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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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Nck1 and Nck2 mRNA expression in human melanocytes and melanoma cells. (A) RT-PCR amplification performed on isolated RNA from human melanocyte and melanoma cells using specific primers for human Nck1, Nck2 and 18S. Shown are PCR products after 20 and 25 cycles of amplification. HEM: human normal melanocytes; WM278: human primary melanoma that rarely metastasis; WM164: human melanoma isolated from metastasis in lymph nodes; 451Lu: lung melanoma metastasis of WM164 injected in mice. 18S is used as control. (B) Ratios of Nck1 and Nck2 mRNA over 18S were calculated from quantification by densitometry of PCR amplification products in the linear range. Asterisk: p ≤ 0.001 compared to HEM, black triangle: p ≤ 0.001 compared to WM278.
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Figure 2: Nck1 and Nck2 mRNA expression in human melanocytes and melanoma cells. (A) RT-PCR amplification performed on isolated RNA from human melanocyte and melanoma cells using specific primers for human Nck1, Nck2 and 18S. Shown are PCR products after 20 and 25 cycles of amplification. HEM: human normal melanocytes; WM278: human primary melanoma that rarely metastasis; WM164: human melanoma isolated from metastasis in lymph nodes; 451Lu: lung melanoma metastasis of WM164 injected in mice. 18S is used as control. (B) Ratios of Nck1 and Nck2 mRNA over 18S were calculated from quantification by densitometry of PCR amplification products in the linear range. Asterisk: p ≤ 0.001 compared to HEM, black triangle: p ≤ 0.001 compared to WM278.

Mentions: To assess whether increased expression of Nck2 protein levels in human metastatic melanoma cells correlates with upregulated transcription of Nck2 gene, we compared Nck1 and Nck2 mRNA levels in three human melanoma cell lines at different stages of progression and in human primary melanocytes (HEM) by performing RT-PCR using Nck isoforms specific primers. In the linear range of PCR amplification, no significant change in Nck1 mRNA levels was detected in all human melanoma cell lines and compared with HEM (Figure 2A). In contrast, we observed a strong increase in Nck2 mRNA levels in all human melanoma cell lines compared with HEM. In addition, compared with primary melanoma cells (WM278), metastatic melanoma cell lines (WM164 and 451LU) showed significant increased Nck2 mRNA levels (Figure 2B). Altogether, our results reveal that Nck2 protein and mRNA levels are significantly increased in different human metastatic melanoma cells compared with human weakly metastatic primary melanoma and melanocyte cells, suggesting Nck2 as a biological marker of human melanoma metastasis that could contribute to melanoma progression.


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)

Nck1 and Nck2 mRNA expression in human melanocytes and melanoma cells. (A) RT-PCR amplification performed on isolated RNA from human melanocyte and melanoma cells using specific primers for human Nck1, Nck2 and 18S. Shown are PCR products after 20 and 25 cycles of amplification. HEM: human normal melanocytes; WM278: human primary melanoma that rarely metastasis; WM164: human melanoma isolated from metastasis in lymph nodes; 451Lu: lung melanoma metastasis of WM164 injected in mice. 18S is used as control. (B) Ratios of Nck1 and Nck2 mRNA over 18S were calculated from quantification by densitometry of PCR amplification products in the linear range. Asterisk: p ≤ 0.001 compared to HEM, black triangle: p ≤ 0.001 compared to WM278.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Nck1 and Nck2 mRNA expression in human melanocytes and melanoma cells. (A) RT-PCR amplification performed on isolated RNA from human melanocyte and melanoma cells using specific primers for human Nck1, Nck2 and 18S. Shown are PCR products after 20 and 25 cycles of amplification. HEM: human normal melanocytes; WM278: human primary melanoma that rarely metastasis; WM164: human melanoma isolated from metastasis in lymph nodes; 451Lu: lung melanoma metastasis of WM164 injected in mice. 18S is used as control. (B) Ratios of Nck1 and Nck2 mRNA over 18S were calculated from quantification by densitometry of PCR amplification products in the linear range. Asterisk: p ≤ 0.001 compared to HEM, black triangle: p ≤ 0.001 compared to WM278.
Mentions: To assess whether increased expression of Nck2 protein levels in human metastatic melanoma cells correlates with upregulated transcription of Nck2 gene, we compared Nck1 and Nck2 mRNA levels in three human melanoma cell lines at different stages of progression and in human primary melanocytes (HEM) by performing RT-PCR using Nck isoforms specific primers. In the linear range of PCR amplification, no significant change in Nck1 mRNA levels was detected in all human melanoma cell lines and compared with HEM (Figure 2A). In contrast, we observed a strong increase in Nck2 mRNA levels in all human melanoma cell lines compared with HEM. In addition, compared with primary melanoma cells (WM278), metastatic melanoma cell lines (WM164 and 451LU) showed significant increased Nck2 mRNA levels (Figure 2B). Altogether, our results reveal that Nck2 protein and mRNA levels are significantly increased in different human metastatic melanoma cells compared with human weakly metastatic primary melanoma and melanocyte cells, suggesting Nck2 as a biological marker of human melanoma metastasis that could contribute to melanoma progression.

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