Abstract 5222: A novel mechanism of microenvironment-mediated drug resistance in BRAF V600E melanoma with loss of PTEN
Authors: Fedorenko Inna V. , Paraiso Kim H.T. , Rebecca Vito W. , Wood Elizabeth , Koomen John , Messina Jane , Smalley Keiran 
Published: August 2012

Abstract
Despite recent success of the BRAF kinase inhibitor vemurafenib in the clinic, nearly all of those treated follow a course in which initial tumor regression was followed by eventual relapse, which constitutes a major barrier to long-term therapeutic management of melanoma. We have identified a novel mechanism of microenvironment-mediated drug resistance that arises from the release of TGFβ from melanomas that lack PTEN expression leading to the recruitment of host fibroblasts and the generation of a protective microenvironment through autocrine and paracrine fibronectin secretion facilitating therapeutic escape. Preliminary studies show that BRAF V600E mutant melanoma cell lines that lacked PTEN expression dramatically increased their secretion of TGF-β following vemurafenib treatment, as detected by western blot, immunofluorescence and ELISA assays. Furthermore, autocrine TGFβ secretion initiated with vemurafinib treatment stimulated the upregulation and secretion of fibronectin in PTEN- cells. The vemurafenib-mediated ECM remodeling in this case was dependent upon autocrine TGFβ signaling and could be attenuated following pre-treatment with the TGFβ receptor kinase inhibitor SB505124 or by siRNA knockdown of TGFβ receptor I. A mass spectrometry based phospho-proteomic screen in conjunction with innovative network modeling approaches suggested that increased integrin α5β1 signaling was a key adaptive response of PTEN- melanoma cells to BRAF inhibitor therapy. The importance of increased fibronectin expression and integrin α5β1 signaling in the therapeutic escape of PTEN- melanoma cells was confirmed by the significant increases in vemurafenib-induced apoptosis observed following siRNA knockdown of either integrin α5, β1, or fibronectin expression. A mass spectrometry screen (LC-MRM) of 18 BH3 proteins demonstrated Mcl-1 and Bak to be critical apoptosis regulators in the microenvironment-mediated drug resistance response. The importance of PTEN loss and Mcl-1 downregulation in therapeutic escape was validated in PTEN- melanoma cell lines expressing doxycycline inducible forms of PTEN, lipid phosphatase mutant PTEN and Mcl-1. Novel organotypic co-culture models were developed to demonstrate that TGF-β released from PTEN- melanoma cell lines activated host fibroblasts leading to the attenuation of drug response in the tumor cells. Our overall hypothesis is that BRAF inhibition remodels both the melanoma and host microenvironments to provide a protective “sanctuary” for the minor populations of melanoma cells that escape therapy. We postulate that tumor eradication can only be achieved by a dual therapeutic strategy that targets both the bulk tumor and the microenvironment.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5222. doi:1538-7445.AM2012-5222