Wnt expression and mechanisms of Wnt signaling remains a poorly investigated area in prostate. We demonstrate that (i) Wnt5A protein expression is increased in malignant compared to benign human prostate tissue and in cancer cell lines compared to normal. (ii) Wnt5A is major transducer of Wnt signaling via activation of Wnt/Ca2+ pathway and not Wnt/β-catenin pathway in prostate cells. (iii) Activation of CaMKII is a novel and critical regulator of cytoskeleton in prostate cancer. (iv) CaMKII inhibition significantly decreases cell motility and the capacity of wound healing in prostate cancer cell lines.
An understanding of the molecular basis of prostate cancer, and the role played by signaling networks such as Wnt-pathway, requires not only a comprehensive description of gene and protein expression in human prostate tissue, but also a cell system with which to investigate the mechanisms of signaling and its functional consequences in disease. Our prostate tissue array investigations show that there was a highly significant (p<0.0001) increase in both the total area (extent) and average size of the labelled particles (intensity) and area fraction ( and Table S2
). These results support the earlier observations of increase in the expression of Wnt5A gene in prostate cancer due to hypomethylation 
and also a very recent report by Yamamoto et a
that used conventional, non-quantiative, histological examination to assess Wnt5A expression prostate. This data is also in agreement with that obtained for normal (1542-NPTX) and cancer (1542-CP3
TX) cell lines (). Taken together the human tissue and cell line data suggest that Wnt5A protein is expressed in normal (or benign) tissue but its expression is dramatically increased in malignant (cancer) prostate tissue and this change is reflected in the cell lines used in this study.
Almost all known transcription targets activated by the Wnt/β-catenin pathway, e.g. CTNNB1, CCCNDs, MMPs, PITX2, CD44, APCDD1, JUN 
, remain unchanged or are down-regulated in prostate cancer tissue or cell lines compared to normal tissue or cell line (Fig. S3
and Table S1
). These results indicate that the cell line dataset largely reflects the gene expression pattern for TCF/LEF regulated genes in both the cell line and the cancer tissue. Furthermore, there was no increase in protein expression or functional activity of downstream targets of canonical Wnt-signaling (e.g. MMP-14). This suggests that 1542-NPTX and 1542-CP3
TX and PC3 cell lines may be a useful model to investigate the mechanisms of Wnt signaling in prostate cancer as the integrity of Wnt signaling elements in prostate cancer are preserved in these cell lines at the gene, protein and functional levels.
CaMKII is a key mediator of Wnt/Ca2+
signaling although a further subset of the non-canonical Wnt signaling via Wnt5A is proposed to be a β-catenin degradation pathway which does not require activation of CaMKII 
. A 3 fold increase in free intracellular calcium concentration was observed after addition of Wnt5A in prostate cells ( and Movie S1
). In addition, we did not see a decrease in β-catenin protein expression and the activity of CaMKII was found to increase in prostate cancer cell lines, indicating that Wnt/Ca2+
pathway was likely to be operative via CaMKII in prostate cancer.
The multiple roles of CamKII in actin remodelling, cell motility and migration of normal keratinocytes, muscle and nerve cells are known 
. However the involvement of CaMKII mediated Wnt/Ca2+
signaling in disease has been less well characterized. Pukrop et al 
suggested that the non-canonical pathway may be involved in the increased migration/invasiveness of MCF7 breast cancer cell lines when recombinant Wnt5A is added to the cell culture. However, the mechanisms or whether Wnt5A mediated its effect through CaMKII activation, were not investigated. Cell motility requires two main types of actin based structures in the cell membrane, lamellipodia and filopodia. It has been proposed, in fibroblasts, that filopodia formation from lamellipodia is a tightly regulated process which is partly controlled by actin binding proteins such as enabled (Ena) / vasodilator-stimulated phosphoprotein (VASP) capping the actin filament 
. Fibroblasts move by extending lamellipodia at the leading edge and formation of fine actin structures at the leading edge causes retrograde movement 
; sequestration of Ena/VASP to mitochondria increases whereas its targeting to cell membrane reduces motility 
. The role of Wnt5A mediated signaling in cell motility 
and other intermediary proteins (e.g. Ena/VASP, Arp2/3) in filopodia formation has been investigated in human 
and mouse 
melanoma cells. Weeraratna and colleagues 
suggested that Wnt5A increased cell motility in human melanoma cell lines by activation of protein kinase C (PKC). Witze et al 
showed that acute response of melanoma cell lines to Wnt5A involves recruitment of major cytoskeletal proteins (actin and myoxin IIB) and Frizzled 3 and melanoma cell adhesion molecule into an intracellular structure via Wnt5A regulation of Rab4 and RhoB guanosine triphosphatases. Another study investigated the role for Ror2 kinase in Wnt5A induced cell migration 
. Unlike the data presented here, these studies 
neither investigated nor directly manipulated CaMKII activity in their experiments or investigated its role in filopodia formation. We used tatCN21a, a specific inhibitor of CaMKII (but not CaMKIV, PKA, PKC, MAPK1, JNK1α1, or Raf). Treatment of prostate cancer cells with tatCN21a also caused a loosening of cell to cell contact and induced filopodia formation (Fig. S4
) similar to the results obtained with AIP (). These results indicate that direct inhibition of CaMKII (and not CaMKIV, PKC, PKA and other kinases) results in the loss of cell to cell contact and filopodia formation in prostate cancer cells.
In prostate cancer cell lines (1542-CP3TX and PC3) a smooth, regular wound edge is observed with close cell to cell contact preceding the lamellipodia like leading edge ( and ). In contrast, treatment with AIP induces an increase in filopodia like protrusions with a loosening of cell to cell contact preceding the wound edge ( and and ). Indeed the rate of wound closure was decreased by 80% after inhibition of CaMKII in cancer cell line (PC3). Conversely, addition of Wnt5A to normal cell line (1542-NPTX) increased the rate of wound closure (µm/h) compared to untreated cells by 25±4%. We suggest that activation of CaMKII via Wnt5A signaling is advantageous to cancer cell mobility as it suppresses formation of fine filopodia that induce retrograde movement thus reducing cell mobility.
Protein expression of Wnt5A in human prostate cancer and mechanisms of Wnt signaling in normal and cancer prostate cell lines, described here, provide the first framework within which the exact participation of Wnt receptors and secretory frizzled related proteins 
and various actin binding proteins and intermediary signaling molecules such as Ena/VASP and ERK1 
, could be investigated in cancer. Further investigations to identify the receptor(s) for Wnt5A binding and other proteins involved in the downstream signaling via CaMKII in cell motility in prostate cancer will help to elucidate the novel role of CaMKII in the suppression of filopodia formation to increase cell motility in prostate cancer. In conclusion, from the results presented here and our recent study 
showing hypomethylation as a regulator of Wnt5A gene transcription in prostate, the following sequence could be envisaged: (i) Gene expression of Wnt5A is increased in cancer compared to normal cells due to hypomethylation of the gene promoter region (ii) Increase in gene expression results in increased protein expression of Wnt5A in human prostate cancer (iii) Wnt/Ca2+
pathway (and not β-catenin pathway) is activated in prostate cancer cells (iv) Signaling via Wnt/Ca2+
pathway activates CaMKII (v) CaMKII activity, most likely via intermediary signaling involving actin binding proteins, causes a major reorganization of cytoskeleton in cancer cells by decreasing the length and frequency of fine, filopodia like actin structures. (vi) Cytoskeletal remodeling due to CaMKII causes an increase in cell motility. Targeting of Wnt/Ca2+
signaling, particularly CaMKII may provide a useful tool in prostate cancer therapy.