BACKGROUND: Although there is extensive evidence for the amoeboid invasiveness of cancer cells in vitro, much less is known about the role of amoeboid invasiveness in metastasis and the importance of Rho/ROCK/MLC signaling in this process. RESULTS: We analyzed the dependence of amoeboid invasiveness of rat and chicken sarcoma cells and the metastatic activity of chicken cells on individual elements of the Rho/ROCK/MLC pathway. In both animal models, inhibition of Rho, ROCK or MLC resulted in greatly decreased cell invasiveness in vitro, while inhibition of extracellular proteases using a broad spectrum inhibitor did not have a significant effect. The inhibition of both Rho activity and MLC phosphorylation by dominant negative mutants led to a decreased capability of chicken sarcoma cells to metastasize. Moreover, the overexpression of RhoA in non-metastatic chicken cells resulted in the rescue of both invasiveness and metastatic capability. Rho and ROCK, unlike MLC, appeared to be directly involved in the maintenance of the amoeboid phenotype, as their inhibition resulted in the amoeboid-mesenchymal transition in analyzed cell lines. CONCLUSION: Taken together, these results suggest that protease-independent invasion controlled by elements of the Rho/ROCK/MLC pathway can be frequently exploited by metastatic sarcoma cells.
- MeSH
- invazivní růst nádoru MeSH
- kinázy asociované s rho metabolismus MeSH
- krysa rodu rattus MeSH
- kur domácí MeSH
- lehké řetězce myosinu metabolismus MeSH
- nádorové buněčné linie MeSH
- pohyb buněk MeSH
- rho proteiny vázající GTP metabolismus MeSH
- sarkom metabolismus patologie MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of this study was to analyze the potential role of NG2 chondroitin sulfate proteoglycan in amoeboid morphology and invasiveness of cancer cells. In the highly metastatic amoeboid cell lines A3 and A375M2, siRNA-mediated down-regulation of NG2 induced an amoeboid-mesenchymal transition associated with decreased invasiveness in 3D collagen and inactivation of the GTPase Rho. Conversely, the expression of NG2 in mesenchymal sarcoma K2 cells as well as in A375M2 cells resulted in an enhanced amoeboid phenotype associated with increased invasiveness and elevated Rho-GTP levels. Remarkably, the amoeboid-mesenchymal transition in A375M2 cells triggered by NG2 down-regulation was associated with increased extracellular matrix-degrading ability, although this was not sufficient to compensate for the decreased invasive capability caused by down-regulated Rho/ROCK signaling. Conversely, in K2 cells with overexpression of NG2, the ability to degrade the extracellular matrix was greatly reduced. Taken together, we suggest that NG2-mediated activation of Rho leading to effective amoeboid invasiveness is a possible mechanism through which NG2 could contribute to tumor cell invasion and metastasis.
- MeSH
- chondroitinsulfát proteoglykany genetika metabolismus MeSH
- down regulace MeSH
- extracelulární matrix metabolismus MeSH
- invazivní růst nádoru MeSH
- kinázy asociované s rho metabolismus MeSH
- kolagen chemie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- malá interferující RNA MeSH
- membránové proteiny genetika metabolismus MeSH
- molekulární konformace MeSH
- nádorové buněčné linie MeSH
- nádory metabolismus patologie ultrastruktura MeSH
- pohyb buněk * MeSH
- rho proteiny vázající GTP metabolismus MeSH
- upregulace MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The newly established breast cancer cell line G3S1, derived from EM-G3 breast cancer progenitors, was analyzed for functional changes related to neoplastic progression manifested by elevated invasiveness and enhanced capability to degrade gelatin. Degradation of gelatin and invasiveness of G3S1 cells was found to be dependent on the activity of matrix proteinases and actin cytoskeletal dynamics. Therefore, the expression and activity of these proteases was compared in G3S1 and EM-G3 cells. Despite enhanced capability of G3S1 cells to degrade gelatin, these cells exhibited lower levels of secreted extracellular matrix degrading proteases than parental EM-G3 cells. However, the expression of membrane-bound MT1-MMP was strongly elevated in G3S1 cells. While the degradation of gelatin was associated with invadopodia-like structures in both EM-G3 and G3S1 cells, the cytoskeletal remodeling dynamics was greatly elevated in G3S1 cells, suggesting that upregulation of MT1-MMP, together with elevation of cytoskeletal remodeling dynamics can effectively cause elevated invasiveness and enhanced matrix degrading capability in G3S1 cells.
