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 tumorinhibitory 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.Rho GTPases | RhoA | cancer-associated fibroblasts | tumor-inhibitory capacity | cytoskeleton

Vimentin Diversity in Health and Disease

“…In support of this, in lung adenocarcinomas, glycosylated vimentin was shown to be frequently downregulated, defining its potential as a biomarker both for treatment and diagnosis [ 253 ]. A more recent study showed that vimentin expression in cancer-associated fibroblasts is required for dissemination of early stage lung adenocarcinoma, which indicates that vimentin might have a key role in the cancer-promoting capacity of cancer-associated fibroblasts in the tumour microenvironment [ 254 ]. Fibroblasts that promote the growth of cancer cells in vivo have also been shown to have reorganised vimentin filaments, compared to normal fibroblasts [ 255 ].…”

Fibroblasts in the Tumor Microenvironment: Shield or Spear?

“…RhoA is a master regulator of cell shape, adhesiveness, contractile behavior, and the configuration of focal adhesions [ 39 ]. Our studies showed that knockout of the RhoA gene in fibroblasts disturbs cytoskeletal organization, increases cellular stiffness, and decreases cellular contractility [ 40 ], which all are important properties required to maintain normal connective tissue structure and function [ 39 , 40 ]. As a result, the fibroblasts lose their tumor suppressive function and, rather, provide a growth stimulatory niche via induction of a cancer propagating phenotype in 3D-collagen culture and xenograft tumors in vivo [ 40 ].…”

LncRNAs regulate the cytoskeleton and related Rho/ROCK signaling in cancer metastasis

“…RhoA is present at the cell membrane when it is active [ 84 ]. RhoA regulates the generation of actomyosin bundles, stress fibers, focal adhesions, and lamellipodia [ 85 ]. RhoB is found in endosomes and at the plasma membrane.…”