The dynamics of Rho GTPase signaling and implications for targeting cancer and the tumor microenvironment

Abstract

Numerous large scale genomics studies have demonstrated that cancer is a molecularly heterogeneous disease, characterized by acquired changes in the structure and DNA sequence of tumor genomes. More recently, the role of the equally complex tumor microenvironment in driving the aggressiveness of this disease is increasingly being realized. Tumor cells are surrounded by activated stroma, creating a dynamic environment that promotes cancer development, metastasis and chemoresistance. The Rho family of small GTPases plays an essential role in the regulation of cell shape, cytokinesis, cell adhesion, and cell motility. Importantly, these processes need to be considered in the context of a complex 3-dimensional (3D) environment, with reciprocal feedback and cross-talk taking place between the tumor cells and host environment. Here we discuss the role of molecular networks involving Rho GTPases in cancer, and the therapeutic implications of inhibiting Rho signaling in both cancer cells and the emerging concept of targeting the surrounding stroma.

Keywords: ECM; FLIM; FRET; Rho GTPase; Rho inhibitors; cancer stroma; chemoresistance; collagen; intravital imaging.

Figure 1

(See previous page). The Rac-FRET mouse is an invaluable tool to assess and quantify Rac1 activity in primary cells (A and B) and in vivo (C and D). (A) Neutrophil harvested from a Rac-FRET mouse migrating toward the chemoattractant N-formyl-methionyl-leucyl-phenyalanine (fMLP) south of the cell. Time series of Rac1 activity was obtained by ratiometric FRET live imaging and illustrated as a heat map of high (yellow to red) and low (blue to green) Rac1 activity. High Rac1 activity was localized to leading edge protrusions (green box) with short-lived bursts at the cell's periphery and at the trailing edge. (B) Maximum Rac1 activity along the longitudinal axis of a neutrophil (blue) migrating toward an fLMP gradient oscillated between the leading (green) and lagging edge (red) illustrated by fitting the experimental data (purple). (C) Intravital FLIM-FRET imaging demonstrated that Cre-mediated loss of APC in intestinal crypts promotes Rac1 activity. Low Rac1 activity in APC wild type mice (left panel) is represented by high fluorescence lifetimes (low FRET-efficiency, green), while loss of APC (right panel) results in high FRET-efficiency (low lifetimes, blue) indicating increased Rac1 activity. Each panel consists of fluorescence images of Rac-FRET expressing crypt cells (blue) and collagen (red, assessed by SHG imaging) on the left and FLIM images on the right. (D) Quantification of Rac1 activity by FLIM-FRET imaging reveals a significant decrease in the fluorescence lifetime upon deletion of APC correlating with an increase in Rac1 activity compared to APC wild type cells (mean± SEM; **P < 0.05). Figure adapted from Johnsson et al. Cell Reports .

Figure 2.

The ROCK inhibitor Y-27632 decreases collagen contraction and crosslinking driven by fibroblasts in 3D organotypic assays. (A) Top panel: Schematic representation and representative pictures of contracting collagen-fibroblast matrices over a 12-day time course. Lower panel: Area of collagen in matrices treated with DMSO (control) and with Y-27632 (10 μM) as a function of time, p* = 0.0268. Scale bar: 10 mm. (B) Collagen coverage (%) quantified by multi-photon SHG imaging as a variable associated with depth (μm) within the 12-day contracted collagen-fibroblast matrices treated with control (DMSO) or with Y-27632 (10 μM) P*** < 0.0001. (C) Maximum intensity projection of SHG signal in representative collagen-fibroblast matrices. D. 3D projection of SHG signal in representative collagen-fibroblast matrices n = 3. Scale bar: 100 μm.