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.

Resistance to currently available targeted therapies significantly hampers the survival of prostate cancer (PCa) patients with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a "resistance niche" adjacent to PCa-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed "osteocrines") found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to newly-formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic PCa.

No abstract

Loss of E-cadherin and up-regulation of mesenchymal cadherins, a hallmark of the epithelialmesenchymal transition, contributes to migration and dissemination of cancer cells. Expression of human cadherin-11 (Cad11), also known as osteoblast cadherin, in prostate cancer increases the migration of prostate cancer cells. How Cad11 mediates cell migration is unknown. Using the human Cad11 cytoplasmic domain in pulldown assays, we identified human angiomotin (Amot), known to be involved in cell polarity, migration, and Hippo pathway, as a component of the Cad11 protein complex. Deletion analysis showed that the last C-terminal 10 amino acids in Cad11 cytoplasmic domain are required for Amot binding. Further, Cad11 preferentially interacts with Amot-p80 than Amot-p130 isoform and binds directly to the middle domain of Amot-p80. Cad11-Amot interaction affects Cad11-mediated cell migration, but not homophilic adhesion, as deletion of Amot binding motif of Cad11 (Cad11-DAmot) did not abolish Cad11-mediated cell-cell adhesion of mouse L cells, but significantly reduced Cad11-mediated cell migration of human C4-2B4 and PC3-mm2 prostate cancer cells and human HEK293T cells. Together, our studies identified Amot-p80 as a novel component of the Cad11 complex and demonstrated that Amot-p80 is critical for Cad11-mediated cell

Osteoblasts communicate both with normal cells in the bone marrow, and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130–140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC-MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (~47%) and plasma membrane (~31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes, based on emPAI ratios of more than 5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions.

Prostate cancer (PCa) has a proclivity to metastasize to bone. The mechanism by which PCa cells are able to survive and progress in the bone microenvironment is not clear. Identification of molecules that play critical roles in the progression of PCa in bone will provide essential targets for therapy. Ribosomal S6 protein kinase (RSKs) have been shown to mediate many cellular functions critical for cancer progression. Whether RSK plays a role in the progression of PCa in bone is unknown. Immunohistochemical (IHC) analysis of human PCa specimens showed increased phosphorylation of RSK in the nucleus of PCa cells in a significant fraction of human PCa bone metastasis specimens, compared to the primary site or lymph node metastasis. Expression of constitutively active myristylated-RSK in C4-2B4 cells (C4-2B4/RSK) increased their survival and anchorage-independent growth compared to C4-2B4/vector cells. Using an orthotopic bone injection model, it was determined that injecting C4-2B4/RSK cells into mouse femurs enhanced their progression in bone compared to control cells. In PC3-mm2 cells, knockdown of RSK1 (RPS6KA1), the predominant RSK isoform, but not RSK2 (RPS6KA2) alone, decreased anchorage-independent growth in vitro and reduced tumor progression in bone and tumor-induced bone remodeling in vivo. Mechanistic studies showed that RSK regulates anchorage-independent growth through transcriptional regulation of factors that modulate cell survival, including ING3, CKAP2 and PTK6. Together, these data provide strong evidence that RSK is an important driver in PCa progression in bone. Implications RSK, an important driver in PCa progression in bone, has promising potential as a therapeutic target for PCa bone metastasis.