Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor.

Targeted therapies that inhibit the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) have shown activity against solid malignancies when used as single agents or in combination with chemotherapy. Although anti-EGFR therapies are active in some patients, eventually disease in nearly all patients will become refractory to therapy. Therefore, a better understanding of the mechanisms of resistance to anti-EGFR therapies is critical to further improve the efficacy of this class of agents. Mechanisms that mediate resistance to anti-EGFR therapies include the presence of redundant tyrosine kinase receptors, increased angiogenesis, and the constitutive activation of downstream mediators. Two recent landmark publications have also shown that specific mutations in the kinase domain of EGFR in some lung carcinomas are associated with markedly improved response rates to an EGFR tyrosine kinase inhibitor. Mutations in the EGFR receptor seem to play a significant role in determining the sensitivity of tumor cells to EGFR inhibitor therapy by altering the conformation and activity of the receptor. As the field of molecular therapeutics continues to evolve, a comprehensive understanding of resistance mechanisms will ultimately lead to refinements in our regimens to provide better care for patients with cancer.     

Fibroblast growth factor receptor 4 increases epidermal growth factor receptor (EGFR) signaling by inducing amphiregulin expression and attenuates response to EGFR inhibitors in colon cancer

Fibroblast growth factor receptor 4 (FGFR4) is known to induce cancer cell proliferation, invasion, and antiapoptosis through activation of RAS/RAF/ERK and PI3K/AKT pathways, which are also known as major molecular bases of colon cancer carcinogenesis related with epidermal growth factor receptor (EGFR) signaling. However, the interaction between FGFR4 and EGFR signaling in regard to colon cancer progression is unclear. Here, we investigated a potential cross‐talk between FGFR4 and EGFR, and the effect of anti‐EGFR therapy in colon cancer treatment. To explore the biological roles of FGFR4 in cancer progression, RNA sequencing was carried out using FGFR4 transfected colon cell lines. Gene ontology data showed the upregulation of genes related to EGFR signaling, and we identified that FGFR4 overexpression secretes EGFR ligands such as amphiregulin (AREG) with consequent activation of EGFR and ErbB3. This result was also shown in in vivo study and the cooperative interaction between EGFR and FGFR4 promoted tumor growth. In addition, FGFR4 overexpression reduced cetuximab‐induced cytotoxicity and the combination of FGFR4 inhibitor (BLU9931) and cetuximab showed profound antitumor effect compared to cetuximab alone. Clinically, we found the positive correlation between FGFR4 and AREG expression in tumor tissue, but not in normal tissue, from colon cancer patients and these expressions were significantly correlated with poor overall survival in patients treated with cetuximab. Therefore, our results provide the novel mechanism of FGFR4 in connection with EGFR activation and the combination of FGFR4 inhibitor and cetuximab could be a promising therapeutic option to achieve the optimal response to anti‐EGFR therapy in colon cancer.     

Nuclear localization of epidermal growth factor receptor (EGFR) in ameloblastomas

Background: Ameloblastoma is a locally invasive neoplasm often associated with morbidity and facial deformities, showing increased Epidermal Growth Factor Receptor (EGFR) expression. Inhibition of EGFR was suggested as a treatment option for a subset of ameloblastomas. However, there are resistance mechanisms that impair anti-EGFR therapies. One important resistance mechanism for EGFR-inhibition is the EGFR nuclear localization, which activates genes responsible for its mitogenic effects, such as Cyclin D1.Methods: We assessed EGFR nuclear localization in encapsulated (unicystic, n = 3) and infiltrative (multicystic, n = 11) ameloblastomas and its colocalization with Cyclin D1 by using anti-EGFR and anti-lamin B1 double labeling immunofluorescence analyzed by confocal microscopy. Oral inflammatory fibrous hyperplasia and oral squamous cell carcinoma samples were used for comparison.Results: Twelve cases of ameloblastoma exhibited nuclear EGFR colocalization with lamin B1. This positive staining was mainly observed in the ameloblast-like cells. The EGFR nuclear localization was also observed in control samples. In addition, nuclear EGFR colocalized with Cyclin D1 in ameloblastomas.Conclusions: Nuclear EGFR occurs in ameloblastomas in association with Cyclin D1 expression, which is important in terms of tumor biology clarification and raises a concern about anti-EGFR treatment resistance in ameloblastomas.     

