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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 cadherins and integrins expression in human melanoma cells. Total cell lysates (50 μg protein) prepared human primary (WM278) or metastatic (WM1617) melanoma cells, as well as WM278 cells overexpressing GFP (C2) or GFP-Nck2 (N7, N14) were probed for cadherins and integrins using specific antibodies. β-tubulin used as loading control.
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Figure 9: Effect of Nck2 on cadherins and integrins expression in human melanoma cells. Total cell lysates (50 μg protein) prepared human primary (WM278) or metastatic (WM1617) melanoma cells, as well as WM278 cells overexpressing GFP (C2) or GFP-Nck2 (N7, N14) were probed for cadherins and integrins using specific antibodies. β-tubulin used as loading control.

Mentions: Protein tyrosine phosphorylation is a critical mechanism regulating focal adhesion dynamics [27,28]. Substantial evidence support a role for protein tyrosine kinases in focal adhesions assembly/disassembly toward the formation of invasive adhesion structures called invadopodia during cancer progression [29-33]. To investigate the mechanism by which Nck2 overexpression impinges on the phenotype of primary melanoma cells, we compared the levels of tyrosine phosphorylated proteins between human melanoma cells expressing different levels of Nck2 protein. To evaluate protein tyrosine phosphorylation, we exposed melanoma cells to pervanadate, a potent protein tyrosine phosphatase inhibitor [34-36] that allows tyrosine phosphorylated proteins to accumulate before harvesting the cells and performing immunoprecipitation. In these conditions, as observed in human metastatic (WM1617, WM164) compared with (WM278) melanoma cells (Figure 8A), WM278 cells overexpressing GFP-Nck2 presented increasing levels of proteins phosphorylated on tyrosine residues than WM278 control cells overexpressing GFP (Figure 8A). In addition, we found that tyrosine phosphorylated proteins coimmunoprecipitated with Nck2 (Nck2 IP) or total Nck (pan-Nck IP) were more abundant in human metastatic melanoma WM1617 cells compared with the counterpart WM278 primary melanoma cells (Figure 8B). We discovered also that like the metastatic WM1617 melanoma cells, the WM278 primary melanoma cells overexpressing high levels GFP-Nck2 (N14) displayed low levels of Integrin β1 and β3, as well as E- and N-Cadherin in Triton X-100 soluble extracts compared either with parental WM278 cells, WM278 cells overexpressing GFP (C2) or low levels of GFP-Nck2 (N7) (Figure 9). Collectively, these results reveal that increased expression of Nck2 in human primary melanoma cells promotes phosphorylation of proteins on tyrosine, concomitant with the assembly of Nck2-dependent pY-proteins containing molecular complexes and downregulation of cell surface adhesion proteins.


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 cadherins and integrins expression in human melanoma cells. Total cell lysates (50 μg protein) prepared human primary (WM278) or metastatic (WM1617) melanoma cells, as well as WM278 cells overexpressing GFP (C2) or GFP-Nck2 (N7, N14) were probed for cadherins and integrins using specific antibodies. β-tubulin used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Effect of Nck2 on cadherins and integrins expression in human melanoma cells. Total cell lysates (50 μg protein) prepared human primary (WM278) or metastatic (WM1617) melanoma cells, as well as WM278 cells overexpressing GFP (C2) or GFP-Nck2 (N7, N14) were probed for cadherins and integrins using specific antibodies. β-tubulin used as loading control.
Mentions: Protein tyrosine phosphorylation is a critical mechanism regulating focal adhesion dynamics [27,28]. Substantial evidence support a role for protein tyrosine kinases in focal adhesions assembly/disassembly toward the formation of invasive adhesion structures called invadopodia during cancer progression [29-33]. To investigate the mechanism by which Nck2 overexpression impinges on the phenotype of primary melanoma cells, we compared the levels of tyrosine phosphorylated proteins between human melanoma cells expressing different levels of Nck2 protein. To evaluate protein tyrosine phosphorylation, we exposed melanoma cells to pervanadate, a potent protein tyrosine phosphatase inhibitor [34-36] that allows tyrosine phosphorylated proteins to accumulate before harvesting the cells and performing immunoprecipitation. In these conditions, as observed in human metastatic (WM1617, WM164) compared with (WM278) melanoma cells (Figure 8A), WM278 cells overexpressing GFP-Nck2 presented increasing levels of proteins phosphorylated on tyrosine residues than WM278 control cells overexpressing GFP (Figure 8A). In addition, we found that tyrosine phosphorylated proteins coimmunoprecipitated with Nck2 (Nck2 IP) or total Nck (pan-Nck IP) were more abundant in human metastatic melanoma WM1617 cells compared with the counterpart WM278 primary melanoma cells (Figure 8B). We discovered also that like the metastatic WM1617 melanoma cells, the WM278 primary melanoma cells overexpressing high levels GFP-Nck2 (N14) displayed low levels of Integrin β1 and β3, as well as E- and N-Cadherin in Triton X-100 soluble extracts compared either with parental WM278 cells, WM278 cells overexpressing GFP (C2) or low levels of GFP-Nck2 (N7) (Figure 9). Collectively, these results reveal that increased expression of Nck2 in human primary melanoma cells promotes phosphorylation of proteins on tyrosine, concomitant with the assembly of Nck2-dependent pY-proteins containing molecular complexes and downregulation of cell surface adhesion proteins.

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