Cadherin-11 promotes the metastasis of prostate cancer cells to bone

Abstract

Bone is the most common site of metastases from prostate cancer. The mechanism by which prostate cancer cells metastasize to bone is not fully understood, but interactions between prostate cancer cells and bone cells are thought to initiate the colonization of metastatic cells at that site. Here, we show that cadherin-11 (also known as osteoblast-cadherin) was highly expressed in prostate cancer cell line derived from bone metastases and had strong homophilic binding to recombinant cadherin-11 in vitro. Down-regulation of cadherin-11 in bone metastasis-derived PC3 cells with cadherin-11-specific short hairpin RNA (PC3-shCad-11) significantly decreased the adhesion of those cells to cadherin-11 in vitro. In a mouse model of metastasis, intracardiac injection of PC3 cells led to metastasis of those cells to bone. However, the incidence of PC3 metastasis to bone in this model was reduced greatly when the expression of cadherin-11 by those cells was silenced. The clinical relevance of cadherin-11 in prostate cancer metastases was further studied by examining the expression of cadherin-11 in human prostate cancer specimens. Cadherin-11 was not expressed by normal prostate epithelial cells but was detected in prostate cancer, with its expression increasing from primary to metastatic disease in lymph nodes and especially bone. Cadherin-11 expression was not detected in metastatic lesions that occur in other organs. Collectively, these findings suggest that cadherin-11 is involved in the metastasis of prostate cancer cells to bone.

Figure 1

Expression of cadherin-11 in human prostate cancer cell lines evaluated by A, northern blotting, B, western blotting, and C, immunocytochemical staining. Cadherin-11 was expressed in cell membranes in areas of cell-cell contact in PC3 and C4–2B cells. D, Binding of prostate cancer cell lines to cadherin-11-Fc–coated wells, indicated by luciferase activity plotted versus cell number. The bone-derived PC3 cells show the strongest binding. E, Analysis of PC3 binding to different substratum. Strong binding of PC3 was observed with cadherin 11-Fc and this binding was inhibited by the addition of 5 mM EDTA. PC3 showed minimal binding to BSA, cadherin 12-Fc, or uncoated well.

Figure 2

Metastasis of PC3 cells in vivo after intracardiac injection. PC3-Luc cells were transduced with genes for luciferase [Luc] and green fluorescent protein [GFP]) and injected into the left ventricles of mice. A, Bioluminescence images of a single mouse at various times after tumor cell inoculation. B, Bioluminescence tracings for two mice show exponential increases in Luc activity in hind leg. C, Ex vivo detection of tumor in mouse femur by bioluminescence (BLI) and GFP imaging. D, Histologic analysis of tumor in bone at low (left) and high (right) magnification.

Figure 3

Knockdown of cadherin-11 in PC3 cells blocks adhesion of those cells to cadherin-11 and reduces the incidence of metastasis to hind legs in a mouse model. A,, Western blotting shows decreased cadherin-11 expression in PC3 cells treated with shRNA to cadherin-11 (quantification shown at right). B, Immuncytochemical staining of cadherin-11 in PC3-shCont and PC3-shCad-11 cells. Cy3 labelled cadherin-11 at the membrane of PC3-shCont and PC3-shCad-11 cells (left). Overlay of Cy3 labelled-cadherin-11 with nuclear staining by DAPI to localize cells (middle). Negative control mouse IgG staining with DAPI overlay (right). C, Proliferation rates were not different for PC3-shCont and PC3-shCad-11 cells in vitro. D, Coculture of PC3-shCont or PC3-shCad-11 with MC3T3-E1 osteoblast cells. The presence of osteoblast does not affect the proliferation of PC3-shCont and PC3-shCad-11 cells. E, A cell-to-substrate assay indicates that knockdown of cadherin-11 reduced adhesion to cadherin-11-Fc. BSA, bovine serum albumin. F, PC3-Luc cells treated with shRNA to cadherin-11 formed fewer skeletal metastases in hind legs of mice after intracardiac injection than did cells with a scrambled shRNA control vector. G, Tartrate-resistant alkaline phosphatase staining of PC3-shCont and PC3-shCad-11 bones showed that both type of cells induced osteolytic bone lesion. Magnified region showing the presence of tartrate-resistant alkaline phosphatase positive cells (purple staining, arrow) were observed on the bone surface adjacent to tumor cells (T).

Figure 4

Staining of human prostate cancer specimens with antibody to cadherin-11. Top row, Osteosarcoma specimens show positive staining for cadherin-11 and negative staining for an IgG control; cadherin-11 is also expressed in osteoblasts. Bottom row, Cadherin-11 is expressed in a primary prostate cancer specimen (Gleason score 5+5). Adjacent prostate intraepithelial neoplasia did not express cadherin-11. Cadherin-11 is also evident in metastatic prostate cancer cells in lymph nodes and, to a much greater extent, in metastatic prostate cancer cells in bone.