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Constitutively activated STAT3 plays a critical role in non-small cell lung carcinoma (NSCLC) progression by mediating proliferation and survival. STAT3 activation in normal cells is transient, making it an attractive target for NSCLC therapy. The therapeutic potential of blocking STAT3 in NSCLC was assessed utilizing a decoy approach by ligating a double-stranded 15-mer oligonucleotide that corresponds to the STAT3 response element of STAT3-target genes, to produce a cyclic STAT3 decoy (CS3D). The decoy was evaluated using NSCLC cells containing either wild-type EGFR (201T) or mutant EGFR with an additional EGFRi resistance mutation (H1975). These cells are resistant to EGFR inhibitors and require an alternate therapeutic approach. CS3D activity was compared with an inactive cyclic control oligonucleotide (CS3M) that differs by a single base pair, rendering it unable to bind to STAT3 protein. Transfection of 0.3 μmol/L of CS3D caused a 50% inhibition in proliferation in 201T and H1975 cells, relative to CS3M, and a 2-fold increase in apoptotic cells. Toxicity was minimal in normal cells. CS3D treatment caused a significant reduction of mRNA and protein expression of the STAT3 target gene c-Myc and inhibited colony formation by 70%. The active decoy decreased the nuclear pool of STAT3 compared with the mutant. In a xenograft model, treatments with CS3D (5 mg/kg) caused a potent 96.5% and 81.7% reduction in tumor growth in 201T ( P < 0.007) and H1975 models ( P < 0.0001), respectively, and reduced c-Myc and p-STAT3 proteins. Targeting STAT3 with the cyclic decoy could be an effective therapeutic strategy for NSCLC. Mol Cancer Ther; 17(9); 1917-26. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: The EGF receptor (EGFR) and COX2 pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic antitumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a nonselective COX inhibitor; versus placebo., Experimental Design: Patients with untreated, operable stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib-sulindac, or placebo. Tumor specimens were collected before and after seven to 14 days of treatment. The primary endpoint was change in Ki67 proliferation index. We hypothesized an ordering effect in Ki67 reduction: erlotinib-sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX2 signaling intermediates., Results: From 2005-2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki67 was significantly decreased by erlotinib or erlotinib-sulindac (omnibus comparison, two-sided Kruskal-Wallis, P = 0.04). Wilcoxon pairwise contrasts confirmed greater Ki67 effect in both erlotinib groups (erlotinib-sulindac vs. placebo, P = 0.043; erlotinib vs. placebo, P = 0.027). There was a significant trend in ordering of Ki67 reduction: erlotinib-sulindac > erlotinib > placebo (two-sided exact Jonckheere-Terpstra, P = 0.0185). Low baseline pSrc correlated with greater Ki67 reduction (R(2) = 0.312, P = 0.024)., Conclusions: Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR-COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target., (©2014 American Association for Cancer Research.)

The BRAF inhibitor vemurafenib is currently used for treating patients with BRAF V600E mutant melanoma. However, the responses to vemurafenib are generally partial and of relatively short duration. Recent evidence suggests that activation of the epidermal growth factor receptor (EGFR)/erbB signaling pathway may be responsible for the development of BRAF inhibitor resistance in melanoma patients. In this study, we characterized the erbB family of receptors and ligands in melanoma cell lines and examined whether targeting both BRAF and erbB provided enhanced antitumor activity in BRAF mutant melanoma. Variable levels of erbB2, erbB3, and truncated erbB4 were expressed in both BRAF wildtype and mutant melanoma cells with no significant differences between wildtype and mutant lines. EGFR was rarely expressed. Neuregulin 3 and neuregulin 4 were the major erbB ligands released by melanoma cells. Multi-erbB targeting with the irreversible tyrosine kinase inhibitor canertinib exerted a more effective growth inhibitory effect in both BRAF wildtype and mutant melanoma cells compared with the single-erbB or dual-erbB targeting inhibitors, gefitinib, erlotinib, and lapatinib. Canertinib inhibited both EGF-induced and neuregulin 1-induced erbB downstream signaling in both mutant and wildtype cell lines. However, canertinib induced apoptosis and sub-G1 arrest only in mutant cells. Canertinib statistically increased the antiproliferative effects of vemurafenib in the BRAF mutant melanoma cell lines while little or no enhanced effect was observed with the combination treatment in the wildtype cell lines. A combined inhibition strategy targeting BRAF together with multiple erbB family kinases is potentially beneficial for treating BRAF V600E mutant melanoma. Wildtype BRAF melanoma may also benefit from a multi-erbB kinase inhibitor.

