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A novel role for Wnt/Ca2+ signaling in actin cytoskeleton remodeling and cell motility in prostate cancer.

Wang Q, Symes AJ, Kane CA, Freeman A, Nariculam J, Munson P, Thrasivoulou C, Masters JR, Ahmed A - PLoS ONE (2010)

Bottom Line: Wnt signaling is a critical regulatory pathway in development and disease.Live cell imaging showed that a functional consequence of CaMKII inhibition was 80% decrease in wound healing capacity and reduced cell motility in cancer cells.We propose that non-canonical Wnt/Ca(2+) signaling via CaMKII acts as a novel regulator of structural plasticity and cell motility in prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Prostate Cancer Research Centre and Division of Surgery, University College London, London, United Kingdom.

ABSTRACT
Wnt signaling is a critical regulatory pathway in development and disease. Very little is known about the mechanisms of Wnt signaling in prostate cancer, a leading cause of death in men. A quantitative analysis of the expression of Wnt5A protein in human tissue arrays, containing 600 prostate tissue cores, showed >50% increase in malignant compared to benign cores (p<0.0001). In a matched pair of prostate cancer and normal cell line, expression of Wnt5A protein was also increased. Calcium waves were induced in prostate cells in response to Wnt5A with a 3 fold increase in Flou-4 intensity. The activity of Ca(2+)/calmodulin dependent protein kinase (CaMKII), a transducer of the non-canonical Wnt/Ca(2+) signaling, increased by 8 fold in cancer cells; no change was observed in beta-catenin expression, known to activate the canonical Wnt/beta-catenin pathway. Mining of publicly available human prostate cancer oligoarray datasets revealed that the expression of numerous genes (e.g., CCND1, CD44) under the control of beta-catenin transcription is down-regulated. Confocal and quantitative electron microscopy showed that specific inhibition of CaMKII in cancer cells causes remodeling of the actin cytoskeleton, irregular wound edges and loose intercellular architecture and a 6 and 8 fold increase in the frequency and length of filopodia, respectively. Conversely, untreated normal prostate cells showed an irregular wound edge and loose intercellular architecture; incubation of normal prostate cells with recombinant Wnt5A protein induced actin remodeling with a regular wound edge and increased wound healing capacity. Live cell imaging showed that a functional consequence of CaMKII inhibition was 80% decrease in wound healing capacity and reduced cell motility in cancer cells. We propose that non-canonical Wnt/Ca(2+) signaling via CaMKII acts as a novel regulator of structural plasticity and cell motility in prostate cancer.

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Scanning electron microscopy of the wound edge in 1542-CP3TX cells.Main pictures are representative images (scale bar 1 µm) of untreated (A) and AIP (10 µM) treated (B) cells. Inset is the low magnification image (scale bar 10 µm) of the main picture. Irregular wound edge and fine filopodia like protrusion are visible after treatment with AIP (B, main picture). Length of filopodia like protrusions was measured using Image J software and converted to distribution histogram (C) for AIP treated (hollow bars) and untreated (hatched bars) cells. An 8-fold increase in the area under the curve was observed for treated compared to untreated cells using a Gaussian fit.
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pone-0010456-g006: Scanning electron microscopy of the wound edge in 1542-CP3TX cells.Main pictures are representative images (scale bar 1 µm) of untreated (A) and AIP (10 µM) treated (B) cells. Inset is the low magnification image (scale bar 10 µm) of the main picture. Irregular wound edge and fine filopodia like protrusion are visible after treatment with AIP (B, main picture). Length of filopodia like protrusions was measured using Image J software and converted to distribution histogram (C) for AIP treated (hollow bars) and untreated (hatched bars) cells. An 8-fold increase in the area under the curve was observed for treated compared to untreated cells using a Gaussian fit.

Mentions: The mechanism by which CaMKII inhibition caused filopodia formation in prostate cancer cells was next considered. We used scanning electron microscopy to quantify the filopodia like structures (Fig. 6). Low magnification scanning electron micrographs of 1542-CP3TX cells show regular and irregular wound leading edges in untreated (inset Fig. 6A) and AIP treated cells (inset Fig. 6B). High magnification images clearly show numerous and extended fine filopodia like projections from the cell membrane (Fig. 6B) in AIP treated 1542-CP3TX cells compared to untreated cells (Fig. 6A). Inhibition of CaMKII with AIP caused a similar effect in PC3 cell line (Fig. 5H and Fig. S5B). The frequency and length of the filopodia was measured manually using ImageJ software. A Gaussian fit of the length distribution histogram of treated and untreated 1542-CP3TX cells revealed an 8 fold increase in the length and a 6 fold increase in the overall frequency of filopodia like protrusions in AIP treated compared to untreated 1542-CP3TX cells (Fig. 6C and Table 1). The histogram could be fitted for at least two lengths of filopodia like protrusions in 1542-CP3TX cells: long (up to 2 µm) and very long (>2 µm). Further analysis showed that there was a 5 fold increase in length but a much greater (15 fold) increase in the frequency of the very long protrusions in AIP treated cells compared to untreated cells (Table 1). These results confirm a major role for CaMKII mediated Wnt signaling in actin remodeling in prostate cancer by decreasing the length and frequency of filopodia.


