Bone metastasis from prostate cancer can occur years after prostatectomy, due to reactivation of dormant disseminated tumor cells (DTC) in the bone, yet the mechanism by which DTCs are initially induced into a dormant state in the bone remains to be elucidated. We show here that the bone microenvironment confers dormancy to C4-2B4 prostate cancer cells, as they become dormant when injected into mouse femurs but not under the skin. Live-cell imaging of dormant cells at the single-cell level revealed that conditioned medium from differentiated, but not undifferentiated, osteoblasts induced C4-2B4 cellular quiescence, suggesting that differentiated osteoblasts present locally around the tumor cells in the bone conferred dormancy to prostate cancer cells. Gene array analyses identified GDF10 and TGFβ2 among osteoblast-secreted proteins that induced quiescence of C4-2B4, C4-2b, and PC3-mm2, but not 22RV1 or BPH-1 cells, indicating prostate cancer tumor cells differ in their dormancy response. TGFβ2 and GDF10 induced dormancy through TGFβRIII to activate phospho-p38MAPK, which phosphorylates retinoblastoma (RB) at the novel N-terminal S249/T252 sites to block prostate cancer cell proliferation. Consistently, expression of dominant-negative p38MAPK in C4-2b and C4-2B4 prostate cancer cell lines abolished tumor cell dormancy both and Lower TGFβRIII expression in patients with prostate cancer correlated with increased metastatic potential and decreased survival rates. Together, our results identify a dormancy mechanism by which DTCs are induced into a dormant state through TGFβRIII-p38MAPK-pS249/pT252-RB signaling and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. These findings provide mechanistic insights into the dormancy of metastatic prostate cancer in the bone and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. .

Summary Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDX) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX conditioned media, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4-overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2cre/Osxf/f/SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX versus in bigenic mice (Osxf/f/SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa.

Cadherin-11 is a member of a superfamily mainly expressed in osteoblasts but not in epithelial cells. However, prostate cancer (PCa) cells with bone metastasis propensity express high levels of cadherin-11 and reduced levels of E-cadherin. Downregulation of cadherin-11 inhibits interaction of PCa cells with osteoblasts in vitro and homing of PCa cells to bone in an animal model of metastasis. These findings raise the possibility that targeting the extracellular domain of cadhein-11 may prevent PCa bone metastasis. To explore this possibility, we generated a panel of monoclonal antibodies (mAbs) against cadherin-11 extracellular domain. From the 21 antibodies obtained, mAbs 2C7 and 1A5 inhibited cadherin-11 mediated cell-cell aggregation in L-cells transfected with cadherin-11 in vitro. Both antibodies were specific to cadherin-11 as they did not recognize E-cadherin or N-cadherin on C4-2B or PC3 cells, respectively. Further, mAb 2C7 inhibited cadherin-11-mediated aggregation between PC3-mm2 cells and MC3T3-E1 osteoblasts. To determine which cadherin domains are critical for PCa and osteoblast interactions, a series of deletion mutants were analyzed. We identified a previously unknown unique motif, aa 343-348, in the cadherin-11 EC3 domain that is recognized by mAb 2C7 and showed that this motif mediated cell-cell adhesion. Consistent with the inhibition of cell-cell aggregation in vitro, application of mAb 2C7 in a prophylactic setting as a single agent effectively prevented dissemination of highly metastatic PC3-mm2 cells to bone in a mouse model of metastasis. These results suggest that targeting the extracellular domain of cadherin-11 may be developed for the prevention of bone metastases.