Purpose: To assess the prognostic value of epidermal growth factor receptor (EGFR) molecular characteristics of head and neck squamous cell carcinoma (HNSCC)., Patients and Methods: HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N = 154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N = 39) were evaluated for EGFR gene amplification by FISH and EGFR protein by immunohistochemical staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (n = 67). Tumor (n = 50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR., Results: EGFR expression by immunohistochemistry (IHC) was significantly higher in the EDRN tumors with EGFR gene amplification (P < 0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (P = 0.019 and P = 0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort [HR = 2.75; 95% confidence interval (CI) = 1.26-6.00 and HR = 3.29; 95% CI = 1.34-8.14, respectively]., Conclusions: In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR., (©2012 AACR.)

The c-Met receptor is a potential therapeutic target for non-small cell lung cancer (NSCLC). Signaling interactions between c-Met and the mutant epidermal growth factor receptor (EGFR) have been studied extensively, but signaling intermediates and biological consequences of lateral signaling to c-Met in EGFR wild-type tumors are minimally understood. Our observations indicate that delayed c-Met activation in NSCLC cell lines is initiated by wild-type EGFR, the receptor most often found in NSCLC tumors. EGFR ligands induce accumulation of activated c-Met, which begins at 8 h and continues for 48 h. This effect is accompanied by an increase in c-Met expression and phosphorylation of critical c-Met tyrosine residues without activation of mitogen-activated protein kinase (MAPK) or Akt. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of hepatocyte growth factor (HGF) or another secreted factor, supporting a ligand-independent mechanism. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activity of both receptors is required for this effect. Prolonged c-Src phosphorylation is observed, and c-Src pathway is essential for EGFR to c-Met communication. Pretreatment with pan-Src family kinase inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared with control, further confirming a c-Src requirement. Inhibition of c-Met with PF2341066 and siRNA decreases EGF-induced phenotypes of invasion by ~86% and motility by ~81%, suggesting that a novel form of c-Met activation is utilized by EGFR to maximize these biological effects. Combined targeting of c-Met and EGFR leads to increased xenograft antitumor activity, demonstrating that inhibition of downstream and lateral signaling from the EGFR-c-Src-c-Met axis might be effective in treatment of NSCLC.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade., Experimental Design: Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with antitumor responses to inhibitors of these pathways., Results: Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion, and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, TGF-α, were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also showed that TGF-α can induce phosphorylation of c-Met over sixfold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared with single agent treatment in vivo., Conclusions: Together, these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC.

Purpose: This study aimed to characterize estrogen receptor expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues, and to evaluate estrogen receptor and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC., Experimental Design: Estrogen receptor expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to estrogen receptor and EGFR ligands or inhibitors. Estrogen receptor and EGFR protein expression in patient tissues was assessed by immunohistochemical staining., Results: Phospho-mitogen-activated protein kinase (P-MAPK) levels were significantly increased following combined estrogen and EGF treatment. Treatment of HNSCC cells with estrogen and EGF significantly increased cell invasion compared with either treatment alone, whereas inhibiting these two pathways resulted in reduced invasion compared with inhibiting either pathway alone. EGFR (P = 0.008) and nuclear estrogen receptor alpha (ER alpha(nuc); P < 0.001) levels were significantly increased in HNSCC tumors (n = 56) compared with adjacent mucosa (n = 30), whereas nuclear estrogen receptor beta (ER beta(nuc)) levels did not differ (P = 0.67). Patients with high ER alpha(nuc) and EGFR tumor levels had significantly reduced progression-free survival compared with patients with low tumor ER alpha(nuc) and EGFR levels (hazards ratio, 4.09; P = 0.01; Cox proportional hazards). In contrast, high ER beta(nuc) tumor levels were not associated with reduced progression-free survival alone or when combined with EGFR., Conclusions: ER alpha and ER beta were expressed in HNSCC, and stimulation with estrogen receptor ligands resulted in both cytoplasmic signal transduction and transcriptional activation. Estrogen receptor and EGFR cross-talk was observed. Collectively, these studies indicate that estrogen receptor and EGFR together may contribute to HNSCC development and disease progression.

