The αvβ6 integrin is an important in vivo activator of TGF-β activation in the lung. Immunohistochemical analysis of αvβ6 protein expression and bronchoalveolar analysis of TGF-β pathway signaling indicates activation of the αvβ6/TGF-β pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvβ6/TGF-β pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvβ6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvβ6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the β6 integrin, TGF-α transgenic mice were mated with β6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the β6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the β6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvβ6/TGF-β pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

HB‐EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a vascular-derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia-inducible proteins released by astrocytes in CNS injuries. It was suggested that HB-EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB-EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB-EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia-like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT-qPCR analysis demonstrated that HB-EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB-EGF-mediated astrocyte response. HB-EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de-differentiation of astrocytes.

Bradykinin-induced asthmatic fibroblast/myofibroblast activities via bradykinin B2 receptor and different MAPK pathways

Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B2 receptor expression by Western blot analysis; cell proliferation by [3H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively.Constitutive bradykinin B2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B2 receptor expression in HBAFb. Bradykinin, via bradykinin B2 receptor, significantly increased fibroblast proliferation at lower concentration (10−11M) and α-SMA expression/polymerization at higher concentration (10−6M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation.These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.

Superposition of an incoherent magnetic field inhibited EGF receptor clustering and phosphorylation induced by a 1.8 GHz pulse-modulated radiofrequency radiation

Purpose: The present study was conducted to investigate the effect of a temporally incoherent (‘noise’) magnetic field (MF) on radiofrequency radiation (RFR)-induced epidermal growth factor (EGF) receptor clustering and phosporylation in cultured cells.Materials and methods: Human amniotic epithelial (FL) cells were exposed for 15 min to either a 1.8 GHz RFR (modulated at 217 Hz), a 2 μT incoherent MF, or concurrently to the RFR and incoherent MF. Epidermal growth factor treatment severed as the positive control. Epidermal growth factor receptor clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining, and phosphorylation of EGF receptors was measured by western blot technology.Results: Exposure of FL cells to the 1.8 GHz RFR at SAR (specific absorption rate) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min induced EGF receptor clustering and enhanced phosphorylation on tyrosine-1173 residue, whereas exposure to RFR at SAR of 0.1 W/kg for 15 min did not significantly cause these effects. Exposure to a 2 μT incoherent MF for 15 min did not significantly affect clustering and phosphorylation of EGF receptor in FL cells. When superimposed, the incoherent MF completely inhibited EGF receptor clustering and phosphorylation induced by RFR at SAR of 0.5, 1.0, and 2.0 W/kg, but did not inhibit the effects induced at SAR of 4.0 W/kg.Conclusion: Based on the data of the experiment, it is suggested that membrane receptors could be one of the main targets by which RFR interacts with cells. An incoherent MF could block the interaction to a certain extent.

P4-01-14: Fulvestrant Regulates Epidermal Growth Factor (EGF) Ligands and Induces EGF Receptor Activation in MCF-7 Breast Cancer Cells.

Abstract Selective Estrogen Receptor Modulators (SERMs) 4 Hydroxy Tamoxifen (4-HT) and fulvestrant (fulv) inhibit estrogen receptor a (ER) positive breast cancer growth. We have observed that treatment (24 to 48 hours) of fulv in MCF-7 cells induced a 190kDa tyrosine phosphorylated band, which was blocked by EGFR and HER-2 tyrosine kinase inhibitors erlotinib and lapatinib. Immunoprecipitation showed EGFR, HER-2, and HER-3 were all phosphorylated after fulv treatment. 4-HT and estradiol did not cause this phosphorylation. No changes in receptor level after fulv treatment were noted. Downstream MAPK signaling was also blocked by erlotinib and lapatinib. Fulv induced activation of EGFR was ER dependent, since fulv treatment in C4-12, an ER negative cell line derived from MCF-7 cells, did not induce EGFR activation. Co-treatment with estradiol and fulv prevented EGFR activation. To explore the possibility that fulv enhanced ligand expression, we collected conditioned media (CM) from MCF-7 cells after 48 hours of treatment. pEGFR was lost when CM was removed, but recurred within 30 minutes. Cycloheximide abolished the ability of fulv to activate EGFR suggesting autorcine production of EGFR ligands was induced by fulv. To detect specific EGFR ligands, we used qPCR to measure various EGFR ligand mRNA levels in MCF-7 and C4-12 cell at 1, 4, 24 and 48 hours after fulv treatment. TGF-α and HB-EGF mRNAs were upregulated over 48 hours, which correlated well with pEGFR activation. 4-HT did not affect mRNA levels of these ligands. In contrast, amphiregulin (AREG) mRNA levels were substantially reduced 48 hours after fulv treatment. A similar trend was seen for AREG mRNA levels in 4HT treated cells but to a much lesser extent. There was no change in any EGFR ligand mRNA levels in ER negative C4-12 cells. These qPCR data suggested differential regulation of EGFR ligands by fulv treatment contributed to EGFR family member activation and was dependent on ER expression. Upon fulv treatment, levels of ER were diminished with decreased detection of S167 and S118 ER phosphorylation sites. Monolayer cell growth analysis showed that while fulv treatment in SFM reduced MCF-7 cell numbers compared to control treatment, erlotinib plus fulv significantly reduced cell numbers below the level of cells maintained in fulv alone. In conclusion, we show that fulv, but not E2 or 4-HT, activates EGFR family members accompanied by upregulation of ligands. Since SERM resistance has been associated with EGFR family member activation, differential control of EGFR ligand gene expression by ER may contribute to the development of fulvestrant resistance in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-14.

