P3.01-038 STAT3 and Src-YAP1 Inhibition Results in Greater Necitumumab Sensitivity in Lung Squamous Cell Carcinoma: Topic: Functional Biology in Lung Cancer

Abstract

The anti-EGFR monoclonal antibody (mAb), necitumumab, has been recently approved in combination with chemotherapy, as 1st-line treatment for advanced lung squamous cell carcinoma (LSCC) patients, but with minimal survival benefit. Evidence continues to accumulate that signal transducer and activator of transcription 3 (STAT3) is a promising molecular target for cancer therapies. STAT3 is activated by tyrosine phosphorylation in response to EGF and interleukin-6 (IL-6). In addition to STAT3, IL-6 activates the Src family kinases, and subsequently YES-associated protein 1 (YAP1). STAT3 and Src-YAP1 activation contributes to EGFR inhibitor resistance and concomitant targeting of EGFR and STAT3-Src may represent an effective treatment strategy for LSCC. RNA was isolated from six LSCC cell lines and the mRNA expression analysis of EGFR, STAT3, Src and YAP1 was performed by TaqMan based qRT-PCR. Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with necitumumab and evodiamine, an alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit that exerts an anticancer effect by inhibiting STAT3 and Src. Western blotting was performed to assess the effect of necitumumab on EGFR downstream signaling pathways. We first evaluated the expression of EGFR in our panel of LSCC cell lines. We found that almost all of them homogeneously express high levels of EGFR. We then assessed the effect of necitumumab on EGFR downstream signaling in the SK-MES1 cell line. Treatment of SK-MES1 cells with 25ug/ml of necitumumab for seven days was unable to ablate STAT3, Src or YAP1 mRNA expression. Consistent with this, we found that necitumumab suppressed EGFR, ERK1/2 and AKT phosphorylation but increased STAT3 phosphorylation on the critical tyrosine residue 705 in a time and dose-dependent manner. We examined the growth inhibitory effect of the necitumumab and evodiamine combination. We performed an MTT cell proliferation assay on SK-MES1 cells and we used a constant ratio drug combination method to determine synergy, additivity, or antagonism. The combination of necitumumab and evodiamine resulted in a clear synergism in SK-MES1 cells as measured by the combination index (CI) analysis, with a CI of 0.74. Experiments in the rest of our LSCC cell lines are ongoing. Herein we have examined the role of STAT3 and Src-YAP1 in the context of treatment with the FDA-approved EGFR mAb, necitumumab. Our data provide initial evidence that co-activation of STAT3 and Src-YAP1 may limit the cellular response to EGFR inhibition in LSCC.