Purpose: Increased expression and/or activation of epidermal growth factor receptor (EGFR) is associated with tumor progression and poor prognosis in many cancers, including head and neck squamous cell carcinoma (HNSCC). Src family kinases, including c-Src, mediate a variety of intracellular or extracellular signals that contribute to tumor formation and progression. This study was undertaken to elucidate the role of c-Src in the growth and invasion of HNSCC and to determine the effects of combined targeting of EGFR and Src kinases in HNSCC cell lines., Experimental Design: HNSCC cells were engineered to stably express a dominant-active form of c-Src and investigated in cell growth and invasion assays. The biochemical effects of combined treatment with the Src inhibitor AZD0530, a potent, orally active Src inhibitor with Bcr/Abl activity, and the EGFR kinase inhibitor gefitinib were examined, as well as the consequences of dual Src/EGFR targeting on the growth and invasion of a panel of HNSCC cell lines., Results: HNSCC cells expressing dominant-active c-Src showed increased growth and invasion compared with vector-transfected controls. Combined treatment with AZD0530 and gefitinib resulted in greater inhibition of HNSCC cell growth and invasion compared with either agent alone., Conclusions: These results suggest that increased expression and activation of c-Src promotes HNSCC progression where combined targeting of EGFR and c-Src may be an efficacious treatment approach.

Purpose: To determine the effect of tyrosine-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) immunoexpression on survival in two independent cohorts of patients with squamous cell carcinoma of the head and neck (SCCHN) and to evaluate pSTAT3, transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), and gastrin-releasing peptide receptor (GRPR) expression in matched tumor and lymph node metastases in one of these cohorts. EXPERIMENTAL TECHNIQUE: Immunostaining for pSTAT3, TGF-alpha, EGFR, and GRPR was done in two SCCHN cohorts (cohort 1, 61 tumors; cohort 2, 69 paired primary tumors and lymph node metastases). Semiquantitative scores derived from the product of staining intensity (scale 0-3) score and percentage of positive tumor cells were correlated with clinical outcome., Results: Immunoexpression of pSTAT3 did not correlate with clinical outcome in either cohort (cohort 1, P = 0.914; cohort 2, P = 0.312). In cohort 2, TGF-alpha and EGFR expression in the primary tumors showed some association with decreased disease-free survival (P = 0.0306 and P = 0.0985, respectively). Both pSTAT3 and EGFR showed a correlation of expression between tumor and matched lymph node metastasis (P < 0.0001 and P = 0.0046, respectively). In addition, the expression of EGFR and GRPR in the primary tumors correlated with TGF-alpha expression in paired nodal metastases (P = 0.0043 and P = 0.0268, respectively). In the nodal metastases, TGF-alpha expression correlated with EGFR expression (P = 0.0069). In primary tumors, GRPR expression correlated with TGF-alpha and EGFR expression (P = 0.0378 and P = 0.0026, respectively)., Conclusions: These findings support an autocrine signaling pathway involving TGF-alpha, EGFR, and pSTAT3 in metastatic SCCHN as well as transactivation of EGFR by GRPR via TGF-alpha, but fails to identify an independent prognostic role for pSTAT3 immunoexpression.

Epidermal growth factor receptor (EGFR) plays an important role in the development and progression of lung cancer. A previous report noted that an increased number of polymorphic CA repeats in the first intron of the EGFR gene results in decreased transcriptional activity. To estimate the association of the length of polymorphic CA repeats in intron 1 of the EGFR gene with lung cancer, a case-control study of 176 lung cancer patients and 161 controls was conducted in Caucasians. This case-control study is based on two existing prospective cohorts: the Early Detection Research Network (EDRN) and the Lung Cancer Specialized Program of Research Excellence (SPORE) at the University of Pittsburgh. The frequencies of the SL (one allele>16 repeats), and SS (both allele36) was 20.5% versus 36.7%, for lung cancer cases versus controls. The lower sum of EGFR-CA repeats associated with the risk of lung cancer; the estimated odds ratio was 2.25 with 95% confidence interval: 1.38-3.66 (P=0.001). Associations involving EGFR-CA repeat genotype and EGFR-CA repeat sum remained significant when adjusted for age, gender, and tobacco exposure. Our study, which is preliminary, demonstrates for the first time that shorter EGFR-CA repeats associate with lung cancer risk. The number of EGFR-CA repeats identifies a possible susceptible population to lung cancer., (Copyright (c) 2007 Wiley-Liss, Inc.)

Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G-protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Because GRPR transactivates the epidermal growth factor receptor (EGFR), we investigated induction by GRP of Akt, an EGFR-activated signaling pathway, and examined effects of GRP on viability of non-small cell lung carcinoma (NSCLC) cells exposed to the EGFR tyrosine kinase inhibitor gefitinib. GRP induced Akt activation primarily through c-Src-mediated transactivation of EGFR. Transfection of dominant-negative c-Src abolished GRP-induced EGFR and Akt activation. GRP induced release of amphiregulin, and pre-incubation with human amphiregulin neutralizing antibody eliminated GRP-induced Akt phosphorylation. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely blocked GRP-initiated Akt phosphorylation. These results suggest that GRP stimulates Akt activation primarily via c-Src activation, followed by extracellular release of the EGFR ligand amphiregulin, leading to the activation of EGFR and PI3K. Pretreatment of NSCLC cells with GRP resulted in an increase in the IC(50) of gefitinib of up to 9-fold; this protective effect was mimicked by the pretreatment of cells with amphiregulin and reversed by Akt or PI3K inhibition. GRP appears to rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of the Akt pathway, suggesting GRPR and/or EGFR autocrine pathways in NSCLC cells may modulate therapeutic response to EGFR inhibitors.

Head and neck squamous cell carcinoma (HNSCC) is characterized by epidermal growth factor receptor (EGFR) overexpression, where EGFR levels correlate with survival. To date, EGFR targeting has shown limited antitumor effects in head and neck cancer when administrated as monotherapy. We previously identified a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) aurocrine regulatory pathway in HNSCC, where GRP stimulates Src-dependent cleavage of EGFR proligands with subsequent EGFR phosphorylation and mitogen-activated protein kinase (MAPK) activation. To determine whether GRPR targeting can enhance the antitumor efficacy of EGFR inhibition, we investigated the effects of a GRPR antagonist (PD176252) in conjunction with an EGFR tyrosine kinase inhibitor (erlotinib). Combined blockade of GRPR and EGFR pathways significantly inhibited HNSCC, but not immortalized mucosal epithelial cell, proliferation, invasion, and colony formation. In addition, the percentage of apoptotic cells increased upon combined inhibition. The enhanced antitumor efficacy was accompanied by increased expression of cleaved poly(ADP-ribose) polymerase (PARP) and decreased phospho-EGFR, phospho-MAPK, and proliferating cell nuclear antigen (PCNA). Using reverse-phase protein microarray (RPPA), we further detected decreased expression of phospho-c-Jun, phospho-p70S6K, and phospho-p38 with combined targeting. Cumulatively, these results suggest that GRPR targeting can enhance the antitumor effects of EGFR inhibitors in head and neck cancer.

G protein-coupled receptors (GPCR) and the epidermal growth factor receptor (EGFR) are often both overexpressed and contribute to the growth of cancers by activating autocrine pathways. GPCR ligands have been reported to trigger EGFR signaling via receptor cross-talk in cancer cells. Here, we show that GPCR ligands prostaglandin E2 (PGE2) and bradykinin (BK) activate EGFR signaling. Inhibition of EGFR using several strategies, including small-molecule inhibitors and an EGFR-specific antibody, resulted in partial attenuation of signaling downstream of EGFR. PGE2 and BK triggered EGFR signaling by increasing selective autocrine release of transforming growth factor-alpha (TGF-alpha). Inhibition of tumor necrosis factor-alpha-converting enzyme abrogated BK- or PGE2-mediated activation of EGFR signaling. Both PGE2 and BK stimulated head and neck squamous cell carcinoma (HNSCC) invasion via EGFR. Treatment of HNSCC cells with the BK antagonist CU201 resulted in growth inhibition. The combination of CU201 with the EGFR small-molecule inhibitor erlotinib resulted in additive inhibitory effects on HNSCC cell growth in vitro. Inhibition of the PGE2 synthesis pathway with sulindac induced HNSCC cytotoxicity at high doses (EC(50), 620 micromol/L). However, combined inhibition of both EGFR with the tyrosine kinase inhibitor erlotinib and GPCR with sulindac at low doses of 6 and 310 micromol/L, respectively, resulted in synergistic killing of HNSCC tumor cells. Combined blockade of both EGFR and GPCRs may be a rational strategy to treat cancers, including HNSCC that shows cross-talk between GPCR and EGFR signaling pathways.