A 1.8-GHz radiofrequency radiation induces EGF receptor clustering and phosphorylation in cultured human amniotic (FL) cells

Purpose: Many studies have shown that exposure to radiofrequency radiation (RFR) could activate cellular signal transduction pathways. In the present research, we investigated the effects of exposure to a 1.8-GHz RFR at different intensities on epidermal growth factor (EGF) receptor clustering and phosphorylation in human amniotic (FL) cells.Materials and methods: Receptor clustering on cellular membrane surface was analyzed using immunofluorescence assessed by confocal microscopy, and phosphorylation of EGF receptors was measured by western blot technology. EGF treatment served as a positive control.Results: The results showed that, compared with sham exposure, exposure to RFR at specific absorption rate (SAR) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min significantly induced EGF receptor clustering and enhanced phosphorylation on the tyrosine-1173 residue in FL cells, whereas exposure to a SAR 0.1 W/kg radiation for 15 min did not cause a significant effect.Conclusion: Based on the results of this experiment, we conclude that membrane receptors could be one of the main targets that RFR interacts with cells, and the dose-rate threshold, in the case of EGF receptors, is between SAR of 0.1 and 0.5 W/kg. The results indicate a sigmoid dependence of RFR effects on intensity.

PTP1B Targets the Endosomal Sorting Machinery: DEPHOSPHORYLATION OF REGULATORY SITES ON THE ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT COMPONENT STAM2

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated STAM2 specifically suppressed Akt activation, and a phosphorylation-deficient STAM2 mutant displayed prolonged localization on endosomes following EGF stimulation. These results reveal a novel link between the dephosphorylation and endocytic machinery and suggest that PTP1B can affect RTK signaling in a previously unrecognized manner.

Epstein-Barr Virus Latent Membrane Protein 1 Modulates Distinctive NF-κB Pathways through C-Terminus-Activating Region 1 To Regulate Epidermal Growth Factor Receptor Expression

Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1) is required for EBV B-lymphocyte transformation, transforms rodent fibroblasts, and can induce lymphoma and epithelial hyperplasia in transgenic mice. Two domains have been identified within the intracellular carboxy terminus that can activate NF-kappaB, C-terminus-activating region 1 (CTAR1) and CTAR2, through interactions with tumor necrosis receptor-associated factors (TRAFs). CTAR1 can activate both the canonical and noncanonical NF-kappaB pathways and has unique effects on cellular gene expression. The epidermal growth factor receptor (EGFR) is highly induced by LMP1-CTAR1 in epithelial cells through activation of a novel NF-kappaB form containing p50 homodimers and Bcl-3. To further understand the regulation of NF-kappaB in CTAR1-induced EGFR expression, we evaluated the ability of CTAR1 to induce EGFR in mouse embryonic fibroblasts (MEFs) defective for different NF-kappaB effectors. CTAR1-mediated EGFR induction required the NF-kappaB-inducing kinase (NIK) but not the IkappaB kinase (IKK) complex components that regulate canonical or noncanonical NF-kappaB pathways. CTAR1-mediated induction of nuclear p50 occurred in IKKbeta-, IKKgamma-, and NIK-defective MEFs, indicating that this induction is not dependent on the canonical or noncanonical NF-kappaB pathways. EGFR and nuclear p50 were expressed at high levels in TRAF2(-/-) fibroblasts and were not induced by CTAR1. In TRAF3(-/-) MEFs, CTAR1 induced nuclear p50 but did not affect basal levels of STAT3 serine phosphorylation or induce EGFR expression. EGFR was induced by LMP1 in TRAF6(-/-) MEFs. These findings suggest that this novel NF-kappaB pathway is differentially regulated by TRAF2 and TRAF3, and that distinct interactions of LMP1 and its effectors regulate LMP1-mediated gene expression.

