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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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Nck expression in human melanoma cell lines at different stages of cancer progression. Equivalent amount of total proteins from human primary and metastatic melanoma cell lysates (30 μg) were subjected to western blot analysis using indicated antibodies. (A) Pan-Nck antibodies recognize Nck1 and Nck2. eIF2α was used as loading control. (B) Nck1 and Nck2 are specific Nck isotypes antibodies (Additional file 1) and β-actin or tubulin was detected as loading controls. Quantification of Nck1 and Nck2 signals, evaluated in the linear range of detection by densitometry, are reported below the blots. Human Melanoma Cell Lines Characteristics: FW2294: normal melanocyte; WM115: poorly metastatic primary melanoma in vertical growth phase and with round morphology; WM164: elongated metastatic melanoma isolated from lymph nodes; WM278: poorly metastatic primary melanoma in vertical growth phase and with mixed morphology; WM1617: elongated metastatic melanoma derived from lymph nodes, sister match of WM278; 451Lu: elongated metastatic melanoma selected in lungs of mice injected with WM164 cells; 1205Lu: round metastatic melanoma selected in mice; WM1232: round metastatic melanoma.
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Figure 1: Nck expression in human melanoma cell lines at different stages of cancer progression. Equivalent amount of total proteins from human primary and metastatic melanoma cell lysates (30 μg) were subjected to western blot analysis using indicated antibodies. (A) Pan-Nck antibodies recognize Nck1 and Nck2. eIF2α was used as loading control. (B) Nck1 and Nck2 are specific Nck isotypes antibodies (Additional file 1) and β-actin or tubulin was detected as loading controls. Quantification of Nck1 and Nck2 signals, evaluated in the linear range of detection by densitometry, are reported below the blots. Human Melanoma Cell Lines Characteristics: FW2294: normal melanocyte; WM115: poorly metastatic primary melanoma in vertical growth phase and with round morphology; WM164: elongated metastatic melanoma isolated from lymph nodes; WM278: poorly metastatic primary melanoma in vertical growth phase and with mixed morphology; WM1617: elongated metastatic melanoma derived from lymph nodes, sister match of WM278; 451Lu: elongated metastatic melanoma selected in lungs of mice injected with WM164 cells; 1205Lu: round metastatic melanoma selected in mice; WM1232: round metastatic melanoma.

Mentions: From western blots performed using a rabbit polyclonal antibody that equally recognizes both Nck isotypes (Pan-Nck, Additional file 1) [21], we observed higher levels of Nck proteins in highly metastatic melanoma (WM164, 451Lu, 1205Lu and WM1232) compared with weakly metastatic primary melanoma (WM115 and WM278) and normal melanocyte (FW2294) cell lines (Figure 1A). Further analyses using in-house generated Nck isoforms specific antibodies (Additional file 1) [15] revealed that increased expression of Nck in metastatic melanoma cells is mainly due to drastic higher expression levels of Nck2 (Figure 1B, compare WM164 and 451Lu with WM278). Additional comparison of highly metastatic (WM1617) and weakly metastatic (WM278) human melanoma cell lines isolated from the same patient (Figure 1B), further confirmed increased expression of Nck2 in human metastatic melanoma. Interestingly, Nck1 protein levels normalized according to actin or tubulin loading control were comparable among the human melanoma cell lines investigated (Figure 1B and Additional file 2). In addition, we also found no change in expression levels of other SH2/SH3 domain-containing signaling proteins, such as PLC-γ1, p85 of PI3K, Grb2 and Crk, as normalized for actin or tubulin loading control (Additional file 2 and data not shown). Altogether, these results suggest a specific role for Nck2 in human 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)

Nck expression in human melanoma cell lines at different stages of cancer progression. Equivalent amount of total proteins from human primary and metastatic melanoma cell lysates (30 μg) were subjected to western blot analysis using indicated antibodies. (A) Pan-Nck antibodies recognize Nck1 and Nck2. eIF2α was used as loading control. (B) Nck1 and Nck2 are specific Nck isotypes antibodies (Additional file 1) and β-actin or tubulin was detected as loading controls. Quantification of Nck1 and Nck2 signals, evaluated in the linear range of detection by densitometry, are reported below the blots. Human Melanoma Cell Lines Characteristics: FW2294: normal melanocyte; WM115: poorly metastatic primary melanoma in vertical growth phase and with round morphology; WM164: elongated metastatic melanoma isolated from lymph nodes; WM278: poorly metastatic primary melanoma in vertical growth phase and with mixed morphology; WM1617: elongated metastatic melanoma derived from lymph nodes, sister match of WM278; 451Lu: elongated metastatic melanoma selected in lungs of mice injected with WM164 cells; 1205Lu: round metastatic melanoma selected in mice; WM1232: round metastatic melanoma.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198724&req=5

Figure 1: Nck expression in human melanoma cell lines at different stages of cancer progression. Equivalent amount of total proteins from human primary and metastatic melanoma cell lysates (30 μg) were subjected to western blot analysis using indicated antibodies. (A) Pan-Nck antibodies recognize Nck1 and Nck2. eIF2α was used as loading control. (B) Nck1 and Nck2 are specific Nck isotypes antibodies (Additional file 1) and β-actin or tubulin was detected as loading controls. Quantification of Nck1 and Nck2 signals, evaluated in the linear range of detection by densitometry, are reported below the blots. Human Melanoma Cell Lines Characteristics: FW2294: normal melanocyte; WM115: poorly metastatic primary melanoma in vertical growth phase and with round morphology; WM164: elongated metastatic melanoma isolated from lymph nodes; WM278: poorly metastatic primary melanoma in vertical growth phase and with mixed morphology; WM1617: elongated metastatic melanoma derived from lymph nodes, sister match of WM278; 451Lu: elongated metastatic melanoma selected in lungs of mice injected with WM164 cells; 1205Lu: round metastatic melanoma selected in mice; WM1232: round metastatic melanoma.
Mentions: From western blots performed using a rabbit polyclonal antibody that equally recognizes both Nck isotypes (Pan-Nck, Additional file 1) [21], we observed higher levels of Nck proteins in highly metastatic melanoma (WM164, 451Lu, 1205Lu and WM1232) compared with weakly metastatic primary melanoma (WM115 and WM278) and normal melanocyte (FW2294) cell lines (Figure 1A). Further analyses using in-house generated Nck isoforms specific antibodies (Additional file 1) [15] revealed that increased expression of Nck in metastatic melanoma cells is mainly due to drastic higher expression levels of Nck2 (Figure 1B, compare WM164 and 451Lu with WM278). Additional comparison of highly metastatic (WM1617) and weakly metastatic (WM278) human melanoma cell lines isolated from the same patient (Figure 1B), further confirmed increased expression of Nck2 in human metastatic melanoma. Interestingly, Nck1 protein levels normalized according to actin or tubulin loading control were comparable among the human melanoma cell lines investigated (Figure 1B and Additional file 2). In addition, we also found no change in expression levels of other SH2/SH3 domain-containing signaling proteins, such as PLC-γ1, p85 of PI3K, Grb2 and Crk, as normalized for actin or tubulin loading control (Additional file 2 and data not shown). Altogether, these results suggest a specific role for Nck2 in human 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