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 Ca2+/calmodulin dependent protein kinase (CaMKII), a transducer of the non-canonical Wnt/Ca2+ signaling, increased by 8 fold in cancer cells; no change was observed in β-catenin expression, known to activate the canonical Wnt/β-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 β-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/Ca2+ signaling via CaMKII acts as a novel regulator of structural plasticity and cell motility in prostate cancer.
Highlights
Wingless/Wnt genes code for a family of secreted glycoproteins that regulate many cellular processes [1,2]
For the first time, that Wnt5A protein expression is increased in malignant human prostate compared to benign tissue and Wnt/Ca2+ signaling mechanism is activated in prostate cancer cells
RNA transcript assessment using real time PCR We measured the mRNA levels for WNT5A (Table S1), previously identified from our oligoarray analysis to be dysregulated in prostate cancer cells [23]
Summary
Wingless/Wnt genes code for a family of secreted glycoproteins that regulate many cellular processes [1,2]. Aberrant signaling through Wnt signaling pathways is linked to a number of diseases including cancer [3,4,5,6]. Signaling via the Wnt/b-catenin pathway activates transcription of many genes, such as cyclin Ds and membrane metalloproteinases (MMPs), via TCF/LEF factors. Wnt/Ca2+ pathway leads to an increase in intracellular calcium [9] and activation of calmodulin dependent protein kinase II (CaMKII) [10] and is known to modulate cell movement and behavior [11,12] and induces structural changes [11,13,14]. The link between increased b-catenin signaling in tumorigenesis and increased transcription of genes involved in cell transformation and proliferation are documented, the role non-canonical Wnt/Ca2+ signaling in cancer is only slowly being elucidated [15,16]
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