Determination of the Biological Significances of Platelet Factor 4 (PF4), a Tumor Suppressor Gene Encoding an Angiogenesis Inhibitor in Multiple Myeloma

Multiple myeloma (MM) is an incurable hematological malignancy characterized by accumulation of clonal plasma cells in bone marrow (BM). The development and progression of MM is a complex multistep tumorigenic event involving both genetic and epigenetic changes in the tumor cell as well as the support by the BM microenvironment. It has been well established that the physical interaction of MM ceils with the BM milieu are crucial for MM pathogenesis, MM cell growth, survival, migration and drug resistance. Platelet factor 4 (PF4), a potent antiangiogenic chemokine, not only inhibits endothelial cell proliferation and migration in vitro but also solid tumor growth in vivo. Our group previously demonstrated loss of PF4 expression in patient MM samples and MM cell lines due to concurrent allelic loss and DNA hypermethylation. In this study, we characterized the effects of PF4 on MM cells and angiogenesis in the BM milieu both in vitro and in vivo and elucidated the mechanism ofPF4 effects on MM.;To characterize the effects of PF4 on MM cells in vitro, assays on cell growth, cell cycle arrest and apoptosis were performed and we found that PF4 inhibited growth and induced apoptosis in both MM cell lines and MM cells from patients. The proapoptotic activity of PF4 is associated with activation of caspase-3 and poly (ADP) ribose polymerase (PARP). We also investigated the effects of PF4 on angiogenesis in MM using endothelial cells isolated from patient's BM aspirates (MMECs). Our results showed that PF4 suppressed MMECs growth and tube formation on matrigel in a dose-dependent manner.;Given the ability of PF4 to suppress MM cell growth and angiogenesis in vitro, we evaluated its tumor suppressive function in vivo. In human subcutaneously matrigel xenograft mouse model, tail vein injection of 200ng PF4 significantly reduced MM tumor growth and prolonged survival. We next used the SCID-rab mouse model which recapitulates the human BM milieu in vivo. In this model, MM cells were directly injected into the rabbit bone which was subcutaneously implanted into the NOD-SCID mice. Two weeks after injection, SCID mice were treated with various dose of PF4 (20 or 200ng per injection, three times per week) or PBS by tail vein injection. ELISA assay for hig (lambda) showed that tumor growth in 200ng PF4-treated mice was markedly reduced by 58% compared with the control group, which was further confirmed by immunohistochemistry analysis of CD138 staining on rabbit bone section. Consistent with the in vitro results, induction of apoptosis in MM cells and inhibition of angiogenesis by PF4 could also be demonstrated in vivo, as evidenced by the findings on ki67, Cleaved caspase-3, CD31 and VEGF staining on rabbit bone sections from treated versus control mice. Our findings thus confirmed that PF4 is a novel tumor suppressor in MM.;However, the molecular mechanism of how PF4 inhibits MM tumorigenesis is still unclear. To identify the signal pathway PF4 involved in MM, Protein/DNA array was performed. We found that PF4 regulated several transcription factors including STAT3 in U266 cells. EMSA and luciferase reporter assay further confirmed that PF4 suppressed STAT3 DNA binding and transcriptional activity. So it is possible that PF4 mediates its tumor suppressive function, through suppressing STAT3 pathway in MM cells. We further found that pre-treatment of PF4 blocked both constitutive and interleukin-6-induced STAT3 activation in a time-dependent manner in human MM cells. PF4 could also down-regulate the STAT3-regulated gene products including Mcl-1, Survivin and vascular endothelial growth factor (VEGF). Moreover, enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis. In SCID-rab mouse model, we also found that PF4 inhibited STAT3 nuclear translocation by immunostaining of rabbit bone sections. When examined further, we found that PF4 induced the expression of one of the STAT3 inhibitor SOCS3, and gene silencing of SOCS3 by small interfering RNA abolished the ability of PF4 to inhibit STAT3 activation, suggesting a critical role of SOCS3 in the action of PF4. Our findings therefore suggest that by inducing SOCS3 expression, PF4 abrogates STAT3 activity, thus induces tumor growth inhibition and anti-angiogenesis.;Together, these novel studies have shown that PF4 is an important regulator of MM tumorigenesis. By abrogating STAT3 signaling it targets cell growth, induces apoptosis, suppresses angiogenesis both in vitro and in vivo in MM. These scientific observations provide the framework for clinical studies of this chemokine, as a novel drug for treatment of MM to improve patient outcome in MM.