Good response to pemetrexed in patients of lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutations

There are no reports about epidermal growth factor receptor (EGFR) mutations and the responsiveness to pemetrexed treatment. In order to understand the influence of EGFR mutations on pemetrexed response, we performed EGFR sequencing of lung adenocarcinoma from patients undergoing pemetrexed treatment and analyzed their response to pemetrexed. The pemetrexed-treated lung adenocarcinoma patients with adequate specimens for EGFR sequencing and measurable target lesions were enrolled for response analysis. Demographic data, EGFR mutation status, response to pemetrexed, and survival data were collected. From January 2004 to December 2008, 156 patients with measurable target lesions and EGFR sequencing results had complete clinical data for analysis. The patients with EGFR mutations (N = 93) had a better response rate (p = 0.016) and longer progression-free survival (PFS) (p = 0.030) than those with wild type EGFR (N = 63). By multivariate analysis, those who had better performance status (p = 0.013) and EGFR mutations (p = 0.021) had a longer PFS. In conclusion, lung adenocarcinoma patients receiving pemetrexed with EGFR mutations have a better response rate and longer PFS than those with wild type EGFR.     

Transforming growth factor alpha expression drives constitutive epidermal growth factor receptor pathway activation and sensitivity to gefitinib (Iressa) in human pancreatic cancer cell lines

The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-alpha (TGF-alpha)] expression (r(2) = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-alpha reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-alpha expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-alpha expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-alpha expression may therefore represent an effective means of identifying EGFR-responsive primary tumors.     

Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA)

The aim of this study was to evaluate the usefulness of EGFR mutation status in serum DNA as a means of predicting a benefit from gefitinib (IRESSA) therapy in Japanese patients with non-small cell lung cancer (NSCLC). We obtained pairs of tumour and serum samples from 42 patients treated with gefitinib. EGFR mutation status was determined by a direct sequencing method and by Scorpion Amplification Refractory Mutation System (ARMS) technology. EGFR mutations were detected in the tumour samples of eight patients and in the serum samples of seven patients. EGFR mutation status in the tumours and serum samples was consistent in 39 (92.9%) of the 42 pairs. EGFR mutations were strong correlations between both EGFR mutation status in the tumour samples and serum samples and objective response to gefitinib (P<0.001). Median progression-free survival time was significantly longer in the patients with EGFR mutations than in the patients without EGFR mutations (194 vs 55 days, P=0.016, in tumour samples; 174 vs 58 days, P=0.044, in serum samples). The results suggest that it is feasible to use serum DNA to detect EGFR mutation, and that it's potential as a predictor of response to, and survival on gefitinib is worthy of further evaluation.     

Expression of the epidermal growth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas

Introduction  

Epidermal growth factor receptor (EGFR) is involved in regulating cell growth in breast carcinomas. Its activated form, phosphorylated EGFR (pEGFR), is correlated with poor prognosis in lung cancer, but it has not yet been fully investigated in breast cancer. The aim of this study was to investigate the expressions of EGFR and pEGFR and their correlation with overall and disease-free survival, clinicopathological parameters and biological markers of invasion and angiogenesis (phosphorylated Akt [pAkt], urokinase plasminogen activator receptor [uPAR], matrix metalloproteinase [MMP]-14, vascular endothelial growth factor receptor [VEGFR]-1/Flt-1).     

Expression of epidermal growth factor receptor (EGFR) in ovarian carcinoma stage III-IV

Hyperexpression of epidermal growth factor receptor (EGFR) is often identified as unfavorable prognosis for different epithelial cancers. The study was concerned with an attempt of establishing a relationship between EGFR expression, on the one hand, and patient's clinico-morphological status, prognosis and efficacy of chemotherapy for stage III-IV serous ovarian carcinoma, on the other. EGFR hyperexpression predominated in advanced aggressive tumors and involved a significantly shorter period preceding tumor progression. Similarly, overall survival median in patients with EGFR hyperexpression (21+/-4 months) appeared lower than without it (42+/-8 months). In serous ovarian carcinoma stage III-IV, EGFR hyperexpression should be considered sufficient for prognosis of chemotherapy efficacy, pre-progression time and survival.     

