Epidermal Growth Factor Induces Internalization But Not Degradation of the Epidermal Growth Factor Receptor in a Human Breast Cancer Cell Line

Abstract

Metabolism of the epidermal growth factor (EGF) receptor was studied in the MDA-MB-231 human breast cancer cell line. As in normal fibroblasts the EGF receptor from MDA-MB-231 cells was synthesized from a M_r =160,000 precursor and tunicamycin treatment of cells resulted in accumulation of a M_r =130,000 polypeptide. Unlike normal fibroblasts in which a M_r =170,000 mature form of the EGF receptor was found, MDA-MB-231 cells contained a M_r =172,000 mature form. Addition of EGF to MDA-MB-231 cells led to rapid internalization of EGF receptors, however, internalization did not affect receptor half-life and receptors did not recycle to the cell surface. EGF receptors could be visualized by immunofluorescence and remained sequestered in intracellular membranous structures following internalization. EGF was degraded slowly by MDA-MB-231 cells relative to degradation of EGF by normal cells. A high endogenous level of in vivo phosphorylation of threonine 654 of the EGF receptor was found in MDA-MB-231 cells and treatment of cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further stimulated phosphorylation of this residue. EGF induced receptor internalization resulted in dephosphorylation of threonine 654. The significance of these unusual properties of EGF receptor metabolism in MDA-MB-231 cells is discussed.     

Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis

Proliferating cell nuclear antigen (PCNA) is an essential component for DNA synthesis upon growth stimulation. It has been shown that phosphorylation of PCNA at Tyr-211 by the EGF receptor (EGFR) protects PCNA from polyubiquitylation and degradation, whereas blocking phosphorylation induces ubiquitylation-mediated degradation of the chromatin-bound, but not the -unbound, PCNA, and suppresses cell proliferation. However, the ubiquitin E3 ligase linking growth signaling to the proteolysis of PCNA and the underlying regulatory mechanism remain to be identified. Here we show that, in the absence of Tyr-211 phosphorylation, PCNA is subject to polyubiquitylation at Lys-164 by the CUL4A E3 ligase, resulting in the degradation of PCNA. Mutation of Lys-164 to arginine prevents PCNA ubiquitylation and rescues the degradation of the K164R/Y211F PCNA double mutant. Activation of EGFR inhibits the interaction of PCNA with CUL4A, whereas inhibition of EGFR leads to increased CUL4A-PCNA interaction and CUL4A-dependent ubiquitin-mediated degradation of PCNA. Substitution of endogenous PCNA with the Y211F mutant PCNA conveys enhanced sensitization to EGFR inhibition. Our findings identify CUL4A as the ubiquitin ligase linking the down-regulation of cell surface receptor tyrosine kinase to the nuclear DNA replication machinery in cancer cells.     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

ZEB1 Mediates Acquired Resistance to the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer

Epithelial-mesenchymal transition (EMT) is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER) by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8), a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin), ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.     

Epidermal Growth Factor Receptor Translocation to the Mitochondria: REGULATION AND EFFECT*

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

Norepinephrine: A Neuromodulator That Boosts the Function of Multiple Cell Types to Optimize CNS Performance

Norepinephrine (NE) is a neuromodulator that in multiple ways regulates the activity of neuronal and non-neuronal cells. NE participates in the rapid modulation of cortical circuits and cellular energy metabolism, and on a slower time scale in neuroplasticity and inflammation. Of the multiple sources of NE in the brain, the locus coeruleus (LC) plays a major role in noradrenergic signaling. Processes from the LC primarily release NE over widespread brain regions via non-junctional varicosities. We here review the actions of NE in astrocytes, microglial cells, and neurons based on the idea that the overarching effect of signaling from the LC is to maximize brain power, which is accomplished via an orchestrated cellular response involving most, if not all cell types in CNS.     

A Potential Peptide Therapeutic Derived from the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is involved in many cancers and EGFR has been heavily pursued as a drug target. Drugs targeting EGFR have shown promising clinical results for several cancer types. However, resistance to EGFR inhibitors often occurs, such as with KRAS mutant cancers, therefore new methods of targeting EGFR are needed. The juxtamembrane (JXM) domain of EGFR is critical for receptor activation and targeting this region could potentially be a new method of inhibiting EGFR. We hypothesized that the structural role of the JXM region could be mimicked by peptides encoding a JXM amino acid sequence, which could interfere with EGFR signaling and consequently could have anti-cancer activity. A peptide encoding EGFR 645–662 conjugated to the Tat sequence (TE-64562) displayed anti-cancer activity in multiple human cancer cell types with diminished activity in non-EGFR expressing cells and non-cancerous cells. In nude mice, TE-64562 delayed MDA-MB-231 tumor growth and prolonged survival, without inducing toxicity. TE-64562 induced non-apoptotic cell death after several hours and caspase-3-mediated apoptotic cell death with longer treatment. Mechanistically, TE-64562 bound to EGFR, inhibited its dimerization and caused its down-regulation. TE-64562 reduced phosphorylated and total EGFR levels but did not inhibit kinase activity and instead prolonged it. Our analysis of patient data from The Cancer Genome Atlas supported the hypothesis that down-regulation of EGFR is a potential therapeutic strategy, since phospho- and total-EGFR levels were strongly correlated in a large majority of patient tumor samples, indicating that lower EGFR levels are associated with lower phospho-EGFR levels and presumably less proliferative signals in breast cancer. Akt and Erk were inhibited by TE-64562 and this inhibition was observed in vivo in tumor tissue upon treatment with TE-64562. These results are the first to indicate that the JXM domain of EGFR is a viable drug target for several cancer types.     

