Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of Nck2 on human primary melanoma-derived tumor growth in vivo. CD-1 Nude mice of 6-week-old were injected subcutaneously with human parental WM278 primary melanoma cells, WM278 cells overexpressing either GFP or GFP-Nck2, or human WM1617 metastatic melanoma cells (5 × 106) into the right flank. (A) The mice were monitored for tumor development. (B). Tumor volume progression assessed with calipers every week in indicated mice. Tumors with volume bigger than 50 mm3 (dashed line) were considered during the course of the study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198724&req=5

Figure 10: Effect of Nck2 on human primary melanoma-derived tumor growth in vivo. CD-1 Nude mice of 6-week-old were injected subcutaneously with human parental WM278 primary melanoma cells, WM278 cells overexpressing either GFP or GFP-Nck2, or human WM1617 metastatic melanoma cells (5 × 106) into the right flank. (A) The mice were monitored for tumor development. (B). Tumor volume progression assessed with calipers every week in indicated mice. Tumors with volume bigger than 50 mm3 (dashed line) were considered during the course of the study.

Mentions: To establish the biological relevance of our findings, we examined whether overexpression of Nck2 in human primary melanoma cells confers some tumorigenic advantage in a xenograft mouse model. To test this, WM278 human primary melanoma cells overexpressing GFP-Nck2 at low (N7) or high (N14) levels, along with parental WM278 cells, WM278 cells overexpressing GFP (C2) and WM1617 human metastatic melanoma cells were injected subcutaneously into CD-1 Nude mice. Two out of 5 mice injected with WM278 human primary melanoma cells overexpressing high levels of GFP-Nck2 (N14) developed tumor at the site of injection between 13-16 weeks post injection (Figure 10A). At the same time, 1 out of 5 mice that received either parental WM278 cells, WM278 melanoma cells overexpressing GFP (C2) or overexpressing low levels of GFP-Nck2 (N7) presented a subcutaneous tumor at the site of injection. In contrast, all mice injected with the metastatic WM1617 cells developed tumors at the site of injection 2-7 weeks post-injection (Figure 10A). As expected, tumors derived from metastatic WM1617 cells grew rapidly and reached maximal volume allowed between 5-11 weeks post-injection. Altogether, these observations suggest that increased expression of Nck2 in human melanoma cells is not sufficient to promote the appearance of subcutaneous tumor derived from melanoma. However, melanoma-derived tumor growth rate in mice injected with WM278 cells overexpressing GFP-Nck2 (N7 and N14) was greatly enhanced compared with tumor found in mice injected with WM278 cells parental or overexpressing GFP (C2) (Figure 10B), suggesting that increased expression level of Nck2 promotes primary melanoma-derived tumor growth rate. Subcutaneous tumors derived from WM278 cells overexpressing GFP-Nck2 (N7 and N14) could not be further monitored than few weeks after their occurrence due to the appearance of important spontaneous tumor necrosis core that required mice to be euthanized. Nevertheless, in line with our in vitro studies (Figure 3B), these results strongly support a role for Nck2 in melanoma-derived tumor growth rate in vivo.


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-derived tumor growth in vivo. CD-1 Nude mice of 6-week-old were injected subcutaneously with human parental WM278 primary melanoma cells, WM278 cells overexpressing either GFP or GFP-Nck2, or human WM1617 metastatic melanoma cells (5 × 106) into the right flank. (A) The mice were monitored for tumor development. (B). Tumor volume progression assessed with calipers every week in indicated mice. Tumors with volume bigger than 50 mm3 (dashed line) were considered during the course of the study.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Effect of Nck2 on human primary melanoma-derived tumor growth in vivo. CD-1 Nude mice of 6-week-old were injected subcutaneously with human parental WM278 primary melanoma cells, WM278 cells overexpressing either GFP or GFP-Nck2, or human WM1617 metastatic melanoma cells (5 × 106) into the right flank. (A) The mice were monitored for tumor development. (B). Tumor volume progression assessed with calipers every week in indicated mice. Tumors with volume bigger than 50 mm3 (dashed line) were considered during the course of the study.
Mentions: To establish the biological relevance of our findings, we examined whether overexpression of Nck2 in human primary melanoma cells confers some tumorigenic advantage in a xenograft mouse model. To test this, WM278 human primary melanoma cells overexpressing GFP-Nck2 at low (N7) or high (N14) levels, along with parental WM278 cells, WM278 cells overexpressing GFP (C2) and WM1617 human metastatic melanoma cells were injected subcutaneously into CD-1 Nude mice. Two out of 5 mice injected with WM278 human primary melanoma cells overexpressing high levels of GFP-Nck2 (N14) developed tumor at the site of injection between 13-16 weeks post injection (Figure 10A). At the same time, 1 out of 5 mice that received either parental WM278 cells, WM278 melanoma cells overexpressing GFP (C2) or overexpressing low levels of GFP-Nck2 (N7) presented a subcutaneous tumor at the site of injection. In contrast, all mice injected with the metastatic WM1617 cells developed tumors at the site of injection 2-7 weeks post-injection (Figure 10A). As expected, tumors derived from metastatic WM1617 cells grew rapidly and reached maximal volume allowed between 5-11 weeks post-injection. Altogether, these observations suggest that increased expression of Nck2 in human melanoma cells is not sufficient to promote the appearance of subcutaneous tumor derived from melanoma. However, melanoma-derived tumor growth rate in mice injected with WM278 cells overexpressing GFP-Nck2 (N7 and N14) was greatly enhanced compared with tumor found in mice injected with WM278 cells parental or overexpressing GFP (C2) (Figure 10B), suggesting that increased expression level of Nck2 promotes primary melanoma-derived tumor growth rate. Subcutaneous tumors derived from WM278 cells overexpressing GFP-Nck2 (N7 and N14) could not be further monitored than few weeks after their occurrence due to the appearance of important spontaneous tumor necrosis core that required mice to be euthanized. Nevertheless, in line with our in vitro studies (Figure 3B), these results strongly support a role for Nck2 in melanoma-derived tumor growth rate in vivo.

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