Limits...
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.


Related in: MedlinePlus

CaMKII expression and phosphorylation at T286 is increased in primary breast cancer and lymph node metastases tissues.(A,D) Normal breast, (B,E) primary breast cancer, and (C,F) lymph node metastases were examined for (A–C) total CaMKII and (D–F) pT286-CaMKII expression by immunohistochemistry. Staining was quantified and expressed as an H-score. (G) Quantification of total CaMKII and (H) pT286-CaMKII expression in 70 primary breast cancer, 40 matched normal breast, and 10 lymph node metastases cores. Photomicrographs are representative of each tissue type. *denotes statistical significance p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001, as determined by one-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5015093&req=5

f2: CaMKII expression and phosphorylation at T286 is increased in primary breast cancer and lymph node metastases tissues.(A,D) Normal breast, (B,E) primary breast cancer, and (C,F) lymph node metastases were examined for (A–C) total CaMKII and (D–F) pT286-CaMKII expression by immunohistochemistry. Staining was quantified and expressed as an H-score. (G) Quantification of total CaMKII and (H) pT286-CaMKII expression in 70 primary breast cancer, 40 matched normal breast, and 10 lymph node metastases cores. Photomicrographs are representative of each tissue type. *denotes statistical significance p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001, as determined by one-way ANOVA.

Mentions: To examine the level of CaMKII phosphorylation at T286 in breast cancer tissues and to confirm that total CaMKII is overexpressed in breast cancer tissues at the protein level, CaMKII protein expression and phosphorylation at T286 was examined in 70 breast cancer, 40 matched normal breast, and 10 lymph node metastases patient samples by immunohistochemistry. Total and phosphorylated CaMKII expression was scored on a scale of 0–300, as previously described27. In contrast to that observed in the established breast cell lines (Fig. 1A,B), total CaMKII expression was significantly increased in primary breast cancer (Fig. 2B,G; p < 0.00001) and lymph node metastases (Fig. 2C,G; p < 0.00001) when compared to normal breast tissue (Fig. 2A). Furthermore, phosphorylation of CaMKII at T286 was also significantly increased in primary breast cancer (Fig. 2E,H; p < 0.001) and metastases (Fig. 2F,H; p < 0.001) when compared to the normal breast tissue (Fig. 2D). Total CaMKII expression (Fig. 2G; p < 0.01) and phosphorylation at T286 (Fig. 2H; p < 0.05) were further increased in lymph node metastases, when compared to the primary breast cancer samples, providing further evidence for a role of CaMKII in breast cancer cell metastases. Taken together, these data demonstrate that both CaMKII expression and phosphorylation at T286 are increased in breast cancer tissue, as well as lymph node metastases, and may be potentially useful biomarkers to predict patient outcome and likelihood of metastasis. However, the functions controlled by CaMKII in breast cancer cells remain largely unexplored.


Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells
CaMKII expression and phosphorylation at T286 is increased in primary breast cancer and lymph node metastases tissues.(A,D) Normal breast, (B,E) primary breast cancer, and (C,F) lymph node metastases were examined for (A–C) total CaMKII and (D–F) pT286-CaMKII expression by immunohistochemistry. Staining was quantified and expressed as an H-score. (G) Quantification of total CaMKII and (H) pT286-CaMKII expression in 70 primary breast cancer, 40 matched normal breast, and 10 lymph node metastases cores. Photomicrographs are representative of each tissue type. *denotes statistical significance p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001, as determined by one-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: CaMKII expression and phosphorylation at T286 is increased in primary breast cancer and lymph node metastases tissues.(A,D) Normal breast, (B,E) primary breast cancer, and (C,F) lymph node metastases were examined for (A–C) total CaMKII and (D–F) pT286-CaMKII expression by immunohistochemistry. Staining was quantified and expressed as an H-score. (G) Quantification of total CaMKII and (H) pT286-CaMKII expression in 70 primary breast cancer, 40 matched normal breast, and 10 lymph node metastases cores. Photomicrographs are representative of each tissue type. *denotes statistical significance p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001, as determined by one-way ANOVA.
Mentions: To examine the level of CaMKII phosphorylation at T286 in breast cancer tissues and to confirm that total CaMKII is overexpressed in breast cancer tissues at the protein level, CaMKII protein expression and phosphorylation at T286 was examined in 70 breast cancer, 40 matched normal breast, and 10 lymph node metastases patient samples by immunohistochemistry. Total and phosphorylated CaMKII expression was scored on a scale of 0–300, as previously described27. In contrast to that observed in the established breast cell lines (Fig. 1A,B), total CaMKII expression was significantly increased in primary breast cancer (Fig. 2B,G; p < 0.00001) and lymph node metastases (Fig. 2C,G; p < 0.00001) when compared to normal breast tissue (Fig. 2A). Furthermore, phosphorylation of CaMKII at T286 was also significantly increased in primary breast cancer (Fig. 2E,H; p < 0.001) and metastases (Fig. 2F,H; p < 0.001) when compared to the normal breast tissue (Fig. 2D). Total CaMKII expression (Fig. 2G; p < 0.01) and phosphorylation at T286 (Fig. 2H; p < 0.05) were further increased in lymph node metastases, when compared to the primary breast cancer samples, providing further evidence for a role of CaMKII in breast cancer cell metastases. Taken together, these data demonstrate that both CaMKII expression and phosphorylation at T286 are increased in breast cancer tissue, as well as lymph node metastases, and may be potentially useful biomarkers to predict patient outcome and likelihood of metastasis. However, the functions controlled by CaMKII in breast cancer cells remain largely unexplored.

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