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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 actin organization and focal adhesions in human primary melanoma cells. (A) WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were submitted to actin staining using phalloidin-coupled to AlexaFluor®555. Pictures were taken at 40X and white bar represent 20 μm. (B) Fluorescence pictures of WM278 human primary melanoma cells stably overexpressing GFP (C2) or GFP-Nck2 (N14) subjected to vinculin staining using anti-vinculin specific antibody and GAM-TRICT (63X, white bar: 20 μm). (C) Manual quantification of focal adhesions as positive vinculin structures in 50 representative cell types. Results normalized for cell size are expressed as the mean of focal adhesion per mm2 or cell ± SD. * p < 0.001 vs C2 using Student's t-test.
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Figure 7: Effect of Nck2 on actin organization and focal adhesions in human primary melanoma cells. (A) WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were submitted to actin staining using phalloidin-coupled to AlexaFluor®555. Pictures were taken at 40X and white bar represent 20 μm. (B) Fluorescence pictures of WM278 human primary melanoma cells stably overexpressing GFP (C2) or GFP-Nck2 (N14) subjected to vinculin staining using anti-vinculin specific antibody and GAM-TRICT (63X, white bar: 20 μm). (C) Manual quantification of focal adhesions as positive vinculin structures in 50 representative cell types. Results normalized for cell size are expressed as the mean of focal adhesion per mm2 or cell ± SD. * p < 0.001 vs C2 using Student's t-test.

Mentions: Because Nck adaptor proteins play an important role in regulating actin cytoskeleton reorganization, we then compared actin staining in WM278 human primary melanoma cells expressing either GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) (Figure 7A). Regardless of Nck2 expression levels, we found no apparent change in actin staining in these cells. This suggests that overexpression of Nck2 has no major effect on actin polymerization and organization, as well as on overall cell morphology in human primary melanoma. In contrast, vinculin staining, which shows that GFP-Nck2 colocalizes with vinculin at focal adhesions (Figure 7B), revealed significant reduced number of focal adhesions in human primary melanoma cells overexpressing Nck2 (N14) compared with control melanoma cells (C2) (Figure 7C). Therefore, these data suggest that increased expression of Nck2 in human primary melanoma cells might facilitate melanoma migration by decreasing focal adhesions.


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 actin organization and focal adhesions in human primary melanoma cells. (A) WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were submitted to actin staining using phalloidin-coupled to AlexaFluor®555. Pictures were taken at 40X and white bar represent 20 μm. (B) Fluorescence pictures of WM278 human primary melanoma cells stably overexpressing GFP (C2) or GFP-Nck2 (N14) subjected to vinculin staining using anti-vinculin specific antibody and GAM-TRICT (63X, white bar: 20 μm). (C) Manual quantification of focal adhesions as positive vinculin structures in 50 representative cell types. Results normalized for cell size are expressed as the mean of focal adhesion per mm2 or cell ± SD. * p < 0.001 vs C2 using Student's t-test.
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Figure 7: Effect of Nck2 on actin organization and focal adhesions in human primary melanoma cells. (A) WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were submitted to actin staining using phalloidin-coupled to AlexaFluor®555. Pictures were taken at 40X and white bar represent 20 μm. (B) Fluorescence pictures of WM278 human primary melanoma cells stably overexpressing GFP (C2) or GFP-Nck2 (N14) subjected to vinculin staining using anti-vinculin specific antibody and GAM-TRICT (63X, white bar: 20 μm). (C) Manual quantification of focal adhesions as positive vinculin structures in 50 representative cell types. Results normalized for cell size are expressed as the mean of focal adhesion per mm2 or cell ± SD. * p < 0.001 vs C2 using Student's t-test.
Mentions: Because Nck adaptor proteins play an important role in regulating actin cytoskeleton reorganization, we then compared actin staining in WM278 human primary melanoma cells expressing either GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) (Figure 7A). Regardless of Nck2 expression levels, we found no apparent change in actin staining in these cells. This suggests that overexpression of Nck2 has no major effect on actin polymerization and organization, as well as on overall cell morphology in human primary melanoma. In contrast, vinculin staining, which shows that GFP-Nck2 colocalizes with vinculin at focal adhesions (Figure 7B), revealed significant reduced number of focal adhesions in human primary melanoma cells overexpressing Nck2 (N14) compared with control melanoma cells (C2) (Figure 7C). Therefore, these data suggest that increased expression of Nck2 in human primary melanoma cells might facilitate melanoma migration by decreasing focal adhesions.

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