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Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis.

No MeSH data available.


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T286D phosphomimic mutation of CaMKII enhances anchorage independent growth.(A) MDA-MB-231 and (B) MCF-7 cells expressing empty vector (EV), wild-type (WT), T286D, or T286V CaMKII were grown for 14 (MDA-MB-231) or 24 (MCF-7) days in soft agar. After this time, colonies were stained with 0.5% crystal violet/PBS overnight, and then colonies >50 μm were counted using an inverted microscope. Photomicrographs are representative of three independent experiments, performed in triplicate. Results are presented as the number of colonies. *denotes statistical significant difference p < 0.05, **p < 0.01, as determined by one-way ANOVA.
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f6: T286D phosphomimic mutation of CaMKII enhances anchorage independent growth.(A) MDA-MB-231 and (B) MCF-7 cells expressing empty vector (EV), wild-type (WT), T286D, or T286V CaMKII were grown for 14 (MDA-MB-231) or 24 (MCF-7) days in soft agar. After this time, colonies were stained with 0.5% crystal violet/PBS overnight, and then colonies >50 μm were counted using an inverted microscope. Photomicrographs are representative of three independent experiments, performed in triplicate. Results are presented as the number of colonies. *denotes statistical significant difference p < 0.05, **p < 0.01, as determined by one-way ANOVA.

Mentions: The ability of cancer cells to grow in the absence of adhesion to extracellular matrix (ECM) is closely correlated with tumourigenicity in animal models28. WT-CaMKII overexpression significantly increased the ability of both breast cancer cell lines to grow in the absence of ECM (Fig. 6A,B; p < 0.01 for both). Furthermore, overexpression of the T286D phosphomimic form of CaMKII further significantly enhanced the ability of both the invasive MDA-MB-231 (Fig. 6A), and the non-invasive MCF-7 (Fig. 6B) breast cancer cells to grow in a semi-solid medium, when compared to the WT and T286V phospho forms of CaMKII. This indicates that phosphorylation of CaMKII at T286 enhances the tumourigenicity of breast cancer cells in vitro.


Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells
T286D phosphomimic mutation of CaMKII enhances anchorage independent growth.(A) MDA-MB-231 and (B) MCF-7 cells expressing empty vector (EV), wild-type (WT), T286D, or T286V CaMKII were grown for 14 (MDA-MB-231) or 24 (MCF-7) days in soft agar. After this time, colonies were stained with 0.5% crystal violet/PBS overnight, and then colonies >50 μm were counted using an inverted microscope. Photomicrographs are representative of three independent experiments, performed in triplicate. Results are presented as the number of colonies. *denotes statistical significant difference p < 0.05, **p < 0.01, as determined by one-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015093&req=5

f6: T286D phosphomimic mutation of CaMKII enhances anchorage independent growth.(A) MDA-MB-231 and (B) MCF-7 cells expressing empty vector (EV), wild-type (WT), T286D, or T286V CaMKII were grown for 14 (MDA-MB-231) or 24 (MCF-7) days in soft agar. After this time, colonies were stained with 0.5% crystal violet/PBS overnight, and then colonies >50 μm were counted using an inverted microscope. Photomicrographs are representative of three independent experiments, performed in triplicate. Results are presented as the number of colonies. *denotes statistical significant difference p < 0.05, **p < 0.01, as determined by one-way ANOVA.
Mentions: The ability of cancer cells to grow in the absence of adhesion to extracellular matrix (ECM) is closely correlated with tumourigenicity in animal models28. WT-CaMKII overexpression significantly increased the ability of both breast cancer cell lines to grow in the absence of ECM (Fig. 6A,B; p < 0.01 for both). Furthermore, overexpression of the T286D phosphomimic form of CaMKII further significantly enhanced the ability of both the invasive MDA-MB-231 (Fig. 6A), and the non-invasive MCF-7 (Fig. 6B) breast cancer cells to grow in a semi-solid medium, when compared to the WT and T286V phospho forms of CaMKII. This indicates that phosphorylation of CaMKII at T286 enhances the tumourigenicity of breast cancer cells in vitro.

View Article: PubMed Central - PubMed

ABSTRACT

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis.

No MeSH data available.


Related in: MedlinePlus