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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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Expression of Nck1 and Nck2 in colon and breast cancer cells. Lysates (50 μg protein) from murine colonic carcinoma cells (A) and human breast cancer cells (B) were subjected to western blot analysis using anti-Nck specific antibodies (Additional file 1). CT36: rarely metastatic; CT51: intermediary; CT26: highly metastatic. MCF-10A: Normal human breast epithelial; MCF7, T47D, MDA-MB-231: invasive metastatic ductal carcinoma. For both cancer cell types, β-actin was probed as loading control.
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Figure 11: Expression of Nck1 and Nck2 in colon and breast cancer cells. Lysates (50 μg protein) from murine colonic carcinoma cells (A) and human breast cancer cells (B) were subjected to western blot analysis using anti-Nck specific antibodies (Additional file 1). CT36: rarely metastatic; CT51: intermediary; CT26: highly metastatic. MCF-10A: Normal human breast epithelial; MCF7, T47D, MDA-MB-231: invasive metastatic ductal carcinoma. For both cancer cell types, β-actin was probed as loading control.

Mentions: To find out whether increased expression of Nck2 during cancer progression is observed in other cancer types than in melanoma skin cancer, we assessed Nck isoforms protein levels in murine colon (CT) and human breast cancer cell lines at different stages of progression (Figure 11). The CT represent mouse tumorigenic colonic carcinoma cell lines established in culture from three transplantable murine tumors of colonic origin at different stages of progression [37]. Based on growth rate and metastatic spreading, CT26 is the most aggressive, while CT51 is intermediary and CT36 is the least and rarely metastasizes. Using these cells lines, we observed that Nck1 protein levels are increased in the metastatic CT26 and CT51, while just barely detected in CT36. However, when Nck1 expression levels were normalized according to actin, these variations were not statistically significant. In contrast, Nck2 that was below detection level in CT36 and CT51, was distinctly detected in CT26, revealing increased expression of Nck2 in aggressive metastatic colon cancer cells. To determine whether expression of Nck isoforms vary during breast cancer progression, we selected few of the widely used human breast cancer cell lines (MCF-7, T-47D and MDA-MB-231) and an immortalized normal human breast epithelial cell line (MCF10A). MCF-7, T-47D and MDA-MB-231 are invasive ductal carcinoma of similar origin with metastatic properties [38]. Independently of estrogen receptor expression (ER+: MCF-7 and T-47D; ER-: MDA-MB-231) or epithelial/mesenchymal phenotype (epithelial-like low invasion: MCF-7 and T-47D; mesenchymal-like high invasion: MDA-MB-231), Nck1 protein levels in breast cancer cells were not consistent with the cancer stage. Considering actin as loading control, Nck1 protein levels were significantly increased in T-47D cells, decreased in MDA-MB-231 cells and not change in MCF7 cells compared to MCF-10A cells, excluding a potential correlation between Nck1 expression levels and breast cancer progression. In contrast, Nck2 was only detected in the most aggressive MDA-MB-231 cells, which are mesenchymal-like ER+ breast cancer cells with strong migratory and metastatic abilities [38,39]. Together these results provide the first evidence that the expression of Nck2 is increased in metastatic cancer cells of various origins and argue for a role of Nck2 in cancer 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)

Expression of Nck1 and Nck2 in colon and breast cancer cells. Lysates (50 μg protein) from murine colonic carcinoma cells (A) and human breast cancer cells (B) were subjected to western blot analysis using anti-Nck specific antibodies (Additional file 1). CT36: rarely metastatic; CT51: intermediary; CT26: highly metastatic. MCF-10A: Normal human breast epithelial; MCF7, T47D, MDA-MB-231: invasive metastatic ductal carcinoma. For both cancer cell types, β-actin was probed as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: Expression of Nck1 and Nck2 in colon and breast cancer cells. Lysates (50 μg protein) from murine colonic carcinoma cells (A) and human breast cancer cells (B) were subjected to western blot analysis using anti-Nck specific antibodies (Additional file 1). CT36: rarely metastatic; CT51: intermediary; CT26: highly metastatic. MCF-10A: Normal human breast epithelial; MCF7, T47D, MDA-MB-231: invasive metastatic ductal carcinoma. For both cancer cell types, β-actin was probed as loading control.
Mentions: To find out whether increased expression of Nck2 during cancer progression is observed in other cancer types than in melanoma skin cancer, we assessed Nck isoforms protein levels in murine colon (CT) and human breast cancer cell lines at different stages of progression (Figure 11). The CT represent mouse tumorigenic colonic carcinoma cell lines established in culture from three transplantable murine tumors of colonic origin at different stages of progression [37]. Based on growth rate and metastatic spreading, CT26 is the most aggressive, while CT51 is intermediary and CT36 is the least and rarely metastasizes. Using these cells lines, we observed that Nck1 protein levels are increased in the metastatic CT26 and CT51, while just barely detected in CT36. However, when Nck1 expression levels were normalized according to actin, these variations were not statistically significant. In contrast, Nck2 that was below detection level in CT36 and CT51, was distinctly detected in CT26, revealing increased expression of Nck2 in aggressive metastatic colon cancer cells. To determine whether expression of Nck isoforms vary during breast cancer progression, we selected few of the widely used human breast cancer cell lines (MCF-7, T-47D and MDA-MB-231) and an immortalized normal human breast epithelial cell line (MCF10A). MCF-7, T-47D and MDA-MB-231 are invasive ductal carcinoma of similar origin with metastatic properties [38]. Independently of estrogen receptor expression (ER+: MCF-7 and T-47D; ER-: MDA-MB-231) or epithelial/mesenchymal phenotype (epithelial-like low invasion: MCF-7 and T-47D; mesenchymal-like high invasion: MDA-MB-231), Nck1 protein levels in breast cancer cells were not consistent with the cancer stage. Considering actin as loading control, Nck1 protein levels were significantly increased in T-47D cells, decreased in MDA-MB-231 cells and not change in MCF7 cells compared to MCF-10A cells, excluding a potential correlation between Nck1 expression levels and breast cancer progression. In contrast, Nck2 was only detected in the most aggressive MDA-MB-231 cells, which are mesenchymal-like ER+ breast cancer cells with strong migratory and metastatic abilities [38,39]. Together these results provide the first evidence that the expression of Nck2 is increased in metastatic cancer cells of various origins and argue for a role of Nck2 in cancer 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