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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 metastatic melanoma cell proliferation. (A) Total cell lysates (30 μg protein) prepared from 451Lu human melanoma cells transfected with control or specific Nck2 siRNA were subjected to western blot analysis using specific antibodies that recognize Nck1 or Nck2 (left panel). RT-PCR was performed to determine Nck1 and Nck2 mRNA levels using total RNA and specific primers for human Nck1 and Nck2. 18S was used as control (right panel). (B) 451Lu human melanoma cells transfected with control or Nck2 siRNA were followed for proliferation 1 and 2 days after siRNA transfection. Results represent cell density corresponding to the mean of Crystal Violet incorporation ± SD from one experiment performed in triplicate. * p < 0.05 vs control siRNA using Student's t-test. Similar results were found in three independent experiments.
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Figure 4: Effect of Nck2 on human metastatic melanoma cell proliferation. (A) Total cell lysates (30 μg protein) prepared from 451Lu human melanoma cells transfected with control or specific Nck2 siRNA were subjected to western blot analysis using specific antibodies that recognize Nck1 or Nck2 (left panel). RT-PCR was performed to determine Nck1 and Nck2 mRNA levels using total RNA and specific primers for human Nck1 and Nck2. 18S was used as control (right panel). (B) 451Lu human melanoma cells transfected with control or Nck2 siRNA were followed for proliferation 1 and 2 days after siRNA transfection. Results represent cell density corresponding to the mean of Crystal Violet incorporation ± SD from one experiment performed in triplicate. * p < 0.05 vs control siRNA using Student's t-test. Similar results were found in three independent experiments.

Mentions: To confirm a role for Nck2 in melanoma cell proliferation, we assessed whether siRNA-mediated downregulation of Nck2 in human metastatic melanoma cells affects cell proliferation. As shown in Figure 4A, Nck2 siRNA transfection of 451Lu metastatic melanoma cells resulted in decreased Nck2 protein and mRNA levels, while Nck1 protein and mRNA levels were not altered. More importantly, we found that cell proliferation was significantly decreased in Nck2 depleted metastatic melanoma cells compared with control siRNA treated cells (Figure 4B). To rule out that this effect was due to increased cell death in Nck2 depleted melanoma cells, 2 days post transfection we evaluated the percentage of cells with nuclei stained by Trypan Blue and observed no difference between metastatic melanoma cells transfected with control (8.7% ± 2.5) and Nck2 (7.7% ± 1.7) siRNA. Altogether, these results indicate that Nck2 contributes to the control of proliferation in human melanoma cells.


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 metastatic melanoma cell proliferation. (A) Total cell lysates (30 μg protein) prepared from 451Lu human melanoma cells transfected with control or specific Nck2 siRNA were subjected to western blot analysis using specific antibodies that recognize Nck1 or Nck2 (left panel). RT-PCR was performed to determine Nck1 and Nck2 mRNA levels using total RNA and specific primers for human Nck1 and Nck2. 18S was used as control (right panel). (B) 451Lu human melanoma cells transfected with control or Nck2 siRNA were followed for proliferation 1 and 2 days after siRNA transfection. Results represent cell density corresponding to the mean of Crystal Violet incorporation ± SD from one experiment performed in triplicate. * p < 0.05 vs control siRNA using Student's t-test. Similar results were found in three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effect of Nck2 on human metastatic melanoma cell proliferation. (A) Total cell lysates (30 μg protein) prepared from 451Lu human melanoma cells transfected with control or specific Nck2 siRNA were subjected to western blot analysis using specific antibodies that recognize Nck1 or Nck2 (left panel). RT-PCR was performed to determine Nck1 and Nck2 mRNA levels using total RNA and specific primers for human Nck1 and Nck2. 18S was used as control (right panel). (B) 451Lu human melanoma cells transfected with control or Nck2 siRNA were followed for proliferation 1 and 2 days after siRNA transfection. Results represent cell density corresponding to the mean of Crystal Violet incorporation ± SD from one experiment performed in triplicate. * p < 0.05 vs control siRNA using Student's t-test. Similar results were found in three independent experiments.
Mentions: To confirm a role for Nck2 in melanoma cell proliferation, we assessed whether siRNA-mediated downregulation of Nck2 in human metastatic melanoma cells affects cell proliferation. As shown in Figure 4A, Nck2 siRNA transfection of 451Lu metastatic melanoma cells resulted in decreased Nck2 protein and mRNA levels, while Nck1 protein and mRNA levels were not altered. More importantly, we found that cell proliferation was significantly decreased in Nck2 depleted metastatic melanoma cells compared with control siRNA treated cells (Figure 4B). To rule out that this effect was due to increased cell death in Nck2 depleted melanoma cells, 2 days post transfection we evaluated the percentage of cells with nuclei stained by Trypan Blue and observed no difference between metastatic melanoma cells transfected with control (8.7% ± 2.5) and Nck2 (7.7% ± 1.7) siRNA. Altogether, these results indicate that Nck2 contributes to the control of proliferation in human melanoma cells.

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