Background: The epidermal growth factor receptor (EGFR) is involved in the development and progression of lung cancer. Somatic EGFR mutations are predictors of response to treatment with EGFR tyrosine kinase (TK) inhibitors (TKIs) for lung cancer, especially among never smokers. EGFR mutations may occur independently of other genetic alterations., Methods: The authors sequenced the EGFR-TK domain and the K-ras and p53 genes from lung tumor tissues from 44 never smokers and 46 smokers. A case-control study also was conducted to examine the relationship between an EGFR single nucleotide polymorphism in the TK domain and the lung cancer through a multivariate logistic regression analysis. In addition, the authors compared cell growth kinetics, EGFR-TKI sensitivity by MTT, and activation of signaling molecules by immunoblot in lung cancer cell lines with and without EGFR-TK mutations., Results: EGFR-TK mutations were more frequently observed in never smokers (25%) than in smokers (2.2%) (p = 0.001). Excluding cases with a K-ras mutation, the frequency of EGFR-TK domain mutation was still significantly higher in never smokers than in smokers, 26.2% versus 4.5% (p = 0.046). EGFR-TK mutations and K-ras mutations (p = 0.015), and p53 and K-ras mutations (p = 0.015) were mutually exclusive, but p53 and EGFR-TK mutations were not (p = 1.00). During sequencing of the EGFR-TK domain in tumors, an EGFR polymorphism (G2607A) was identified. The genotype AA and AA + AG occurred at a significantly higher frequency in lung cancer cases (n = 122) when compared with controls (n = 147) (odds ratio, 3.39 and 2.67; 95% confidence interval, 1.41-8.17 and 1.17-6.08, p = 0.006 and p = 0.02, respectively). This polymorphism was found independently of EGFR-TK mutations in lung cancer cases, indicating that it does not predispose to mutations. In vitro, lung cancer cell lines with EGFR-TK mutations also did not contain K-ras mutations and displayed a lower growth rate (50%, p = 0.013) than EGFR-TK wild-type cell lines. EGFR-TK mutant cell lines were more sensitive to both gefitinib and erlotinib, although relative sensitivity to erlotinib compared with wild-type was less pronounced than for gefitinib. Cell lines with a lower growth rate also expressed higher levels of E-cadherin than faster growing cell lines., Conclusions: EGFR-TK mutation frequency is high in never-smoking lung cancer patients and is exclusive of mutation in K-ras but not p53. In addition to somatic EGFR-TK mutations that arise in lung tumors, germline variation in the EGFR-TK domain might also be associated with an increased risk of lung cancer. Somatic EGFR-TK mutations alter cell biology and response to EGFR-TKIs and may be mutation specific.

G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation and invasion of cancer cells. Elucidation of the mechanism of EGFR activation by G protein-coupled receptors may identify new signaling paradigms. A gastrin-releasing peptide (GRP)/GRP receptor-mediated autocrine pathway was previously described in squamous cell carcinoma of head and neck. In the present study, we demonstrate that TNF-alpha converting enzyme (TACE), a disintegrin and metalloproteinse-17, undergoes a Src-dependent phosphorylation that regulates release of the EGFR ligand amphiregulin upon GRP treatment. Further investigation reveals the phosphatidylinositol 3-kinase (PI3-K) as the intermediate of c-Src and TACE, contributing to their association and TACE phosphorylation. Phosphoinositide-dependent kinase 1 (PDK1), a downstream target of PI3-K, has been identified as the previously undescribed kinase to directly phosphorylate TACE upon GRP treatment. These findings suggest a signaling cascade of GRP-Src-PI3-K-PDK1-TACE-amphiregulin-EGFR with multiple points of interaction, translocation, and phosphorylation. Furthermore, knockdown of PDK1 augmented the antitumor effects of the EGFR inhibitor erlotinib, indicating PDK1 as a therapeutic target to improve the clinical response to EGFR inhibitors.