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.

Protein phosphatase 2A PR130/B”α:1 subunit binds to the SH2 domain‐containing inositol polyphosphate 5‐phosphatase 2 and prevents epidermal growth factor (EGF)‐induced EGF receptor degradation sustaining EGF‐mediated signaling

To elucidate novel cell biological functions of specific protein phosphatase 2A (PP2A) holoenzymes, we identified and biochemically characterized a complex between the Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2) and a PP2A holoenzyme comprising PR130/B''alpha1 as a regulatory subunit (PP2A(T130)) in several mammalian cell lines. PR130/B''alpha1 and SHIP2 partially colocalize in untreated HeLa cells, and both translocate to the cell membrane on epidermal growth factor (EGF) stimulation. Concomitantly, a transient EGF-dependent interaction of PR130/B''alpha1 with the EGF receptor (EGFR) was observed, whereas the SHIP2-PR130 interaction remained constitutive. As previously reported for SHIP2, RNA interference-mediated knockdown of PR130 in COS-7 cells resulted in increased EGF-induced proteasome-dependent EGFR degradation, and an increased interaction of EGFR with the E3 ligase c-Cbl. In concordance with faster EGFR clearance or desensitization, intrinsic EGFR kinase activity (phospho-Tyr-1068) and downstream protein kinase B and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways were more rapidly inactivated in PR130-knockdown cells. Notably, these effects could be rescued by reintroduction of RNA interference-resistant Myc-PR130, excluding any off-target effect. These data highlight a novel biological role of the PP2A(T130) holoenzyme in EGF signaling through interaction with EGFR and the phosphatidylinositol (3,4,5)-trisphosphate 5-phosphatase SHIP2. This interaction may be of clinical relevance as dysfunction of EGF-mediated signaling has been linked to various human cancers.

An Incoherent Magnetic Field Inhibited EGF Receptor Clustering and Phosphorylation Induced by a 50-Hz Magnetic Field in Cultured FL Cells

Previously, we found that exposure to a 50-Hz magnetic field (MF) at 0.4 mT could induce epidermal growth factor (EGF) receptor clustering in Chinese hamster lung (CHL) fibroblast cells and superposition of an incoherent MF with the same intensity could inhibit the effect. In the present experiment, we investigated the effects of 50-Hz MF exposure at different intensities on EGF receptor clustering and phosphorylation in human amniotic cells (FL), and explored the interaction effect of an incoherent MF. Clustering and phosphorylation of EGF receptors on cellular membrane surface were analyzed using immunofluorescence assessed by confocal microscopy and western blot technology, respectively. EGF treatment served as a positive control. The results showed that, compared with sham exposure, exposure to a 50-Hz MF at 0.1, 0.2 or 0.4 mT for 15 min could significantly induce EGF receptor clustering and enhance phosphorylation on tyrosine-1173 residue in FL cells, whereas exposure to a 0.05 mT field for 15 min did not caused a significant effect. Exposure to an incoherent MF (frequency range between 30 to 90 Hz) at 0.2 mT for the same time neither induced EGF receptor clustering nor enhanced phosphorylation of EGF receptor in FL cells. When superposed, the incoherent MF at 0.2 mT completely inhibited EGF receptor clustering and phosphorylation induced by a 50-Hz MF at 0.1 and 0.2 mT. However, the incoherent MF could not completely eliminate the effects induced by a 0.4 mT 50-Hz MF. Based on the results of this experiment, we conclude that membrane receptors could be one of the main targets where extremely-low frequency (ELF) MF interacts with cells, and the intensity threshold, in the case of EGF receptors, is between 0.05 and 0.1 mT. An incoherent MF could completely inhibit the effects induced by an ELF-MF of equal or lower intensity.