Rho/MRTF Pathway Signaling and Small-Molecule Inhibitor Development in Systemic Sclerosis and Metastatic Melanoma

Abstract

Rho GTPases regulate multiple biological functions; most notably, they stimulate formation of F-actin stress fiber formations. Through their modulation of the actin cytoskeleton Rho GTPases also activate gene transcription through myocardin-related transcription factors (MRTF) and the serum response factor (SRF). Recent evidence suggests MRTF/SRF-regulated gene transcription represent a novel target for the treatment of systemic sclerosis and melanoma. The work of this thesis details mechanisms of Rho GTPase and MRTF/SRF activation in these diseases, and provides in vitro and in vivo efficacy studies for a small-molecule inhibitor of the MRTF pathway developed in our lab, CCG-203971. Tissue fibrosis disorders, including systemic sclerosis are characterized by activated myofibroblasts within the affected tissue. These myofibroblasts are stimulated by multiple microenvironment factors. Transforming growth factor beta (TGF-beta) signaling involves cross talk between multiple mediators including Rho GTPase. However, the mechanism by which TGF-beta activates Rho GTPase and subsequent MRTF/SRF-regulated transcription is poorly understood. Based on the evidence here we outline a mechanism where TGF-beta stimulates expression of connective tissue growth factor (CTGF) and endothelin-1 (ET-1), which then mediate the activation of the Rho/MRTF pathway. MRTF/SRF transcription is a convergent mechanism downstream of multiple receptors that stimulate fibrosis. We tested our MRTF pathway inhibitor, CCG-203971 in models of systemic sclerosis and showed inhibition of multiple markers of fibrosis in vitro, as well as efficacy in a mouse model of dermal fibrosis in vivo. Melanoma is the most deadly form of skin cancer, with the majority of deaths attributed to metastasis. Rho GTPases have been implicated in cancer metastasis for many years, however recently it has been shown that MRTF/SRF-regulated gene transcription is essential for melanoma metastasis. Here we show that in highly invasive and metastatic melanoma cells, overexpression of RhoC leading to constitutively active MRTF. CCG-203971 treatment in these cells reduced cellular migration and invasion in vitro and blocked lung colonization in a mouse model of melanoma metastasis in vivo.