Dickkopf-1 is regulated by the mevalonate pathway in breast cancer.

INTRODUCTION: Amino-bisphosphonates and statins inhibit the mevalonate pathway, and may exert anti-tumor effects. The Wnt inhibitor dickkopf-1 (DKK-1) promotes osteolytic bone lesions by inhibiting osteoblast functions and has been implicated as an adverse marker in multiple cancers. We assessed the effects of mevalonate pathway inhibition on DKK-1 expression in osteotropic breast cancer. METHODS: Regulation of DKK-1 by bisphosphonates and statins was assessed in human breast cancer cell lines, and the role of the mevalonate pathway and downstream targets was analyzed. Moreover, the potential of breast cancer cells to modulate osteoblastogenesis via DKK-1 was studied in mC2C12 cells. Clinical relevance was validated by analyzing DKK-1 expression in the tissue and serum of women with breast cancer exposed to bisphosphonates. RESULTS: DKK-1 was highly expressed in receptor-negative breast cancer cell lines. Patients with receptor-negative tumors displayed elevated levels of DKK-1 at the tissue and serum level compared to healthy controls. Zoledronic acid and atorvastatin potently suppressed DKK-1 in vitro by inhibiting geranylgeranylation of CDC42 and Rho. Regulation of DKK-1 was strongest in osteolytic breast cancer cell lines with abundant DKK-1 expression. Suppression of DKK-1 inhibited the ability of breast cancer cells to block WNT3A-induced production of alkaline phosphates and bone-protective osteoprotegerin in preosteoblastic C2C12 cells. In line with the in vitro data, treatment of breast cancer patients with zoledronic acid decreased DKK-1 levels by a mean of 60% after 12 months of treatment. CONCLUSION: DKK-1 is a novel target of the mevalonate pathway that is suppressed by zoledronic acid and atorvastatin in breast cancer.

Dickkopf-1 as a mediator and novel target in malignant bone disease.

Bone metastases are a common problem of many malignancies, including myeloma, breast and prostate cancer. The Wnt inhibitor Dickkopf-1 has been shown to be involved in the process of bone lesions by impairing osteoblast activity. This review will focus on the role of Dickkopf-1 as a mediator of malignant bone disease and discuss its potential as a novel therapeutic target.

Targeting syndecan-1 in breast cancer inhibits osteoclast functions through up-regulation of osteoprotegerin.

BACKGROUND: Breast cancer often metastasizes into bone and leads to osteolytic lesions. The underlying mechanisms, however, are complex and not fully understood. Syndecan-1 is a proteoglycan that has various functions relevant for tumor progression including cell-cell communication and cell-matrix interactions. Moreover, its two glycosaminoglycan-binding sites suggest that it may interfere with glycoproteins such as osteoprotegerin, a potent inhibitor of osteoclastogenesis. Thus, we hypothesize that tumor-derived syndecan-1 alters osteoclast biology by modulating osteoprotegerin. METHODS: Syndecan-1 expression was down-regulated via siRNA and the cell fate of the breast cancer cell lines MCF-7, T-47D, and MDA-MB-231 was investigated. Furthermore, we determined the regulation of syndecan-1 by dexamethasone, a commonly used antiemetic in breast cancer therapy. Additionally, we analyzed the genesis and activity of osteoclasts in indirect co-culture experiments using supernatants from MCF-7 cells with deficient and sufficient levels of syndecan-1. RESULTS: Dexamethasone time- and dose-dependently increased syndecan-1 expression up to 4-fold but did not alter cell behavior. Syndecan-1 up-regulation did not affect the survival or migration of breast cancer cells. Depletion of syndecan-1 using siRNA led to decreased vitality of progesterone receptor-positive cell lines. In MCF-7 cells osteoprotegerin production was up-regulated 2.5-fold after syndecan-1 knock-down. The culture of osteoclast precursors with the supernatant of MCF-7 cells with reduced syndecan-1 levels suppressed osteoclast formation and activity by 21% and 23%, respectively. Adding neutralizing antibodies to osteoprotegerin to the breast cancer supernatants reversed osteoclastogenesis. CONCLUSION: Thus, we identified tumor-derived syndecan-1 as a novel positive regulator of osteoclastogenesis and new player in the tumor-bone dialog.

Zoledronic acid and atorvastatin inhibit αvß3-mediated adhesion of breast cancer cells.

Bone metastases represent common long term complications of patients with breast cancer. Zoledronic acid, an amino-bisphosphonate and mevalonate pathway inhibitor, is an established agent for the treatment of bone metastases. Direct antitumor effects of zoledronic acid have been proposed in breast cancer. Statins are another group of mevalonate pathway inhibitors that have been repeatedly discussed for potential anti-tumor activity. In this study, we tested the hypothesis, whether these agents regulate adhesion of breast cancer cells to extracellular matrix components. Treatment of breast cancer cells with zoledronic acid and atorvastatin, significantly impaired MDA-MB-231 breast cancer cell adhesion on the αvß3 ligands gelatin and vitronectin, but had no effect on collagen type 1 (α2ß1-ligand) and fibronectin (α5ß1-ligand). Anti-adhesive effects of zoledronic acid were fully reversed by geranylgeranyl pyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Furthermore, effects of zoledronic acid and atorvastatin were mimicked by a specific inhibitor of geranylgeranylation GGTI-298. Functional (using integrin array) and quantitative (using FACS) integrin analyses on MDA-231 cells following zoledronic acid exposure revealed decreased levels of αv and αvß3 expression. In addition to its effects on integrin mediated adhesion of breast cancer cells, the presence of zoledronic acid caused pronounced morphological changes in MDA-231 cells as seen by F-actin and vinculin rearrangement. Furthermore, phosphorylation of the focal adhesion kinase was inhibited by zoledronic acid. In both cases, changes were fully reversed by GGPP. These results emphasize the role of mevalonate pathway mediated impairment of geranylgeranylation in the anti-adhesive effects of zoledronic acid in breast cancer cells.

Regulation of VEGF by mevalonate pathway inhibition in breast cancer.

Aminobisphosphonates are used for the treatment of benign and malignant bone disorders. As inhibitors of the mevalonate pathway they exert direct anti-tumor effects in vitro and in preclinical models of bone metastases. Bisphosphonates are thought to have an anti-angiogenic activity as decreased levels of VEGF have been reported in some, although not all patients, following treatment with bisphosphonates. Direct effects of bisphosphonates on tumor derived VEGF have not been examined in detail. We therefore investigated VEGF expression in breast cancer cell lines following mevalonate pathway inhibition. Treatment of cell lines with increasing doses of zoledronic acid and atorvastatin resulted in increased levels of VEGF production. Similar results were seen with the geranylgeranyltransferase I inhibitor GGTI-298. The induction of VEGF was reversed by the supplementation of geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate indicating that this effect is mediated by inhibited geranylgeranylation. Previous reports have reported decreased VEGF levels in patients following BP treatment in vivo. We assessed VEGF levels in patients with non-metastatic breast cancer following repeated treatment with zoledronic acid. In contrast to our in vitro findings, VEGF serum levels decreased in all patients after 6-9 months of treatment (by an average of 41%) as assessed in a small pilot trial. These results indicate that tissues other than breast tumors contribute to the serum pool of circulating VEGF and may be responsible for the observed VEGF decreases. The increases of VEGF in the cancer cells may provide a rationale for the combined treatment with VEGF inhibitors.