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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 proliferation. (A) Total cell lysates from stable WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were subjected to western blot analysis using GFP or Nck2 specific antibodies. Cell proliferation of (B) WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7 and N14), as well as (C) parental primary (WM278) and metastatic (WM1617) melanoma cells was determined by quantification of Crystal Violet incorporation each day during 4 days in culture. Results are shown as the ratio of the mean of cell density over day 1 ± SD (n = 4). * p < 0.02, ** p < 0.006 and ***p ≤ 0.0001compared with C2 (B) or parental WM278 cells (C) using Student's t-test. (D) Nck1 and CrkII expression was evaluated using total cell lysates (30 μg protein) prepared from WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7, and N14). β-tubulin was used as loading control.
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Figure 3: Effect of Nck2 on human primary melanoma cell proliferation. (A) Total cell lysates from stable WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were subjected to western blot analysis using GFP or Nck2 specific antibodies. Cell proliferation of (B) WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7 and N14), as well as (C) parental primary (WM278) and metastatic (WM1617) melanoma cells was determined by quantification of Crystal Violet incorporation each day during 4 days in culture. Results are shown as the ratio of the mean of cell density over day 1 ± SD (n = 4). * p < 0.02, ** p < 0.006 and ***p ≤ 0.0001compared with C2 (B) or parental WM278 cells (C) using Student's t-test. (D) Nck1 and CrkII expression was evaluated using total cell lysates (30 μg protein) prepared from WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7, and N14). β-tubulin was used as loading control.

Mentions: To demonstrate a role for Nck2 in melanoma progression, we first determined whether Nck2 regulates cell proliferation in WM278 human primary melanoma cells, which endogenously express low levels of Nck2 protein and rarely metastasis compared with its metastatic counterpart WM1617 melanoma cells. For this, we created WM278 cell lines stably overexpressing increasing levels of GFP-Nck2 (N15 < N7 < N14) or GFP as control (C2) (Figure 3A). Using these WM278 stable cell lines, we found that overexpressing high levels of Nck2 significantly enhanced cell proliferation (Figure 3B, left panel, compare clone N14 with C2). It is interesting to note though that the effect of Nck2 on WM278 primary melanoma cells proliferation seems to parallel the levels of Nck2 overexpressed (Figure 3A and 3B, compared N15, N7 and N14). In agreement, the WM1617 human metastatic melanoma cells that endogenously express higher levels of Nck2 compared with human primary melanoma cells, also show higher proliferative abilities than its paired WM278 primary melanoma cells that rarely metastasis (Figure 3C). As expected, we found no change in the protein levels of Nck1 or CrkII, a SH2-SH3 domain-containing adaptor protein previously identify as an oncogene [25] and recently reported to regulate sarcoma cell proliferation [26] (Figure 3C).


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 proliferation. (A) Total cell lysates from stable WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were subjected to western blot analysis using GFP or Nck2 specific antibodies. Cell proliferation of (B) WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7 and N14), as well as (C) parental primary (WM278) and metastatic (WM1617) melanoma cells was determined by quantification of Crystal Violet incorporation each day during 4 days in culture. Results are shown as the ratio of the mean of cell density over day 1 ± SD (n = 4). * p < 0.02, ** p < 0.006 and ***p ≤ 0.0001compared with C2 (B) or parental WM278 cells (C) using Student's t-test. (D) Nck1 and CrkII expression was evaluated using total cell lysates (30 μg protein) prepared from WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7, and N14). β-tubulin was used as loading control.
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Figure 3: Effect of Nck2 on human primary melanoma cell proliferation. (A) Total cell lysates from stable WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were subjected to western blot analysis using GFP or Nck2 specific antibodies. Cell proliferation of (B) WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7 and N14), as well as (C) parental primary (WM278) and metastatic (WM1617) melanoma cells was determined by quantification of Crystal Violet incorporation each day during 4 days in culture. Results are shown as the ratio of the mean of cell density over day 1 ± SD (n = 4). * p < 0.02, ** p < 0.006 and ***p ≤ 0.0001compared with C2 (B) or parental WM278 cells (C) using Student's t-test. (D) Nck1 and CrkII expression was evaluated using total cell lysates (30 μg protein) prepared from WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7, and N14). β-tubulin was used as loading control.
Mentions: To demonstrate a role for Nck2 in melanoma progression, we first determined whether Nck2 regulates cell proliferation in WM278 human primary melanoma cells, which endogenously express low levels of Nck2 protein and rarely metastasis compared with its metastatic counterpart WM1617 melanoma cells. For this, we created WM278 cell lines stably overexpressing increasing levels of GFP-Nck2 (N15 < N7 < N14) or GFP as control (C2) (Figure 3A). Using these WM278 stable cell lines, we found that overexpressing high levels of Nck2 significantly enhanced cell proliferation (Figure 3B, left panel, compare clone N14 with C2). It is interesting to note though that the effect of Nck2 on WM278 primary melanoma cells proliferation seems to parallel the levels of Nck2 overexpressed (Figure 3A and 3B, compared N15, N7 and N14). In agreement, the WM1617 human metastatic melanoma cells that endogenously express higher levels of Nck2 compared with human primary melanoma cells, also show higher proliferative abilities than its paired WM278 primary melanoma cells that rarely metastasis (Figure 3C). As expected, we found no change in the protein levels of Nck1 or CrkII, a SH2-SH3 domain-containing adaptor protein previously identify as an oncogene [25] and recently reported to regulate sarcoma cell proliferation [26] (Figure 3C).

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