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Nck2 promotes human melanoma cell proliferation, migration and invasion in vitro and primary melanoma-derived tumor growth in vivo.

Labelle-Côté M, Dusseault J, Ismaïl S, Picard-Cloutier A, Siegel PM, Larose L - BMC Cancer (2011)

Bottom Line: We found that expression of Nck2 is consistently increased in various metastatic cancer cell lines compared with primary counterparts.Particularly, we observed significant higher levels of Nck2 protein and mRNA, as opposed to no change in Nck1, in human metastatic melanoma cell lines compared with non-metastatic melanoma and normal melanocytes.This study provides new insights regarding cancer progression that could impact on the therapeutic strategies targeting cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Programmes de biologie moléculaire, Faculté de Médecine, Université deMontréal, Montréal, Québec, Canada.

ABSTRACT

Background: Nck1 and Nck2 adaptor proteins are involved in signaling pathways mediating proliferation, cytoskeleton organization and integrated stress response. Overexpression of Nck1 in fibroblasts has been shown to be oncogenic. Through the years this concept has been challenged and the consensus is now that overexpression of either Nck cooperates with strong oncogenes to transform cells. Therefore, variations in Nck expression levels in transformed cells could endorse cancer progression.

Methods: Expression of Nck1 and Nck2 proteins in various cancer cell lines at different stages of progression were analyzed by western blots. We created human primary melanoma cell lines overexpressing GFP-Nck2 and investigated their ability to proliferate along with metastatic characteristics such as migration and invasion. By western blot analysis, we compared levels of proteins phosphorylated on tyrosine as well as cadherins and integrins in human melanoma cells overexpressing or not Nck2. Finally, in mice we assessed tumor growth rate of human melanoma cells expressing increasing levels of Nck2.

Results: We found that expression of Nck2 is consistently increased in various metastatic cancer cell lines compared with primary counterparts. Particularly, we observed significant higher levels of Nck2 protein and mRNA, as opposed to no change in Nck1, in human metastatic melanoma cell lines compared with non-metastatic melanoma and normal melanocytes. We demonstrated the involvement of Nck2 in proliferation, migration and invasion in human melanoma cells. Moreover, we discovered that Nck2 overexpression in human primary melanoma cells correlates with higher levels of proteins phosphorylated on tyrosine residues, assembly of Nck2-dependent pY-proteins-containing molecular complexes and downregulation of cadherins and integrins. Importantly, we uncovered that injection of Nck2-overexpressing human primary melanoma cells into mice increases melanoma-derived tumor growth rate.

Conclusions: Collectively, our data indicate that Nck2 effectively influences human melanoma phenotype progression. At the molecular level, we propose that Nck2 in human primary melanoma promotes the formation of molecular complexes regulating proliferation and actin cytoskeleton dynamics by modulating kinases or phosphatases activities that results in increased levels of proteins phosphorylated on tyrosine residues. This study provides new insights regarding cancer progression that could impact on the therapeutic strategies targeting cancer.

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Effect of Nck2 on human primary melanoma cell migration. Human primary melanoma cell migration was evaluated using wound healing assays on WM278 cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14). Pictures from the same area were taken at time 0 and 8 hours after the wound. Magnification: 10X and white bar = 200 μm. Quantification of migration is expressed as percentage of closed wound ± SD. * p < 0.001 vs C2 using Student's t-test.
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Figure 5: Effect of Nck2 on human primary melanoma cell migration. Human primary melanoma cell migration was evaluated using wound healing assays on WM278 cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14). Pictures from the same area were taken at time 0 and 8 hours after the wound. Magnification: 10X and white bar = 200 μm. Quantification of migration is expressed as percentage of closed wound ± SD. * p < 0.001 vs C2 using Student's t-test.

Mentions: Cancer progression involves that transformed cells must acquire motility and invasive activities. Therefore, we next determined whether Nck2 was critical to melanoma cell migration and invasion. We compared migration of WM278 primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) in wound healing assays. As shown in Figure 5, increasing levels of GFP-Nck2 in WM278 melanoma cells promoted migration and this was significant in cell line N14, which expresses higher levels of Nck2 proteins compared with N5 and N7 cell lines (Figure 3A). To exclude that a clonal effect is responsible of increased migration of WM278 melanoma cells overexpressing GFP-Nck2, we transiently overexpressed HA-Nck2 in WM278 primary melanoma cells using retroviral infection (Additional file 3). In this context, we still observed a significant increase in migration of WM278 human primary melanoma cells overexpressing HA-Nck2 compared to control infected WM278 melanoma cells in wound healing assays. It is interesting to note though that the effect of Nck2 on migration was already observed 8 hours after wounding, suggesting that cell proliferation is not involved.


