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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 derived spheroid in type 1 collagen and invasion. (A) Spheroids of human primary WM278 melanoma cells overexpressing GFP (C2) or GFP-Nck2 (N14) and human metastatic WM1617 melanoma cells embedded in 3D type I collagen gel were analyzed for morphology and invading cells. Pictures (10X) were taken at time 0 and after 48 hours of culture. White bars = 200 μm or 50 μm for zoomed pictures. (B) Invasion of human primary WM278 melanoma cells overexpressing GFP (C2) or GFP-Nck2 (N14) was evaluated using Matrigel™ transwell assays. Results are expressed as the mean of invaded cells ± SD from one experiment performed in triplicate. * p < 0.01 vs C2 using Student's t-test. Similar results were observed in three independent experiments.
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Figure 6: Effect of Nck2 on human primary melanoma cell derived spheroid in type 1 collagen and invasion. (A) Spheroids of human primary WM278 melanoma cells overexpressing GFP (C2) or GFP-Nck2 (N14) and human metastatic WM1617 melanoma cells embedded in 3D type I collagen gel were analyzed for morphology and invading cells. Pictures (10X) were taken at time 0 and after 48 hours of culture. White bars = 200 μm or 50 μm for zoomed pictures. (B) Invasion of human primary WM278 melanoma cells overexpressing GFP (C2) or GFP-Nck2 (N14) was evaluated using Matrigel™ transwell assays. Results are expressed as the mean of invaded cells ± SD from one experiment performed in triplicate. * p < 0.01 vs C2 using Student's t-test. Similar results were observed in three independent experiments.

Mentions: To determine whether overexpression of Nck2 in primary melanoma cells promotes invasion in a tumor-like context, we evaluated migration of melanoma cells at the edge of multicellular spheroids embedded into a collagen type I matrix (Figure 6A). As expected, spheroids of human primary melanoma cells overexpressing or not GFP (WM278, C2) grew as compact units devoid of cells migrating outward after 48 h in culture. In contrast, WM1617 human metastatic melanoma cells after 48 h in culture formed open-wave fragile spheroids with cells in periphery sending long projections invading the surrounding collagen. These observations are in agreement with the established invasive phenotype of WM1617 melanoma cells. Interestingly, WM278 human primary melanoma cells overexpressing GFP-Nck2 (N14) apparently did not show similar extensions from spheroid border cells, but form less compact spheroids than WM278-GFP with individual cells that detached form the mother spheroid after 48 h of culture into collagen gel. This observation reveals that overexpression of Nck2 in primary melanoma cells may contribute to invasiveness by promoting cell detachment and migration from primary melanoma lesion in vivo. In agreement, we found that Nck2 overexpression significantly promoted primary melanoma cells invasion through Matrigel™ matrix in transwells assays (Figure 6B). Altogether, these results suggest that Nck2 promotes cell migration and invasion 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 primary melanoma cell derived spheroid in type 1 collagen and invasion. (A) Spheroids of human primary WM278 melanoma cells overexpressing GFP (C2) or GFP-Nck2 (N14) and human metastatic WM1617 melanoma cells embedded in 3D type I collagen gel were analyzed for morphology and invading cells. Pictures (10X) were taken at time 0 and after 48 hours of culture. White bars = 200 μm or 50 μm for zoomed pictures. (B) Invasion of human primary WM278 melanoma cells overexpressing GFP (C2) or GFP-Nck2 (N14) was evaluated using Matrigel™ transwell assays. Results are expressed as the mean of invaded cells ± SD from one experiment performed in triplicate. * p < 0.01 vs C2 using Student's t-test. Similar results were observed in three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Effect of Nck2 on human primary melanoma cell derived spheroid in type 1 collagen and invasion. (A) Spheroids of human primary WM278 melanoma cells overexpressing GFP (C2) or GFP-Nck2 (N14) and human metastatic WM1617 melanoma cells embedded in 3D type I collagen gel were analyzed for morphology and invading cells. Pictures (10X) were taken at time 0 and after 48 hours of culture. White bars = 200 μm or 50 μm for zoomed pictures. (B) Invasion of human primary WM278 melanoma cells overexpressing GFP (C2) or GFP-Nck2 (N14) was evaluated using Matrigel™ transwell assays. Results are expressed as the mean of invaded cells ± SD from one experiment performed in triplicate. * p < 0.01 vs C2 using Student's t-test. Similar results were observed in three independent experiments.
Mentions: To determine whether overexpression of Nck2 in primary melanoma cells promotes invasion in a tumor-like context, we evaluated migration of melanoma cells at the edge of multicellular spheroids embedded into a collagen type I matrix (Figure 6A). As expected, spheroids of human primary melanoma cells overexpressing or not GFP (WM278, C2) grew as compact units devoid of cells migrating outward after 48 h in culture. In contrast, WM1617 human metastatic melanoma cells after 48 h in culture formed open-wave fragile spheroids with cells in periphery sending long projections invading the surrounding collagen. These observations are in agreement with the established invasive phenotype of WM1617 melanoma cells. Interestingly, WM278 human primary melanoma cells overexpressing GFP-Nck2 (N14) apparently did not show similar extensions from spheroid border cells, but form less compact spheroids than WM278-GFP with individual cells that detached form the mother spheroid after 48 h of culture into collagen gel. This observation reveals that overexpression of Nck2 in primary melanoma cells may contribute to invasiveness by promoting cell detachment and migration from primary melanoma lesion in vivo. In agreement, we found that Nck2 overexpression significantly promoted primary melanoma cells invasion through Matrigel™ matrix in transwells assays (Figure 6B). Altogether, these results suggest that Nck2 promotes cell migration and invasion 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