Epidermal Growth Factor Enhances the Expression of an Endogenous Lectin in Aggregating Fetal Brain Cell Cultures

Aggregating cell cultures prepared from fetal rat telencephalon express the two subunits [cerebellar soluble lectins (CSL) 1 and 2] of a soluble, mannose-specific endogenous lectin (CSL) in a development-dependent manner. Increased CSL synthesis was found at an early postmitotic stage as well as during the period of maximal myelination. Repetitive treatment of early cultures with epidermal growth factor (EGF, 3nM) caused a great stimulation of CSL biosynthesis. Immunocytochemical studies revealed particularly intense CSL-specific staining in small, EGF-responsive cells, presumably glial cells. Large quantities of CSL-immunoreactive material were found also in the extracellular space and on the external side of the plasma membrane, indicating abundant release of CSL. The present findings suggest that EGF or EGF-related factors in the brain are able to regulate the expression of an endogenous lectin, affecting brain ontogeny.     

Prognostic Value of Epidermal Growth Factor Receptor Mutations in Resected Non-Small Cell Lung Cancer: A Systematic Review with Meta-Analysis

Background

The prognostic value of epidermal growth factor receptor (EGFR) mutations in resected non-small cell lung cancer (NSCLC) remains controversial. We performed a systematic review with meta-analysis to assess its role.

Methods

Studies were identified via an electronic search on PubMed, Embase and Cochrane Library databases. Pooled hazard ratio (HR) for disease-free survival (DFS) and overall survival (OS) were calculated for meta-analysis.

Results

There were 16 evaluated studies (n = 3337) in the meta-analysis. The combined HR evaluating EGFR mutations on disease free survival was 0.96 (95% CI [0.79–1.16] P = 0.65). The combined HR evaluating EGFR mutations on overall survival was 0.86 (95% CI [0.72–1.04] P = 0.12). The subgroup analysis based on univariate and multivariate analyses in DFS and OS showed no statistically significant difference. There was also no statistically significant difference in DFS and OS of stage I NSCLC patients.

Conclusion

The systematic review with meta-analysis showed that EGFR mutations were not a prognostic factor in patients with surgically resected non-small cell lung cancer. Well designed prospective study is needed to confirm the result.     

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Methods

We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.

Results

In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC₅₀ concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.

Conclusion

Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.     

Diagnostic Value of Mutation-Specific Antibodies for Immunohistochemical Detection of Epidermal Growth Factor Receptor Mutations in Non-Small Cell Lung Cancer: A Meta-Analysis

Background

Various studies have assessed the diagnostic accuracy of EGFR mutation-specific antibodies in non-small cell lung cancer (NSCLC). We performed a meta-analysis of existing data to investigate the diagnostic value of mutation-specific antibodies for detection of EGFR mutations in NSCLC.

Methods

We systematically retrieved relevant studies from PubMed, Web of Knowledge, and Google Scholar. Data from studies that met the inclusion criteria were extracted for further exploration of heterogeneity, including calculation of the average sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and analysis of SROC(summary receiver operating characteristic) curves.

Results

Fifteen studies met our inclusion criteria. A summary of the meta-analysis of the efficacy of the anti-E746-A750 antibody was as follows: sensitivity, 0.60 (95% CI, 0.55–0.64); specificity, 0.98 (95% CI, 0.97–0.98); PLR, 33.50 (95% CI, 13.96–80.39); NLR, 0.39 (95% CI, 0.30–0.51) and DOR, 111.17 (95% CI, 62.22–198.63). A similar meta-analysis was performed for the anti-L858R antibody with results as follows: sensitivity, 0.76 (95% CI, 0.71–0.79); specificity, 0.96 (95% CI, 0.95–0.97); PLR, 24.42 (95% CI, 11.66–51.17); NLR, 0.22 (95% CI, 0.12–0.39) and DOR, 126.66 (95% CI, 54.60–293.82).

Conclusion

Immunohistochemistry alone is sufficient for the detection of EGFR mutations if the result is positive. Molecular-based analyses are necessary only if the anti-E746-A750 antibody results are negative. Immunohistochemistry seems more suitable for clinical screening for EGFR mutations prior to molecular-based analysis.     

Removal of the Membrane-anchoring Domain of Epidermal Growth Factor Leads to Intracrine Signaling and Disruption of Mammary Epithelial Cell Organization

Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti–EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti–EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.     

