The effects of the histone deacetylase inhibitor, LBH589, on breast cancer in bone and on physiological bone remodelling.

Abstract

Histone Deacetylase Inhibitors (HDIs) are emerging as an exciting new class of potential anticancer agents for the treatment of solid and haematological malignancies. Despite the infancy of the field, there is now an impressive body of data describing the ability of these molecules to modulate a wide variety of cellular functions, including cell differentiation, cell cycle progression, apoptosis, cytoskeletal modifications, and angiogenesis. Over the past few years, results obtained from clinical trials and pre-clinical animal experiments demonstrate the ability of HDIs to selectively kill cancer cells with limited or no toxicity to normal tissues and organs. Amongst the HDIs now in clinical trials, SAHA (Vorinostat) is the first of its class to get approval from the U.S.A. Food and Drug Administration (FDA) for the treatment of cutaneous manifestations in patients with cutaneous T-cell lymphoma (CTCL). Although a number of early-phase clinical trials using different HDIs have demonstrated promising antitumour responses for a variety of cancer types, the effect of HDIs on skeletal malignancies, or the consequence of HDI treatment on the bone microenvironment, and in the context of osteoclast and osteoblast function, has not been reported. The studies undertaken in this thesis aimed to: 1). Investigate the anticancer efficacy of the HDI LBH589 in animal models of primary breast cancer, and on bone destruction caused by breast cancer growth in bone. 2). Investigate the effects by which LBH589 regulates normal bone metabolism in the context of osteoclast and osteoblast function both in vitro and in vivo. In vitro, LBH589 treatment resulted in a dose and time-dependent increase in apoptosis in a panel of well established breast cancer cell lines. This was associated with the processing and activation of caspases-8, and -3, concomitant with the activation of the Bcl2 family protein, Bid and cleavage of the apoptosis target protein, PARP. LBH589 treatment leads to a marked increase in acetylated histone-H3 and induction of the p21 protein. The highly aggressive MDA-MB231-TXSA human breast cancer cell line was used to evaluate the antitumour activity of LBH589 in murine models of breast cancer development and progression at both the orthotopic site and in bone. MDA-MB231-TXSA cells form aggressive, rapidly growing tumours when injected into the orthotopic site of the mammary fat pad of nude mice, and stimulate the formation of osteolytic lesions when injected into the tibial marrow cavity of nude mice. MDA-MB231-TXSA breast cancer cells were tagged with a triple reporter gene construct, which allows real-time monitoring of tumour growth in live animals. Tumour progression with and without LBH589 treatment was monitored in live animals, and in real-time using bioluminescence imaging (BLI). The development of breast cancer-induced osteolysis was measured using high resolution μ-CT and histology. In vivo, LBH589 had no effect on tumour growth in the mammary fat pad or in bone, as demonstrated by BLI and histology. However, high resolution μ-CT analyses of the tibiae demonstrated significant protection from breast cancer-induced osteolysis with LBH589 treatment, associated with a marked reduction in the number of TRAP⁺ osteoclasts lining the trabecular bone surface. Furthermore, the bone volume of the contralateral, non-tumour bearing tibiae was significantly increased with LBH589 treatment compared to the vehicle-treated group of animals. This effect was also seen in the tibiae of mice bearing mammary tumours, suggesting potential anabolic actions of LBH589.