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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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Pharmacological inhibition of CaMKII activity prevents breast cancer cell migration and invasion.Confluent monolayers of parental MDA-MB-231 cells were treated with 20 μM KN-92 or KN-93, or 10 μM myr-AIP for 30 min, and a wound was made by scratching the monolayer with a pipette tip. (A) Wounds were photographed hourly for 72 h to measure wound closure over time. Photomicrographs are representative of three independent experiments performed in triplicate. Wound widths are expressed as % of 0h wound width. *denotes statistical significance from untreated cells, p < 0.05, as determined by one-way ANOVA. (B) Following this treatment, cells were placed in the upper chamber of a Transwell, and allowed to migrate through the uncoated membrane (8 μm pore) for 4 h. n = 3. **denotes statistical significance p < 0.01, ***p < 0.001, as determined by one-way ANOVA. (C) Parental MDA-MB-231 cells were treated with 40 μM KN-92 or KN-93, or 10 μM myr-AIP for 30 min, and the ability of cells to invade through Matrigel plugs was examined. *denotes statistical significance p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way ANOVA.
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f7: Pharmacological inhibition of CaMKII activity prevents breast cancer cell migration and invasion.Confluent monolayers of parental MDA-MB-231 cells were treated with 20 μM KN-92 or KN-93, or 10 μM myr-AIP for 30 min, and a wound was made by scratching the monolayer with a pipette tip. (A) Wounds were photographed hourly for 72 h to measure wound closure over time. Photomicrographs are representative of three independent experiments performed in triplicate. Wound widths are expressed as % of 0h wound width. *denotes statistical significance from untreated cells, p < 0.05, as determined by one-way ANOVA. (B) Following this treatment, cells were placed in the upper chamber of a Transwell, and allowed to migrate through the uncoated membrane (8 μm pore) for 4 h. n = 3. **denotes statistical significance p < 0.01, ***p < 0.001, as determined by one-way ANOVA. (C) Parental MDA-MB-231 cells were treated with 40 μM KN-92 or KN-93, or 10 μM myr-AIP for 30 min, and the ability of cells to invade through Matrigel plugs was examined. *denotes statistical significance p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way ANOVA.

Mentions: Taken together, our data suggest that activation of CaMKII can enhance breast cancer cell motility, invasiveness and tumourigenicity. To investigate whether pharmacological inhibition could potentially decrease breast cancer cell motility and invasion in vitro, we inhibited CaMKII activity using two different pharmacological inhibitors with varying mechanisms of action. KN-93 prevents the activation of CaMKII by calcium/calmodulin, but does not inhibit CaMKII that is already autonomously active. However, KN-93 can also inhibit molecules unrelated to CaMKII, such as ion channels. CaMKII specific effects can be determined when the effects of KN-93 are compared to its inactive analogue, KN-92. Myristoylated autocamtide-2-related autoinhibitory peptide (myr-AIP), competes with substrates at the active site of CaMKII, and inhibits activity of CaMKII irrespective of whether CaMKII is autonomously active or not10. Both AIP and KN-93, but not KN-92, significantly decreased migration (Fig. 7A,B) and invasion (Fig. 7C) of MDA-MB-231 cells. These findings demonstrate that pharmacological inhibition of CaMKII can significantly inhibit migration and invasion of highly aggressive breast cancer cells in vitro.


Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells
Pharmacological inhibition of CaMKII activity prevents breast cancer cell migration and invasion.Confluent monolayers of parental MDA-MB-231 cells were treated with 20 μM KN-92 or KN-93, or 10 μM myr-AIP for 30 min, and a wound was made by scratching the monolayer with a pipette tip. (A) Wounds were photographed hourly for 72 h to measure wound closure over time. Photomicrographs are representative of three independent experiments performed in triplicate. Wound widths are expressed as % of 0h wound width. *denotes statistical significance from untreated cells, p < 0.05, as determined by one-way ANOVA. (B) Following this treatment, cells were placed in the upper chamber of a Transwell, and allowed to migrate through the uncoated membrane (8 μm pore) for 4 h. n = 3. **denotes statistical significance p < 0.01, ***p < 0.001, as determined by one-way ANOVA. (C) Parental MDA-MB-231 cells were treated with 40 μM KN-92 or KN-93, or 10 μM myr-AIP for 30 min, and the ability of cells to invade through Matrigel plugs was examined. *denotes statistical significance p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way ANOVA.
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f7: Pharmacological inhibition of CaMKII activity prevents breast cancer cell migration and invasion.Confluent monolayers of parental MDA-MB-231 cells were treated with 20 μM KN-92 or KN-93, or 10 μM myr-AIP for 30 min, and a wound was made by scratching the monolayer with a pipette tip. (A) Wounds were photographed hourly for 72 h to measure wound closure over time. Photomicrographs are representative of three independent experiments performed in triplicate. Wound widths are expressed as % of 0h wound width. *denotes statistical significance from untreated cells, p < 0.05, as determined by one-way ANOVA. (B) Following this treatment, cells were placed in the upper chamber of a Transwell, and allowed to migrate through the uncoated membrane (8 μm pore) for 4 h. n = 3. **denotes statistical significance p < 0.01, ***p < 0.001, as determined by one-way ANOVA. (C) Parental MDA-MB-231 cells were treated with 40 μM KN-92 or KN-93, or 10 μM myr-AIP for 30 min, and the ability of cells to invade through Matrigel plugs was examined. *denotes statistical significance p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way ANOVA.
Mentions: Taken together, our data suggest that activation of CaMKII can enhance breast cancer cell motility, invasiveness and tumourigenicity. To investigate whether pharmacological inhibition could potentially decrease breast cancer cell motility and invasion in vitro, we inhibited CaMKII activity using two different pharmacological inhibitors with varying mechanisms of action. KN-93 prevents the activation of CaMKII by calcium/calmodulin, but does not inhibit CaMKII that is already autonomously active. However, KN-93 can also inhibit molecules unrelated to CaMKII, such as ion channels. CaMKII specific effects can be determined when the effects of KN-93 are compared to its inactive analogue, KN-92. Myristoylated autocamtide-2-related autoinhibitory peptide (myr-AIP), competes with substrates at the active site of CaMKII, and inhibits activity of CaMKII irrespective of whether CaMKII is autonomously active or not10. Both AIP and KN-93, but not KN-92, significantly decreased migration (Fig. 7A,B) and invasion (Fig. 7C) of MDA-MB-231 cells. These findings demonstrate that pharmacological inhibition of CaMKII can significantly inhibit migration and invasion of highly aggressive 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