- MeSH
- aktiny metabolismus MeSH
- aprotinin farmakologie MeSH
- cytoskelet účinky léků enzymologie patologie MeSH
- dipeptidy farmakologie MeSH
- inhibitory matrixových metaloproteinas MeSH
- inhibitory proteas farmakologie MeSH
- invazivní růst nádoru MeSH
- leucin analogy a deriváty farmakologie MeSH
- lidé MeSH
- matrixová metaloproteinasa 14 metabolismus MeSH
- metaloproteinasy secernované do matrix metabolismus MeSH
- mořské toxiny farmakologie MeSH
- nádorové buněčné linie MeSH
- nádorové kmenové buňky enzymologie patologie MeSH
- nádory prsu enzymologie patologie MeSH
- pohyb buněk účinky léků MeSH
- progrese nemoci MeSH
- pseudopodia enzymologie MeSH
- upregulace MeSH
- želatina metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress and treatments with 12-O-tetradecanoylphorbol-13-acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM-G3 cells. Elevated invasiveness of G3S1 cells was accompanied by higher incidence of myzitiras morphotype (sucker-like) and newly observed vthela morphotype (leech-like) both inducible in Hanks' Balanced Salt Solution test. Time-lapse phase contrast microscopy showed a capacity of G3S1 cells to form lobopodial protrusions already 20 min after seeding on gelatin. These protrusions could make contact with the dish and possibly produce the vthela shape. The possible relationship of mysitiras and vthela morphotypes to an increase in malignant potential marked by enhanced invasiveness was thus indicated. Copyright 2009 Wiley Periodicals, Inc.
Tumor cell invasion is the most critical step of metastasis. Determination of the mode of invasion within the particular tumor is critical for effective cancer treatment. Protease-independent amoeboid mode of invasion has been described in carcinoma cells and more recently in sarcoma cells on treatment with protease inhibitors. To analyze invasive behavior, we compared highly metastatic sarcoma cells with parental nonmetastatic cells. The metastatic cells exhibited a functional up-regulation of Rho/ROCK signaling and, similarly to carcinoma cells, an amoeboid mode of invasion. Using confocal and traction force microscopy, we showed that an up-regulation of Rho/ROCK signaling leads to increased cytoskeletal dynamics, myosin light chain localization, and increased tractions at the leading edge of the cells and that all of these contributed to increased cell invasiveness in a three-dimensional collagen matrix. We conclude that cells of mesenchymal origin can use the amoeboid nonmesenchymal mode of invasion as their primary invading mechanism and show the dependence of ROCK-mediated amoeboid mode of invasion on the increased capacity of cells to generate force.
- MeSH
- aktiny metabolismus MeSH
- buněčná adheze fyziologie MeSH
- čipová analýza proteinů MeSH
- cytoskelet metabolismus patologie MeSH
- faktory depolymerizující aktin metabolismus MeSH
- financování organizované MeSH
- fluorescenční mikroskopie MeSH
- fosforylace MeSH
- invazivní růst nádoru MeSH
- kinázy asociované s rho genetika metabolismus MeSH
- kolagen metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lehké řetězce myosinu metabolismus MeSH
- Lim-kinasy genetika metabolismus MeSH
- magnetismus MeSH
- matrixová metaloproteinasa 2 metabolismus MeSH
- mezenchymální kmenové buňky metabolismus patologie MeSH
- pohyb buněk fyziologie MeSH
- rho proteiny vázající GTP genetika metabolismus MeSH
- sarkom metabolismus patologie MeSH
- upregulace MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