Intratumor heterogeneity of epidermal growth factor receptor mutations in lung cancer and its correlation to the response to gefitinib

Somatic mutations introduced into the epidermal growth factor receptor (EGFR) gene in non-small-cell lung cancer (NSCLC) are important factors to determine therapeutic responses to gefitinib. The current diagnostic test measures the overall EGFR mutation status of the cancer tissue, and may ignore the presence of non-mutated, gefitinib-unresponsive cancer cells. Twenty-one NSCLC patients with EGFR mutations were recruited for the study. All patients were treated with gefitinib after surgical treatment. Fifty to sixty areas of NSCLC tumors were sampled from each tissue, and their EGFR mutation states were determined by a primer extension assay. This assay discriminates between EGFR mutation-positive and -negative cancer cells within a single tumor tissue. Fifteen tissues consisted only of cells with EGFR mutations, but the remaining six tissues contained both mutated and non-mutated cells. Time to disease progression and overall survival after gefitinib treatment were significantly shorter in those patients with EGFR heterogeneity ( P =  0.009 and P  = 0.003, respectively). A considerable proportion of NSCLC contains a heterogeneous population of both EGFR mutated and non-mutated cancer cells, resulting in a reduced response to gefitinib. The intratumor genetic heterogeneity of a target molecule such as EGFR would be an important factor to consider when treating patients with molecular target agents. ( Cancer Sci 2008; 99: 929–935)     

Increased epidermal growth factor receptor (EGFR) expression in malignant mammary phyllodes tumors

Mammary phyllodes tumors are uncommon stromal-epithelial neoplasms, and are divided into benign, borderline malignant and frankly malignant groups on the basis of their histological features. Accumulating evidence shows that epidermal growth factor receptor (EGFR) is involved in the pathogenesis and progression of many malignancies. This study investigated 453 phyllodes tumors (296 benign, 98 borderline, 59 malignant) for EGFR expression using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for gene amplification. The staining was correlated to tumor margin status, degree of malignancy, stromal cellularity, mitotic activity, nuclear pleomorphism and stromal overgrowth. Cases with strong positive IHC staining were selected for FISH. The overall positive rate for EGFR was 16.2% (48/296), 30.6% (30/98) and 56% (33/59) for benign, borderline malignant and frankly malignant phyllodes tumors, respectively. FISH demonstrated egfr gene amplification in 8% of immunohistochemically positive cases. The results of this study provide strong evidence that EGFR overexpression is involved in the pathogenesis of phyllodes tumors, although gene amplification may not be the major underlying mechanism for overexpression.     

Inhibition of Hsp90 down-regulates mutant epidermal growth factor receptor (EGFR) expression and sensitizes EGFR mutant tumors to paclitaxel

Mutations in the kinase domain of the epidermal growth factor receptor (EGFR) are found in a subset of patients with lung cancer and correlate with response to EGFR tyrosine kinase inhibitors (TKI). Resistance to these agents invariably develops, and current treatment strategies have limited efficacy in this setting. Hsp90 inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), induce the degradation of EGFR and other Hsp90 interacting proteins and may thus have utility in tumors dependent upon sensitive Hsp90 clients. We find that the EGFR mutations found most commonly in patients with lung adenocarcinoma who respond to EGFR TKIs are potently degraded by 17-AAG. Although the expression of wild-type EGFR was also down-regulated by 17-AAG, its degradation required higher concentrations of drug and a longer duration of drug exposure. In animal models, a single dose of 17-AAG was sufficient to induce degradation of mutant EGFR and inhibit downstream signaling. 17-AAG treatment, at its maximal tolerated dose, caused a significant delay in H3255 (L858R EGFR) xenograft growth but was less effective than the EGFR TKI gefitinib. 17-AAG alone delayed, but did not completely inhibit, the growth of H1650 and H1975 xenografts, two EGFR mutant models which show intermediate and high levels of gefitinib resistance. 17-AAG could be safely coadministered with paclitaxel, and the combination was significantly more effective than either drug alone. These data suggest that Hsp90 inhibition in combination with chemotherapy may represent an effective treatment strategy for patients whose tumors express EGFR kinase domain mutations, including those with de novo and acquired resistance to EGFR TKIs.     