A novel role for Wnt/Ca2+ signaling in actin cytoskeleton remodeling and cell motility in prostate cancer.

Wang Q, Symes AJ, Kane CA, Freeman A, Nariculam J, Munson P, Thrasivoulou C, Masters JR, Ahmed A - PLoS ONE (2010)

Scanning electron microscopy of the wound edge in 1542-CP3TX cells.Main pictures are representative images (scale bar 1 µm) of untreated (A) and AIP (10 µM) treated (B) cells. Inset is the low magnification image (scale bar 10 µm) of the main picture. Irregular wound edge and fine filopodia like protrusion are visible after treatment with AIP (B, main picture). Length of filopodia like protrusions was measured using Image J software and converted to distribution histogram (C) for AIP treated (hollow bars) and untreated (hatched bars) cells. An 8-fold increase in the area under the curve was observed for treated compared to untreated cells using a Gaussian fit.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864254&req=5

pone-0010456-g006: Scanning electron microscopy of the wound edge in 1542-CP3TX cells.Main pictures are representative images (scale bar 1 µm) of untreated (A) and AIP (10 µM) treated (B) cells. Inset is the low magnification image (scale bar 10 µm) of the main picture. Irregular wound edge and fine filopodia like protrusion are visible after treatment with AIP (B, main picture). Length of filopodia like protrusions was measured using Image J software and converted to distribution histogram (C) for AIP treated (hollow bars) and untreated (hatched bars) cells. An 8-fold increase in the area under the curve was observed for treated compared to untreated cells using a Gaussian fit.
Mentions: The mechanism by which CaMKII inhibition caused filopodia formation in prostate cancer cells was next considered. We used scanning electron microscopy to quantify the filopodia like structures (Fig. 6). Low magnification scanning electron micrographs of 1542-CP3TX cells show regular and irregular wound leading edges in untreated (inset Fig. 6A) and AIP treated cells (inset Fig. 6B). High magnification images clearly show numerous and extended fine filopodia like projections from the cell membrane (Fig. 6B) in AIP treated 1542-CP3TX cells compared to untreated cells (Fig. 6A). Inhibition of CaMKII with AIP caused a similar effect in PC3 cell line (Fig. 5H and Fig. S5B). The frequency and length of the filopodia was measured manually using ImageJ software. A Gaussian fit of the length distribution histogram of treated and untreated 1542-CP3TX cells revealed an 8 fold increase in the length and a 6 fold increase in the overall frequency of filopodia like protrusions in AIP treated compared to untreated 1542-CP3TX cells (Fig. 6C and Table 1). The histogram could be fitted for at least two lengths of filopodia like protrusions in 1542-CP3TX cells: long (up to 2 µm) and very long (>2 µm). Further analysis showed that there was a 5 fold increase in length but a much greater (15 fold) increase in the frequency of the very long protrusions in AIP treated cells compared to untreated cells (Table 1). These results confirm a major role for CaMKII mediated Wnt signaling in actin remodeling in prostate cancer by decreasing the length and frequency of filopodia.

Bottom Line: Wnt signaling is a critical regulatory pathway in development and disease.Live cell imaging showed that a functional consequence of CaMKII inhibition was 80% decrease in wound healing capacity and reduced cell motility in cancer cells.We propose that non-canonical Wnt/Ca(2+) signaling via CaMKII acts as a novel regulator of structural plasticity and cell motility in prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Prostate Cancer Research Centre and Division of Surgery, University College London, London, United Kingdom.

ABSTRACT
Wnt signaling is a critical regulatory pathway in development and disease. Very little is known about the mechanisms of Wnt signaling in prostate cancer, a leading cause of death in men. A quantitative analysis of the expression of Wnt5A protein in human tissue arrays, containing 600 prostate tissue cores, showed >50% increase in malignant compared to benign cores (p<0.0001). In a matched pair of prostate cancer and normal cell line, expression of Wnt5A protein was also increased. Calcium waves were induced in prostate cells in response to Wnt5A with a 3 fold increase in Flou-4 intensity. The activity of Ca(2+)/calmodulin dependent protein kinase (CaMKII), a transducer of the non-canonical Wnt/Ca(2+) signaling, increased by 8 fold in cancer cells; no change was observed in beta-catenin expression, known to activate the canonical Wnt/beta-catenin pathway. Mining of publicly available human prostate cancer oligoarray datasets revealed that the expression of numerous genes (e.g., CCND1, CD44) under the control of beta-catenin transcription is down-regulated. Confocal and quantitative electron microscopy showed that specific inhibition of CaMKII in cancer cells causes remodeling of the actin cytoskeleton, irregular wound edges and loose intercellular architecture and a 6 and 8 fold increase in the frequency and length of filopodia, respectively. Conversely, untreated normal prostate cells showed an irregular wound edge and loose intercellular architecture; incubation of normal prostate cells with recombinant Wnt5A protein induced actin remodeling with a regular wound edge and increased wound healing capacity. Live cell imaging showed that a functional consequence of CaMKII inhibition was 80% decrease in wound healing capacity and reduced cell motility in cancer cells. We propose that non-canonical Wnt/Ca(2+) signaling via CaMKII acts as a novel regulator of structural plasticity and cell motility in prostate cancer.

Show MeSH
Related in: MedlinePlus