Nck2 promotes human melanoma cell proliferation, migration and invasion in vitro and primary melanoma-derived tumor growth in vivo.

Labelle-Côté M, Dusseault J, Ismaïl S, Picard-Cloutier A, Siegel PM, Larose L - BMC Cancer (2011)

Effect of Nck2 on human primary melanoma cell migration. Human primary melanoma cell migration was evaluated using wound healing assays on WM278 cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14). Pictures from the same area were taken at time 0 and 8 hours after the wound. Magnification: 10X and white bar = 200 μm. Quantification of migration is expressed as percentage of closed wound ± SD. * p < 0.001 vs C2 using Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198724&req=5

Figure 5: Effect of Nck2 on human primary melanoma cell migration. Human primary melanoma cell migration was evaluated using wound healing assays on WM278 cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14). Pictures from the same area were taken at time 0 and 8 hours after the wound. Magnification: 10X and white bar = 200 μm. Quantification of migration is expressed as percentage of closed wound ± SD. * p < 0.001 vs C2 using Student's t-test.
Mentions: Cancer progression involves that transformed cells must acquire motility and invasive activities. Therefore, we next determined whether Nck2 was critical to melanoma cell migration and invasion. We compared migration of WM278 primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) in wound healing assays. As shown in Figure 5, increasing levels of GFP-Nck2 in WM278 melanoma cells promoted migration and this was significant in cell line N14, which expresses higher levels of Nck2 proteins compared with N5 and N7 cell lines (Figure 3A). To exclude that a clonal effect is responsible of increased migration of WM278 melanoma cells overexpressing GFP-Nck2, we transiently overexpressed HA-Nck2 in WM278 primary melanoma cells using retroviral infection (Additional file 3). In this context, we still observed a significant increase in migration of WM278 human primary melanoma cells overexpressing HA-Nck2 compared to control infected WM278 melanoma cells in wound healing assays. It is interesting to note though that the effect of Nck2 on migration was already observed 8 hours after wounding, suggesting that cell proliferation is not involved.

Bottom Line: We found that expression of Nck2 is consistently increased in various metastatic cancer cell lines compared with primary counterparts.Particularly, we observed significant higher levels of Nck2 protein and mRNA, as opposed to no change in Nck1, in human metastatic melanoma cell lines compared with non-metastatic melanoma and normal melanocytes.This study provides new insights regarding cancer progression that could impact on the therapeutic strategies targeting cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Programmes de biologie moléculaire, Faculté de Médecine, Université deMontréal, Montréal, Québec, Canada.

ABSTRACT

Background: Nck1 and Nck2 adaptor proteins are involved in signaling pathways mediating proliferation, cytoskeleton organization and integrated stress response. Overexpression of Nck1 in fibroblasts has been shown to be oncogenic. Through the years this concept has been challenged and the consensus is now that overexpression of either Nck cooperates with strong oncogenes to transform cells. Therefore, variations in Nck expression levels in transformed cells could endorse cancer progression.

Methods: Expression of Nck1 and Nck2 proteins in various cancer cell lines at different stages of progression were analyzed by western blots. We created human primary melanoma cell lines overexpressing GFP-Nck2 and investigated their ability to proliferate along with metastatic characteristics such as migration and invasion. By western blot analysis, we compared levels of proteins phosphorylated on tyrosine as well as cadherins and integrins in human melanoma cells overexpressing or not Nck2. Finally, in mice we assessed tumor growth rate of human melanoma cells expressing increasing levels of Nck2.

Results: We found that expression of Nck2 is consistently increased in various metastatic cancer cell lines compared with primary counterparts. Particularly, we observed significant higher levels of Nck2 protein and mRNA, as opposed to no change in Nck1, in human metastatic melanoma cell lines compared with non-metastatic melanoma and normal melanocytes. We demonstrated the involvement of Nck2 in proliferation, migration and invasion in human melanoma cells. Moreover, we discovered that Nck2 overexpression in human primary melanoma cells correlates with higher levels of proteins phosphorylated on tyrosine residues, assembly of Nck2-dependent pY-proteins-containing molecular complexes and downregulation of cadherins and integrins. Importantly, we uncovered that injection of Nck2-overexpressing human primary melanoma cells into mice increases melanoma-derived tumor growth rate.

Conclusions: Collectively, our data indicate that Nck2 effectively influences human melanoma phenotype progression. At the molecular level, we propose that Nck2 in human primary melanoma promotes the formation of molecular complexes regulating proliferation and actin cytoskeleton dynamics by modulating kinases or phosphatases activities that results in increased levels of proteins phosphorylated on tyrosine residues. This study provides new insights regarding cancer progression that could impact on the therapeutic strategies targeting cancer.

Show MeSH
Related in: MedlinePlus