Sprouty2 protein enhances the response to gefitinib through epidermal growth factor receptor in colon cancer cells

Sprouty2 (Spry2) is known to increase the expression of epidermal growth factor receptors (EGFR) by conjugating with c‐Casitas B‐lineage lymphoma (C‐Cbl) to decrease protein degradation. The effect of Spry2 on the treatment of gefitinib, a tyrosine kinase inhibitor of EGFR, with regards to colon cancer is still unclear. The half maximal inhibitory concentration (IC50) values of gefitinib in six colon cancer cell lines were assessed. HCT116 and C2BBel cells expressed lower levels of Spry2 protein and were less sensitive to gefitinib, whereas HT29 cells that expressed high levels of Spry2 protein were more sensitive to gefitinib. The sensitivity to gefitinib was increased after overexpression of Spry2 in HCT116 cells, whereas it was decreased after Spry2 knockdown in HT29 cells. The levels of both phosphorylated and total EGFR were increased when HCT116 cells ectopically overexpressed Spry2, with concomitant increase in phosphatase and tensin homolog (PTEN) expression. Inhibition of EGFR by cetuximab reduced sensitivity to gefitinib in HCT116 cells overexpressing Spry2. However, knockdown of PTEN or K‐ras failed to diminish the effect of Spry2 on gefitinib sensitivity. Of note, Spry2 enhanced the antitumor effect of gefitinib in a xenograft model of HCT116 tumors, which harbored K‐ras codon 13 mutation. In conclusion, Spry2 can enhance the response of colon cancer cells to gefitinib by increasing the expression of phosphorylated and total EGFR. These results suggest that Spry2 may be a potential biomarker in predicting the response to anti‐EGFR treatment in colon cancer and that it is necessary to conduct clinical studies to incorporate Spry2 into the network of cancer treatment. (Cancer Sci 2010)     

Effect of epidermal growth factor receptor mutations on the response to epidermal growth factor receptor tyrosine kinase inhibitors: target-based populations for target-based drugs

The recent identification of somatic mutations in the epidermal growth factor receptor (EGFR)-tyrosine kinase (TK) domain of tumor samples from patients with non-small-cell lung cancer (NSCLC) that portend robust response to small-molecule inhibitors of EGFR TK is a watershed event in the fields of lung cancer genetics and therapeutic agents. In addition to paralleling what is already known about c-kit mutations that drive the proliferation of gastrointestinal stromal tumors and their response to imatinib, and providing the possibility of prospectively selecting patients with NSCLC who have a high probability of responding to EGFR TK inhibitors, these reports will likely have much broader implications with regard to the optimal and most expeditious means to develop rationally designed, target-based therapeutic agents--first establishing proof of principle in patients whose malignancies are dependent or driven by aberrations of the therapeutic's target. These new findings will undoubtedly lead to a greater understanding of the biology of EGFR-mutant NSCLC and the mechanism of action of EGFR TK inhibitors. This further validates EGFR as a target for anticancer therapy, particularly in tumors with activating mutations of the target, which lead to sequencing of genes that govern other relevant proteins that are currently being targeted with novel therapeutic agents. The result should be more efficient and scientifically founded clinical development strategies for rationally designed target-based therapeutic agents.     