Epidermal Growth Factor Induces PC12 Cell Differentiation in the Presence of the Protein Kinase Inhibitor K-252a

Abstract: The protein kinase inhibitors K-252a and K-252b have been shown earlier to block the actions of nerve growth factor and other neurotrophins and, at lower concentrations, to selectively potentiate neurotrophin-3 actions. In the present study we show that K-252a, but not K-252b, enhances epidermal growth factor (EGF)- and basic fibroblast growth factor (bFGF)-induced neurite outgrowth of PC12 cells at higher concentrations than required for neurotrophin inhibition. In parallel, tyrosine phosphorylation of extracellular signal-regulated kinases (Erks) elicited by EGF or bFGF was also increased in the presence of K-252a, and this signal was prolonged for 6 h. EGF- and bFGF-induced phosphorylation of phospholipase C-γ1 were not changed. The effect of K-252a on Erks was resistant to chronic treatment with phorbol ester, indicating that protein kinase C is not involved in this potentiation. In partial contrast to the actions of K-252a, the neurotrophin-3-potentiating effect of K-252b was accompanied by an increase in tyrosine phosphorylation of the Erks and of phospholipase C-γ1. Finally, although K-252a alone did not induce neurite outgrowth or tyrosine phosphorylation of Erks or phospholipase C-γ1, this compound alone stimulated phosphatidylinositol hydrolysis. Our findings identify activities of K-252a besides the direct interaction with neurotrophin receptors and suggest that a K-252a-sensitive protein kinase or phosphatase might be involved in signal transduction for EGF and bFGF. Our results are further compatible with the hypothesis that sustained activation of Erks may be important in PC12 differentiation.     

Differential Responses of the Phosphorylation of Ribosomal Protein S6 to Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.     

Binding of Erythroagglutinating Phytohemagglutinin Lectin from Phaseolus vulgaris to the Epidermal Growth Factor Receptor Inhibits Receptor Function in the Human Glioma Cell Line, U373 MG

Abstract: Little is known about the role of the N -linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N -linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N -linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.     

Time-Dependent Effect of Orchidectomy on Vascular Nitric Oxide and Thromboxane A2 Release. Functional Implications to Control Cell Proliferation through Activation of the Epidermal Growth Factor Receptor

This study analyzes whether the release of nitric oxide (NO) and thromboxane A₂ (TXA₂) depends on the time lapsed since gonadal function is lost, and their correlation with the proliferation of vascular smooth muscle cells (VSMC) mediated by the epidermal growth factor receptor (EGFR). For this purpose, aortic and mesenteric artery segments from control and 6-weeks or 5-months orchidectomized rats were used to measure NO and TXA₂ release. The results showed that the basal and acetylcholine (ACh)-induced NO release were decreased 6 weeks post-orchidectomy both in aorta and mesenteric artery, but were recovered 5 months thereafter up to levels similar to those found in arteries from control rats. The basal and ACh-induced TXA₂ release increased in aorta and mesenteric artery 6 weeks post-orchidectomy, and was maintained at high levels 5 months thereafter. Since we previously observed that orchidectomy, which decreased testosterone level, enlarged the muscular layer of mesenteric arteries, the effect of testosterone on VSMC proliferation was analyzed. The results showed that treatment of cultured VSMC with testosterone downregulated mitogenic signaling pathways initiated by the ligand-dependent activation of the EGFR. In contrast, the EGFR pathways were constitutively active in mesenteric arteries of long-term orchidectomized rats. Thus, the exposure of mesenteric arteries from control rats to epidermal growth factor (EGF) induced the activation of EGFR signaling pathways. However, the addition of EGF to arteries from orchidectomized rats failed to induce a further activation of these pathways. In conclusion, this study shows that the release of NO depends on the time lapsed since the gonadal function is lost, while the release of TXA₂ is already increased after short periods post-orchidectomy. The alterations in these signaling molecules could contribute to the constitutive activation of the EGFR and its downstream signaling pathways after long period post-orchidectomy enhancing the proliferation of the vascular muscular layer.     

Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.     

Interaction between Glucocorticoids and Epidermal Growth Factor In Vitro in the Growth of Palatal Mesenchymal Cells from the Human Embryo

Previous studies of ours have shown that palatal mesenchymal cells from the human embryo (HEPM cells) are responsive both to the glucocorticoid dexamethasone (DEX) and epidermal growth factor (EGF) through mechanisms associated with cytoplasmic and cell surface receptors, respectively. HEPM cell growth was inhibited by DEX and was stimulated by EGF. In the present study, the interactions between DEX and EGF were investigated. DEX (10^–6 M) enhanced EGF-stimulated HEPM cell growth as assessed by an increase in cell number and ornithine decarboxylase activity under serum-free cell culture conditions. DEX also enhanced the specific binding of ¹²⁵I-EGF to these cells, which was reflected in an increase in both the number and the affinity of EGF receptors. EGF (1 ng/ml), on the other hand, decreased the number of sites per cell which specifically bind ³H-DEX. EGF completely prevented the inhibition by DEX of HEPM cell growth. These results indicate that DEX and EGF interact with each other in the process(es) regulating HEPM cell growth. This interaction may be partially influenced by direct modulation of existing receptors for DEX and EGF present in the cells.