Prostate cancer metastasizes to bone in more than 80% of patients suffering from advanced disease [2
]. Unlike breast and lung cancer, the nature of bone lesions is predominantly osteoblastic resulting in hypocalcaemia, pain, bone fractures and nerve compression syndromes. We have used a novel human prostate cancer cell line, PacMetUT1, in our study. Our results indicate that the PacMetUT1 cells have the potential for skeletal metastasis and bone formation in a nude mice model after intracardiac inoculation. To elucidate the molecular mechanism that mediates PacMetUT1-induced bone formation, we investigated the role of TGFβ signaling because PacMetUT1 secretes large amounts of TGFβ1. TGFβ regulates a broad range of biological processes, including cell proliferation, survival, differentiation, migration, and production of extracellular matrix (ECM). However, TGFβ acts as a double-edged sword during tumor progression by inhibiting cell proliferation during early stage and stimulating metastasis in the later stages [35
]. TGFβ suppresses cancer progression by stimulating cyclin-dependent kinase inhibitors through its canonical signaling pathway involving Smad-2/3 proteins. We observed a diminished Smad2 protein activation after TGFβ1 knockdown suggesting that the downstream Smad signaling pathway is getting altered after TGFβ blockade in PacMetUT1 cells. However, we observed a moderate decrease in cell proliferation after knocking down TGFβ1 in PacMetUT1 cells in vitro
. On the other hand, TGFβ1 knockdown did not affect the tumorigenicity of PacMetUT1 cells in vitro
as shown in our soft agar clonogenic assay. As such, its signaling in the cells does not appear to confer tumor suppression.
TGFβ is secreted in the bone matrix by osteoblasts and is a critical regulator of osteogenesis. TGFβ plays a key role in osteoblast differentiation, bone development and remodeling. It has been shown to stimulate proliferation and early osteoblast differentiation, while inhibiting terminal differentiation [6
]. TβR1 inhibitors were shown to increase osteoblast differentiation and bone formation while reducing osteoclast differentiation and bone resorption in the post-natal skeleton [26
]. TGFβ released during osteolytic resorption from bone matrix has been shown to stimulate osteolytic metastasis and osteoclast activation in breast cancer by activating bone resorbing cytokines [36
]. But the role of TGFβ signaling in prostate cancer-induced bone metastatic lesions is not very clear in part due to a lack of model system which can mimic osteoblastic bone metastasis process. In this study, we have evaluated the role of TGFβ pathway in osteoblastic bone tumor formation by PacMetUT1 cells. Because of the low incidence of bone metastasis in intracardiac injection, we used direct intra-tibia injection route for studying the effect of TGFβ signaling abrogation on tumor growth in the tibias of nude mice. We have observed a decrease in tumor incidence and osteoblastic lesions in vivo
in the TGFβ1 knockdown group as compared to the control group. Systemic administration of the TβRI-KI and a pan-TGFβ inhibitor BGE
RII also resulted in a decreased bone tumor growth. This result is in contrast to what has previously been observed with TGFβ mediated osteolysis in breast cancer model system. However, a recent report shows that TGFβ1 induces osteogenic differentiation of murine bone marrow stromal cells by increasing osteoblast differentiation markers (Runx-2, osteopontin, collagen-1) as well as alkaline phosphatase activity as compared to control cells [37
]. The decreased osteoblastic bone formation by inhibition of TGFβ signaling in tumor cells may be because of reduced expression of osteoblast specific factors like alkaline phosphatase, collagen, osteonectin and osteopontin [38
]. TGFβ1 after being activated by prostate specific antigen (PSA), which is an osteoblast stimulating factor, can result in an increase in mature osteoblasts and a decrease in osteoclast population [40
]. However, PacMetUT1 cells do not express PSA, suggesting that the increase in bone formation was mediated by a PSA-independent mechanism. Bone cells such as osteoblasts express aromatase, which is the enzyme required for estrogen biosynthesis in humans [42
]. TGFβ1 was shown to stimulate expression of the aromatase gene in osteoblast-like cells [44
]. The autocrine or paracrine secreted TGFβ1 from PacMetUT1 cells might result in an increased aromatase gene expression and activity leading to an enhanced estradiol synthesis which would in turn lead to an increase in bone formation as estrogen is a known osteogenic factor.
TGFβ has been shown to be a potential modifier of receptor activator of NF-kappaB ligand (RANK)-dependent osteoclast activation and osteolysis in breast cancer [36
]. TGFβ can also activate osteoclastic differentiation and activity [26
]. In our study, we observed a decrease in osteoblastic bone lesions with a moderate decrease in osteoclast number by the systemic administration of TGFβ signaling inhibitors in the nude mice. This rules out the possibility that the decreased bone formation is due to an increase in osteoclast number. We also observed that in the tumor-containing tibias, osteoclasts migrated to the area where new bone formation was taking place, which is consistent with the dogma that new bone formation occurs in the area of osteolytic resorption [45
]. Further studies are required to reveal the mechanism of TGFβ mediated increase in bone formation in PacMetUT1 cells.
Pulmonary metastasis commonly develops after bone metastasis of prostate cancer. Few patients with prostate cancer present initially with symptomatic metastatic lung lesions without any other concomitant distant dissemination [46
]. TGFβ has been shown to cause lung metastasis in several animal models of breast cancer [34
]. In our study, we have observed that systemic administration of TGFβ inhibitors, TβRI-KI and BGE
RII, reduced the number of lung metastatic colonies from intratibia PacMetUT1 tumors. One possible mechanism for the reduced incidence of bone tumor-derived lung metastasis by TGFβ inhibitors may be because of the inhibition of TGFβ-induced migration of tumor cells in the bone marrow, similar to the TGFβ-induced extravasation in the pulmonary metastasis model of breast cancer [48
In summary, our study revealed the predominant osteoblastic nature of a novel human prostate cancer cell line, PacMetUT1. Our results indicated for the first time that abrogation of TGFβ signaling by the systemic administration of TβRI-KI and BGERII or by knockdown of TGFβ1 with shRNA’s can effectively inhibit the tumor incidence and osteoblastic lesion formation by this novel prostate cancer model in the bone microenvironment. Our long-term goal is to determine whether TGFβ antagonists alone or in combinatorial therapy will be suitable as therapeutic agents for the treatment and prevention of prostate cancer-induced bone metastasis.