Publicação
The role of KRAS mutations signalling in autophagy control in Colorectal Carcinomas
| Resumo: | Colorectal carcinoma (CRC) is one of the most common types of cancer, leading to a high rate of mortality in Europe. Current therapies for CRC patients present poor outcomes, being mandatory new therapeutic approaches. Autophagy is an essential process for protein turn-over and cellular homeostasis being tightly implicated in cancer. This process has been described as fundamental in CRC cells chemoresistance emerging as a crucial target to anticancer therapy. One of the most frequent events found in CRC is the presence of activating mutations in KRAS (KRASG13D, KRASG12D and KRASG12V) which have a role in the genesis and progression of these tumours. Several studies have already demonstrated the importance of KRASG12V in autophagy induction either in fibroblasts and epithelial cells. However, little is known concerning the precise role of the different KRAS mutations in autophagy regulation in CRC cells. This project aimed to determine the role of KRAS mutations signalling in cellular survival/proliferation and autophagy regulation in CRC. For such purpose, a CRC derived cell line harbouring KRASG12V mutation (SW480) and a kidney derived cell line (HEK 293) stably expressing Flag-KRASWT, Flag-KRASG13D, Flag-KRASG12D, and Flag-KRASG12V were used as in vitro models. Downregulation of KRAS was performed by RNA interference (RNAi); the levels of autophagic markers were accessed by Western-blot analysis; cellular survival and proliferation were evaluated by cell cycle analysis and colony formation assay technique. Our preliminary results showed that in SW480 cells, upon KRAS inhibition by RNAi, there was a decrease in autophagic markers levels namely LC3 II, Atg5 and p62 as well as a decrease in ERK phosphorylation. Down-regulation of KRAS led to a decrease in S-phase and an increase of sub-G1 phase of the cell cycle. Moreover, treatment with the autophagy inhibitor, Bafilomycin A1, also led to an increase in cell death. In contrast, the results observed in HEK 293 cells do not support the obtained in SW480 cells, which might indicate that HEK 293 cells are not a suitable model to study the role of typical CRC KRAS mutations in autophagy regulation. Altogether the results indicate that KRASG12V protein may play a crucial role in autophagy regulation, survival and cell proliferation, possibly involving RAS–RAF–MEK–ERK signalling pathway, though further studies are required. The results also showed that CRC cells harbouring KRASG12V mutation seem to be dependent on autophagy for survival which can have important implications in CRC therapy. |
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| Autores principais: | Castro, Paula Alexandra Martins Cosme Vieira de |
| Ano: | 2012 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Colorectal carcinoma (CRC) is one of the most common types of cancer, leading to a high rate of mortality in Europe. Current therapies for CRC patients present poor outcomes, being mandatory new therapeutic approaches. Autophagy is an essential process for protein turn-over and cellular homeostasis being tightly implicated in cancer. This process has been described as fundamental in CRC cells chemoresistance emerging as a crucial target to anticancer therapy. One of the most frequent events found in CRC is the presence of activating mutations in KRAS (KRASG13D, KRASG12D and KRASG12V) which have a role in the genesis and progression of these tumours. Several studies have already demonstrated the importance of KRASG12V in autophagy induction either in fibroblasts and epithelial cells. However, little is known concerning the precise role of the different KRAS mutations in autophagy regulation in CRC cells. This project aimed to determine the role of KRAS mutations signalling in cellular survival/proliferation and autophagy regulation in CRC. For such purpose, a CRC derived cell line harbouring KRASG12V mutation (SW480) and a kidney derived cell line (HEK 293) stably expressing Flag-KRASWT, Flag-KRASG13D, Flag-KRASG12D, and Flag-KRASG12V were used as in vitro models. Downregulation of KRAS was performed by RNA interference (RNAi); the levels of autophagic markers were accessed by Western-blot analysis; cellular survival and proliferation were evaluated by cell cycle analysis and colony formation assay technique. Our preliminary results showed that in SW480 cells, upon KRAS inhibition by RNAi, there was a decrease in autophagic markers levels namely LC3 II, Atg5 and p62 as well as a decrease in ERK phosphorylation. Down-regulation of KRAS led to a decrease in S-phase and an increase of sub-G1 phase of the cell cycle. Moreover, treatment with the autophagy inhibitor, Bafilomycin A1, also led to an increase in cell death. In contrast, the results observed in HEK 293 cells do not support the obtained in SW480 cells, which might indicate that HEK 293 cells are not a suitable model to study the role of typical CRC KRAS mutations in autophagy regulation. Altogether the results indicate that KRASG12V protein may play a crucial role in autophagy regulation, survival and cell proliferation, possibly involving RAS–RAF–MEK–ERK signalling pathway, though further studies are required. The results also showed that CRC cells harbouring KRASG12V mutation seem to be dependent on autophagy for survival which can have important implications in CRC therapy. |
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