RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo.
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.
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden.|Department of Biology, College of Science, Salahaddin University, 44002, Irbil, Kurdistan-Iraq.
Twana
T
Alkasalias
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden.|National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Karolinska Institute, 17177 Stockholm, Sweden.
Andrey
A
Alexeyenko
Laboratoire Interdisciplinaire de Physique, Université Joseph Fourier (Grenoble 1), 38402 Saint Martin d'Hères Cedex 9, France.
Katharina
K
Hennig
National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Karolinska Institute, 17177 Stockholm, Sweden.
Frida
F
Danielsson
Department of Medical Microbiology, University Medical Centre Utrecht, 3584 CX Utrecht, The Netherlands.
Robert Jan
RJ
Lebbink
Department of Applied Physics, Nanostructure Physics, Kungliga Tekniska Högskolan Royal Institute of Technology, Albanova University Center, 106 91 Stockholm, Sweden.
Matthew
M
Fielden
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
S Pauliina
SP
Turunen
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden.|Research Programmes Unit, Genome-Scale Biology, University of Helsinki, Finnish Cancer Institute, F1-00014 Helsinki, Finland.
Kaisa
K
Lehti
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden.|Department of Molecular Oncogenetics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 01004 Kiev, Ukraine.
Vladimir
V
Kashuba
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
Harsha S
HS
Madapura
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
Benedek
B
Bozoky
Cell Profiling Facility, Science for Life Laboratory, Kungliga Tekniska Högskolan Royal Institute of Technology, 17177 Stockholm, Sweden.
Emma
E
Lundberg
Laboratoire Interdisciplinaire de Physique, Université Joseph Fourier (Grenoble 1), 38402 Saint Martin d'Hères Cedex 9, France.
Martial
M
Balland
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
Hayrettin
H
Guvén
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden; georg.klein@ki.se Annica.Gad@ki.se tatiana.pavlova@ki.se.
George
G
Klein
Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden georg.klein@ki.se Annica.Gad@ki.se tatiana.pavlova@ki.se.
Annica K B
AK
Gad
Department of Microbiology, Tumour, and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden; georg.klein@ki.se Annica.Gad@ki.se tatiana.pavlova@ki.se.
Tatiana
T
Pavlova
D000199:Actins / Q000378:metabolism; D000818:Animals; D000072645:Cancer-Associated Fibroblasts / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology*; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D005260:Female; D022001:Focal Adhesions / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009369:Neoplasms / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D022502:Stress Fibers / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D003094:Collagen; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
Rho GTPases; RhoA; cancer-associated fibroblasts; cytoskeleton; tumor-inhibitory capacity