PGE2 induces sustained activation of Erk signaling in erlotinib-resistant proliferation of NSCLC cells.

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The Epidermal Growth Factor Receptor (EGFR) is frequently overexpressed in a variety of malignancies and plays an important role in tumor cell growth, proliferation and metastasis. Because of its important role in tumor progression, EGFR is a promising target for cancer therapy. However, only a limited population of cancer patients responds to EGFR inhibitor therapy. Recent studies have linked sensitivity to EGFR TK inhibitors to activating somatic mutations in the TK domain observed in some non-small cell lung cancer (NSCLC) patients. Other studies have focused on the cross-talk between the downstream components of the EGFR pathway and other signal transduction pathways that can overcome pharmacological inhibition of EGFR. Cyclooxygenase 2 (COX-2) is often upregulated in a wide variety of human cancers including lung cancer and is linked to all stages of tumorigenesis. We have found that the COX-2 metabolite prostaglandin E2 (PGE2) is able to activate the MAPK/Erk pathway in a subset of NSCLC cell lines in an EGFR-independent manner via intracellular cross-talk between EP1 receptor and protein kinase C signal transduction pathways. This activation is resistant to EGFR inhibitors including erlotinib and not evident in bronchial epithelial cells. The functional manifestation of such trans-activation was an enhanced NSCLC cell proliferation in response to PGE2 treatment that was resistant to EGFR inhibition, suggesting that COX-2 overexpression may contribute to EGFR inhibitor resistance in NSCLC. We have found that combination treatment of NSCLC cells with EGFR and COX-2 inhibitors significantly reduces cell proliferation compared to either drug alone. Analysis of temporal patterns of Erk phosphorylation in NSCLC cells in response to PGE2 or EGF alone or in combination revealed that PGE2 induces sustained Erk phosphorylation and stimulates accumulation of critical cell cycle modulators such as c-fos, c-myc and cyclin D1. This finding is in accord with previously reported data suggesting that cellular proliferative responses depend significantly on the duration of Erk phosphorylation following MAPK/Erk pathway activation. By immunohistochemistry we found that ten out of 16 lung adenocarcinoma specimens stained positively for both p-Erk and COX-2, one stained with neither of the 2 antibodies, three were COX-2-positive and p-Erk-negative and two - p-Erk-positive and COX-2-negative. Thus, in accord with our in vitro data these findings demonstrate that constitutive Erk activation is associated with COX-2 overexpression in a subset of lung adenocarcinomas. Overall, these results provide a strong rationale to evaluate the combined use of COX-2 and EGFR inhibition in NSCLC therapy. Erlotinib was generously provided by OSI Pharmaceuticals (Farmingdale, NY).