Gastrin-releasing peptide receptor (GRPR) and the epidermal growth factor receptor (EGFR) are expressed in several cancers including non-small cell lung cancer (NSCLC). Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP), in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-A or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP) inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18 microM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.

Identifying new effective therapeutic treatments for lung cancer is critical to improving overall patient survival. We have targeted both the estrogen receptor (ER) and the epidermal growth factor receptor (EGFR) pathways using an ER antagonist, fulvestrant ("Faslodex"), and the selective EGFR tyrosine kinase inhibitor, gefitinib ("Iressa"), in non-small cell lung cancer (NSCLC) cells. Rapid activation of phospho-EGFR and phospho-p44/p42 mitogen-activated protein kinase by estrogen was observed, indicating nonnuclear ER transactivation of EGFR. Additionally, EGFR protein expression was down-regulated in response to estrogen and up-regulated in response to fulvestrant in vitro, suggesting that the EGFR pathway is activated when estrogen is depleted in NSCLC cells. Cell growth and apoptosis were examined in several NSCLC lines that express varying amounts of ERbeta, EGFR, and Neu but no full-length ERalpha. One cell line contained an EGFR mutation. Cells were exposed to 10 nmol/L estrogen and 10 ng/mL EGF and either 1 mumol/L fulvestrant or 1 mumol/L gefitinib alone or in combination. In all cell lines, the drug combination decreased cell proliferation up to 90% and increased apoptosis 2-fold. The relative responses to gefitinib and fulvestrant were similar regardless of ER and EGFR expression and mutation status. In an in vivo lung tumor xenograft model, the drug combination decreased tumor volume in severe combined immunodeficient mice by approximately 60% compared with 49% and 32% for gefitinib and fulvestrant treatment alone, respectively. Antitumor effects of the combination therapy were accompanied by biochemical and histologic evidence of increased apoptosis, decreased phospho-p44/p42 mitogen-activated protein kinase expression, and increased Ki-67 expression compared with individual treatment. These studies provide evidence of a functional interaction between the ER and the EGFR pathways in NSCLC.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by up-regulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR and p42/44 mitogen-activated protein kinase (MAPK) activation in part via extracellular release of transforming growth factor alpha (TGF-alpha) by matrix metalloproteinases (MMPs). It has been reported that Src family kinases are activated by G-protein-coupled receptors (GPCRs), followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using an Src-specific tyrosine kinase inhibitor A-419259 decreased GRP-induced EGFR phosphorylation and MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src-transfected HNSCC cells. Invasion and growth assays showed that c-Src was required for GRP-induced proliferation or invasion of HNSCC cells. In addition to TGF-alpha release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor marimastat. TGF-alpha and amphiregulin secretion by GRP stimulation also was inhibited by blockade of Src family kinases. These results suggest that Src family kinases contribute to GRP-mediated EGFR growth and invasion pathways by facilitating cleavage and release of TGF-alpha and amphiregulin in HNSCC.

Head and neck squamous cell carcinomas (HNSCC) are characterized by upregulation of the epidermal growth factor receptor (EGFR), where EGFR serves as a potential therapeutic target. We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. In the present study, we examined the mechanism of EGFR activation by GRP/GRPR in HNSCC proliferation. In HNSCC cells that express elevated levels of both GRPR and EGFR, we found that GRP induced rapid phosphorylation of EGFR as well as p44/42-MAPK activation. Using several EGFR-specific tyrosine kinase inhibitors and cells derived from EGFR knockout mice, we demonstrated that GRP-induced p44/42-MAPK activation was dependent upon EGFR activation. Further investigation demonstrated that cleavage of transforming growth factor-alpha (TGF-alpha) by matrix metalloproteinases mediated GRP-induced MAPK activation. In addition, HNSCC proliferation stimulated by GRP was eliminated upon specific inhibition of EGFR or MEK, and GRP failed to stimulate proliferation in EGFR-deficient cells. These results imply that the mitogenic effects of GRP in HNSCC are mediated by extracellular release of TGF-alpha and require the activation of an EGFR-dependent MEK/MAPK-dependent pathway.