Activating epidermal growth factor receptor (EGFR) mutations have been linked with sensitivity to gefitinib and erlotinib; however, there are no established predictive markers for response to the combination of EGFR inhibitors with standard chemotherapy in non -small cell lung cancer (NSCLC) patients. In this study, we characterized a panel of human EGFR wild-type and mutant NSCLC cells for their sensitivity to gefitinib alone and in combination with cisplatin or Taxol. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays. Cell cycle distribution was measured by flow cytometry. EGFR expression was measured by flow cytometry, real-time PCR, and Western blotting. EGFR/ Her2/Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation were measured by Western blotting. Two of nine EGFR wild type and one of two EGFR mutant NSCLC cells were sensitive to gefitinib, and this was associated with a decrease in phospho (p) -Akt and pErk1/2 following gefitinib exposure. There was no correlation between constitutive EGFR expression or activity and sensitivity to gefitinib nor was there a correlation between Her2/Akt and Erk1/2 activity and gefitinib sensitivity. However, in cells displaying a synergistic interaction between gefitinib and chemotherapy (cisplatin or Taxol), a dose-dependent increase in pEGFR was observed following chemotherapy exposure. In contrast, in cells where no change or a decrease in pEGFR following drug treatment was observed, we found an antagonistic or (at best) an additive interaction between the two compounds. Furthermore, the nature of this interaction was not dependent on the presence of a mutant EGFR. These novel findings suggest that modulation of EGFR activity following drug treatment determines response to gefitinib in combination with chemotherapy in NSCLC cells.

Purpose: Up to now, there have been no established predictive markers for response to epidermal growth factor receptor (EGFR/HER1/erbB1) inhibitors alone and in combination with chemotherapy in colorectal cancer. To identify markers that predict response to EGFR-based chemotherapy regimens, we analyzed the response of human colorectal cancer cell lines to the EGFR-tyrosine kinase inhibitor, gefitinib (Iressa, AstraZeneca, Wilmington, DE), as a single agent and in combination with oxaliplatin and 5-fluorouracil (5-FU). Experimental Design: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays and analyzed by ANOVA. Apoptosis was measured by flow cytometry, poly(ADP-ribose) polymerase, and caspase 3 cleavage. EGFR protein phosphorylation was detected by Western blotting. Results: Cell lines displaying high constitutive EGFR phosphorylation (a surrogate marker for EGFR activity) were more sensitive to gefitinib. Furthermore, in cell lines exhibiting low constitutive EGFR phosphorylation, an antagonistic interaction between gefitinib and oxaliplatin was observed, whereas in cell lines with high basal EGFR phosphorylation, the interaction was synergistic. In addition, oxaliplatin treatment increased EGFR phosphorylation in those cell lines in which oxaliplatin and gefitinib were synergistic but down-regulated EGFR phosphorylation in those lines in which oxaliplatin and gefitinib were antagonistic. In contrast to oxaliplatin, 5-FU treatment increased EGFR phosphorylation in all cell lines and this correlated with synergistic decreases in cell viability when 5-FU was combined with gefitinib. Conclusions: These results suggest that phospho-EGFR levels determine the sensitivity of colorectal cancer cells to gefitinib alone and that chemotherapy-mediated changes in phospho-EGFR levels determine the nature of interaction between gefitinib and chemotherapy.Colorectal cancer is the second most common cause of cancer death in the United States and Europe. Advances in first-line treatment of metastatic colorectal cancer have been achieved by addition of agents such as the topoisomerase I inhibitor CPT-11 and the DNA-damaging agent oxaliplatin to 5-fluorouracil (5-FU) -based chemotherapy with response rates in the 40% to 50% range (1 -4). Despite these improvements, the overall clinical effect of these therapies remains modest with the majority of patients relapsing with median survival times of 20 months (1 -4). The generation of selective agents, targeting malignant angiogenesis (5), cell cycle regulation (6), and transduction of growth stimulatory signals (7), has heralded an era of new therapeutic opportunities.Epidermal growth factor receptor (EGFR) is a member of the HER subfamily of four closely related receptors: EGFR (erbB1/HER1), ErbB2 (HER-2/neu), ErbB3 (HER3), and ErbB4 (HER4). The receptor is a 170-kDa transmembrane glycoprotein, with an extracellular ligand-binding domain and an intracellular region containing the tyrosine k...

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-A (TGF-A) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-A was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-A also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-A, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL. [Cancer Res 2008;68(20):8312-21]

Supplementary Figure 3 from Src and ADAM-17–Mediated Shedding of Transforming Growth Factor-α Is a Mechanism of Acute Resistance to TRAIL

Supplementary Figure 4 from Src and ADAM-17–Mediated Shedding of Transforming Growth Factor-α Is a Mechanism of Acute Resistance to TRAIL