In this study, we show that inhibiting α6 integrin cleavage on the tumor cell surface, either through antibody engagement or integrin mutation, will substantially delay the appearance of osseous metastases in a mouse xenograft model. The results reported here support the hypothesis that α6 integrin cleavage permits extravasation of tumor cells from the circulation since subcutaneous injection or direct injection of PC3N-RR mutant cells into bone has no affect on tumor growth at either location (28
). This is significant in the course of the human disease since extravasation from the circulation is a critical factor of metastatic spread (8
The influence of antibody engagement to delay the metastatic phenotype suggests that providing circulating levels of the integrin specific antibody may be beneficial in preventing bone metastasis. The ability of J8H to reversibly delay the appearance of metastases by one week is significant since this corresponds to the expected half life of therapeutic type antibodies in the SCID mouse (45
). Toxicity of J8H in the normal tissue is not expected since this antibody does not block cell adhesion to laminin (27
). Previous work has shown that inhibiting the A6 adhesion function will block experimental metastasis to the lung (14
). However, the utility of this approach as a therapeutic strategy appears limited. Circulating levels of immunoglobulin specific for blocking α6 integrin adhesion function in humans results in the formation of blisters and erosive lesions in the oral mucosa (25
). This underscores the potential therapeutic benefits of the J8H antibody, a reagent that inhibits α6 cleavage and not α6 dependent adhesion.
A6 integrin is utilized by hematopoetic stem cells to target the bone (46
). The ability of the α6 integrin RR mutation to reduce the metastatic potential of tumor cells homing to bone occurs in the presence of endogenous, wild type α6 integrin. This leads to speculation that cleavage of the receptor has a dominant role in the process. The results indicate that both the time to metastasis and the number of mice developing bone lesions were substantially reduced in the PC3B1-RR group. In contrast to the antibody blocking experiments, the majority of the animals did not develop bone lesions over the 6 week course of the study. Necropsies of the animals receiving tumor cell injections did not reveal other common sites of metastasis (i.e. lung, liver or adrenal gland), suggesting that circulating tumor cells were either eliminated from the mouse or achieved a level of dormancy (47
) in the animal. Further experiments utilizing labeled cells and sensitive imaging technology as developed by other groups (49
), could distinguish these possibilities.
It is also interesting to note that after week 6, approximately 40% of the animals injected with PC3B1-RR mutant cells developed a detectable metastatic lesion in bone. Although these lesions were progressive, the rate of progression compared to PC3B1-WT was slow as determined by the radiographic features of observed lesions. Termination of these animals was not required because metastases did not produce aggressive lesions leading to pathologic fractures. This suggests that tumor cells that possess uncleavable α6 integrin may eventually adapt to and colonize an osseous microenvironment to produce lytic lesions, but remain less aggressive in nature. This result is consistent with the reported less aggressive phenotype of the RR mutant tumor cells directly injected into the distal end of a mouse femur (28
We note that endogenous levels of α6p observed for cell lines in culture do not correlate with secreted levels of uPA and uPA activity. PC3 cells express minimal levels of α6p, while DU145 cells convert a majority of α6 integrin into the cleaved product (). However, PC3 cells secrete at least 2 fold more active uPA when compared to DU145 (50
). This suggests that uPA concentration is not the limiting factor in the regulation of integrin α6 cleavage. The ability to block α6 integrin cleavage by extracellular engagement of the receptor points to the possibility that lateral membrane associations with surface expressed proteins like tetraspanins (51
) and urokinase receptor (uPAR) (53
), could influence uPA mediated integrin cleavage in a physiologically relevant manner. Current work investigating this possibility may reveal other potential cell surface targets for disruption of extravasation of prostate cancer to bone.
The amount of α6p in vitro is not prognostic for bone metastasis in vivo. However, the inability to produce α6p will significantly hinder bone metastasis development in mice. It will be important to have a method to detect cleaved α6p in vivo to determine if the cleaved integrin could serve as a prognostic factor. We are currently developing an ELISA to determine if the released extracellular fragment of α6 integrin is detectable in blood. We consider it likely that α6 cleavage may add to the multiple molecular features required to reliably detect tumor cells with metastatic potential.