Background:Treatment with epidermal growth factor receptor (EGFR) inhibitors can result in clinical response in non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) for some unselected patients. EGFR and KRAS mutation status, amplification of EGFR, or gene expression predictors of response can forecast sensitivity to EGFR inhibition.Methods:Using an NSCLC cell line model system, we identified and characterised microRNA (miRNA) gene expression that predicts response to EGFR inhibition.Results:Expression of 13 miRNA genes predicts response to EGFR inhibition in cancer cell lines and tumours, and discriminates primary from metastatic tumours. Signature genes target proteins that are enriched for epithelial-to-mesenchymal transition (EMT) genes. Epithelial-to-mesenchymal transition predicts EGFR inhibitor resistance and metastatic behaviour. The EMT transcription factor, ZEB1, shows altered expression in erlotinib-sensitive NSCLC and PDAC, where many signature miRNA genes are upregulated. Ectopic expression of mir-200c alters expression of EMT proteins, sensitivity to erlotinib, and migration in lung cells. Treatment with TGFβ1 changes expression of signature miRNA and EMT proteins and modulates migration in lung cells.Conclusion:From these data, we hypothesise that the tumour microenvironment elicits TGFβ1 and stimulates a miRNA gene expression program that induces resistance to anti-EGFR therapy and drives lung tumour cells to EMT, invasion, and metastasis.

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Purpose: The intent of the study described herein is to develop and characterize a microRNA gene expression signature of response to EGFR inhibition portable to solid tumors for which treatment with an EGFR inhibitor is indicated. Experimental Design: Treatment with inhibitors of the epidermal growth factor receptor (EGFR) can result in clinical response in both non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC), but only in a minority of unselected patients. Identification of biomarkers that can direct treatment to the responsive population is essential and here we describe generation of a microRNA gene signature of response to EGFR inhibition. Using a Taqman MicroRNA Array of 381 probes, we demonstrate that a 20-gene microRNA signature derived from NSCLC cell lines can predict sensitivity of both lung and pancreatic cancer cell lines to erlotinib, a small molecule inhibitor of EGFR. Overlap of independently generated microRNA expression signatures of NSCLC and pancreatic cell lines produces a fourgene signature. We characterize a target of the reduced signature, ZEB1, in NSCLC and pancreatic cancer cell lines by evaluation of mRNA and protein expression. We evaluated ZEB1 protein and the ZEB1 target, E-cadherin, after transfection of mir-200c into inhibitor-resistant NSCLC cell line, A549. Results: We have identified a 20-gene microRNA signature of response to EGFR inhibition that predicts both inhibitor-sensitive NSCLC and pancreatic cell lines. By overlapping independent signatures of response in NSCLC and pancreatic cancer cell lines, a four gene microRNA signature is identified that targets proteins responsible for epithelial to mesenchymal transition (EMT). Of those proteins, ZEB1 is a transcription factor responsible for repression of E-cadherin expression. Both ZEB1 mRNA and protein expression are down-regulated in erlotinib-sensitive NSCLC in which the microRNA genes 200b, 200c, and 141 are up-regulated. Ectopic expression of mir-200c in inhibitor-resistant lines induces cell death, suggesting that down-regulation of this microRNA is essential for viability in these NSCLC lines. Conclusions: The 20-gene miRNA signature of sensitivity provides a means for defining response that can be generated from FFPE samples of tumor rather than fresh tumor samples. Whereas mRNA is difficult to isolate from FFPE samples due to degradation, microRNA may survive the fixation process. Thus, response to EGFR inhibition in the second- or third-line can be predicted from fixed tumor samples collected early in the treatment of an individual patient, eliminating the need for acquisition of fresh tumor. Further, the microRNA signature identifies the well-studied EMT pathway indicative of EGFR-inhibitor sensitivity and provides biological significance for the members of the signature. Citation Information: Cancer Res 2009;69(23 Suppl):B74.