Pathogenic Role of Connective Tissue Growth Factor (CTGF/CCN2) in Osteolytic Metastasis of Breast Cancer

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Abstract
The role of CTGF/CCN2 in osteolytic metastasis by breast cancer cells and its mechanism of action were studied. Osteolytic metastasis accompanied by CCN2 and PTHrP overproduction was efficiently inhibited by an anti‐CCN2 antibody. Furthermore, we found that CCN2 was induced by PTHrP through PKA‐, PKC‐, and ERK‐mediated pathways therein. Introduction: Connective tissue growth factor (CTGF/CCN2) is a mediator of local angiogenesis induced by breast cancer, but its role in osteolytic metastasis has not been evaluated. PTH‐related peptide (PTHrP) is another critical factor in the development of the osteolytic metastasis. Using both in vivo and in vitro approaches, we studied whether/how neutralization of CCN2 prevented bone metastasis and how PTHrP signaling is related. Materials and Methods: A mouse model of bone metastasis by human breast cancer cell line MDA231 was treated with a CCN2‐neutralizing antibody, and osteolytic bone metastases were assessed on radiographs and immunohistochemistry. Ccn2 gene expression and transcription were examined by Northern blot and luciferase analysis. Immunoblot analysis and kinase inhibitors were used to identify the signaling pathways implicated. Anti‐angiogenic/osteoclastogenic effects of ccn2 downregulation were also evaluated. Results: Treatment of mice with a CCN2‐neutralizing antibody greatly decreased osteolytic bone metastasis, microvasculature, and osteoclasts involved. The antibody also suppressed the growth of subcutaneous tumor in vivo and proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. Downregulation of ccn2 also repressed osteoclastogenesis. CCN2 expression was specifically observed in cancer cells producing PTHrP and type I PTH/PTHrP receptor (PTH1R) invaded the bone marrow, and PTHrP strongly upregulated ccn2 in MDA231 cells in vitro. Activation of protein kinase C (PKC) and protein kinase A (PKA) was necessary and sufficient for the stimulation of ccn2 by PTHrP. Indeed, inhibition of the extracellular signal‐regulated kinase (ERK1/2), PKC, or PKA by specific inhibitors counteracted the stimulation of ccn2 expression. Incubation of MDA231 cells with PTHrP induced the activation of ERK1/2. Consistent with these findings, inhibition of PKC prevented PTHrP‐induced ERK1/2 activation, whereas 12‐O‐tetradecanoylphorbol13‐acetate (TPA), a stimulator of PKC, upregulated it. Conclusions: CCN2 was critically involved in osteolytic metastasis and was induced by PKA‐ and PKC‐dependent activation of ERK1/2 signaling by PTHrP. Thus, CCN2 may be a new molecular target for anti‐osteolytic therapy to shut off the PTHrP–CCN2 signaling pathway.