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Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model
BACKGROUND: Previously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival,...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135494/ https://www.ncbi.nlm.nih.gov/pubmed/21714887 http://dx.doi.org/10.1186/1475-2867-11-22 |
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author | Rybko, Vera A Knizhnik, Anna V Komelkov, Andrei V Aushev, Vasily N Trukhanova, Lyubov S Tchevkina, Elena M |
author_facet | Rybko, Vera A Knizhnik, Anna V Komelkov, Andrei V Aushev, Vasily N Trukhanova, Lyubov S Tchevkina, Elena M |
author_sort | Rybko, Vera A |
collection | PubMed |
description | BACKGROUND: Previously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts. METHODS: Retroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography. RESULTS: We have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation. CONCLUSIONS: Presented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression. |
format | Online Article Text |
id | pubmed-3135494 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-31354942011-07-14 Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model Rybko, Vera A Knizhnik, Anna V Komelkov, Andrei V Aushev, Vasily N Trukhanova, Lyubov S Tchevkina, Elena M Cancer Cell Int Primary Research BACKGROUND: Previously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts. METHODS: Retroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography. RESULTS: We have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation. CONCLUSIONS: Presented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression. BioMed Central 2011-06-29 /pmc/articles/PMC3135494/ /pubmed/21714887 http://dx.doi.org/10.1186/1475-2867-11-22 Text en Copyright ©2011 Rybko et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Primary Research Rybko, Vera A Knizhnik, Anna V Komelkov, Andrei V Aushev, Vasily N Trukhanova, Lyubov S Tchevkina, Elena M Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model |
title | Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model |
title_full | Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model |
title_fullStr | Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model |
title_full_unstemmed | Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model |
title_short | Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model |
title_sort | different metastasis promotive potency of small g-proteins rala and ralb in in vivo hamster tumor model |
topic | Primary Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135494/ https://www.ncbi.nlm.nih.gov/pubmed/21714887 http://dx.doi.org/10.1186/1475-2867-11-22 |
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