RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo.

Abstract

Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins overexpressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of α-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.

• Authors: Twana Alkasalias, Andrey Alexeyenko, Katharina Hennig, Frida Danielsson, Robert Jan Lebbink, Matthew Fielden, S Pauliina Turunen, Kaisa Lehti, Vladimir Kashuba, Harsha S Madapura, Benedek Bozoky, Emma Lundberg, Martial Balland, Hayrettin Guvén, George Klein, Annica K B Gad, Tatiana Pavlova
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 114 Issue 8 Pg. E1413-E1421 (02 21 2017) ISSN: 1091-6490 [Electronic] United States
• PMID: 28174275 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Actins
• Collagen
• rho-Associated Kinases
• rhoA GTP-Binding Protein

Topics

• Actins (metabolism)
• Animals
• Cancer-Associated Fibroblasts (metabolism)
• Cell Line, Tumor
• Cell Movement (physiology)
• Cell Proliferation (physiology)
• Cells, Cultured
• Collagen (metabolism)
• Female
• Focal Adhesions (metabolism)
• HEK293 Cells
• Humans
• Mice
• Mice, SCID
• Neoplasms (metabolism)
• Signal Transduction (physiology)
• Stress Fibers (metabolism)
• rho-Associated Kinases (metabolism)
• rhoA GTP-Binding Protein (metabolism)