Activation of downstream epidermal growth factor receptor (EGFR) signaling provides gefitinib-resistance in cells carrying EGFR mutation

Patients with pulmonary adenocarcinoma carrying the epidermal growth factor receptor (EGFR) mutation tend to display dramatic clinical response to treatment with the EGFR tyrosine kinase inhibitor gefitinib. Unfortunately, in many cases the cancer cells eventually acquire resistance, and this limits the duration of efficacy. To gain insight into these acquired resistance mechanisms, we first prepared HEK293T cell line stably transfected with either wild-type (WT) or mutant (L858R) EGFR, and then expressed oncogenic K-Ras12V mutant in the latter transfectant. Although 293T cells expressing wild-type EGFR did not show any growth inhibition by gefitinib treatment similarly to the non-transfected cells, the cells expressing the EGFR-L858R were exquisitely sensitive. Consistently, phospho-Akt levels were decreased in response to gefitinib in cells expressing EGFR-L858R but not in cells with EGFR-WT. In contrast, 293T cells expressing both EGFR-L858R and oncogenic K-Ras were able to proliferate even in the presence of high concentration of gefitinib probably by inducing Erk1/2 activation. We also expressed K-Ras12V in the gefitinib-sensitive pulmonary adenocarcinoma cell line PC-9, which harbors an in-frame deletion in the EGFR gene. The activated K-Ras inhibited the effects of gefitinib treatment on cell growth, cell death induction and levels of phospho-Akt, as well as phospho-Erk. These data indicate that activated Ras could substitute most of the upstream EGFR signal, and are consistent with the hypothesis that mutational activation of targets immediately downstream from the EGFR could induce the secondary resistance to gefitinib in patients with lung cancer carrying EGFR mutation.     

Analysis of epidermal growth factor receptor expression as a predictive factor for response to gefitinib ('Iressa', ZD1839) in non-small-cell lung cancer

Gefitinib ('Iressa', ZD1839) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has demonstrated antitumour activity and favourable tolerability in Phase II studies. We investigated whether EGFR expression levels could predict for response to gefitinib in patients with advanced non-small-cell lung cancer (NSCLC), who received gefitinib (250 mg day(-1)) as part of a worldwide compassionate-use programme. Tissue samples were analysed by immunohistochemistry to assess membrane EGFR immunoreactivity. Of 147 patients enrolled in our institution, 50 patients were evaluable for assessment of both clinical response and EGFR expression. The objective tumour response rate was 10% and disease control was achieved in 50% of patients. Although high EGFR expression was more common in squamous-cell carcinomas than adenocarcinomas, all objective responses were observed in patients with adenocarcinoma. Response and disease control with gefitinib were not associated with high EGFR expression. Overall, median survival was 4 months, and the 1-year survival rate was 18%. Strong EGFR staining correlated with shorter survival time for all patients. Gefitinib demonstrated promising clinical activity in this group of patients with NSCLC. These results have also shown that EGFR expression is not a significant predictive factor for response to gefitinib.     

Epidermal growth factor receptor dynamics influences response to epidermal growth factor receptor targeted agents

Analysis of gene expression of cancer cell lines exposed to erlotinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), showed a marked increase in EGFR mRNA in resistant cell lines but not in susceptible ones. Because cetuximab induces EGFR down-regulation, we explored the hypothesis that treatment with cetuximab would interfere with erlotinib-induced EGFR up-regulation and result in antitumor effects. Exposure of the resistant biliary tract cancer cell line HuCCT1 but not the susceptible A431 epidermoid cell line to erlotinib induced EGFR mRNA and protein expression. Combined treatment with cetuximab blunted the erlotinib-induced EGFR up-regulation and resulted in inhibition of cell proliferation and apoptosis in the HuCCT1 cells. Blockage of erlotinib-induced EGFR synthesis in HuCCT1 cells by small interfering RNA resulted in identical antitumor effects as cetuximab, providing mechanistic specificity. In mice xenografted with A431, HuCCT1, and the pancreatic cancer cell line Panc430, maximal growth arrest and decrease in Ki67 proliferation index were documented with combined therapy, and EGFR down-regulation was observed in cetuximab-treated tumors. These results may indicate that resistance to EGFR kinase inhibition may be, at least in part, mediated by a highly dynamic feedback loop consisting of up-regulation of the EGFR upon exposure to EGFR kinase inhibitors. Abrogation of this response by small interfering RNA-mediated EGFR mRNA down-regulation and/or by cetuximab-mediated protein clearance induced tumor arrest across several cancer models with different EGFR expression levels, suggesting that resistance and sensitivity are dynamic events where proportional decrease in the target rather than absolute content dictates outcome.