Elucidating the Molecular Mechanisms of Zoledronic Acid in Breast Cancer

Abstract

Zoledronic acid (ZA) has been shown to improve disease free survival of postmenopausal breast cancer patients when used alone or in combination with endocrine therapy. These effects have been corroborated by many preclinical studies. Furthermore, ZA has been shown to reduce metastatic disease in patients, which has also been reproduced in the preclinical setting. However, the molecular mechanisms explicating these pharmacological effects of ZA remain unknown. As such, the objective of this dissertation was to elucidate the molecular mechanisms of ZA on cell viability, the epithelial-mesenchymal transition (EMT), and the tumor initiating cell population. Experiments investigating the combination of ZA and aromatase inhibitor letrozole showed that the combination inhibited cell viability in an additive manner. Further examination into the mechanism of this combination effect unveiled the inhibitory effects of ZA on the aromatase enzyme. Immunoprecipitation experiments confirmed that ZA inhibited serine phosphorylation of the enzyme important for its enzymatic activity. Rescue experiments showed that estradiol treatment could prevent loss of cell viability after ZA treatment, suggesting that ZA's inhibition of the aromatase enzyme contributes to the anti-tumor mechanism of ZA. Other mechanisms of ZA's anti-tumor effect were also investigated. Although treatment with ZA inhibited the expression and enzymatic activity of cyclooxygenase-2, as well as the transcriptional activation of nuclear factor kappa B, neither were involved in ZA's inhibition of cell viability. However, treatment with ZA modulated EMT in triple negative breast cancer cell lines, as evidenced by decreased mesenchymal cellular morphology, decreased mesenchymal mRNA and protein expression. This was accompanied by increased E-cadherin protein, mRNA, and CD24 cell surface expression. Furthermore, this reversal in EMT was attributed to inhibition of nuclear factor kappa B transcriptional control. Additionally, ZA treatment decreased the self renewal capability of triple negative breast cancer cells, as evidenced by decreased self renewal protein expression. Furthermore, treatment with ZA both prevented mammosphere formation and decreased the number of existing mammospheres. These results suggest that treatment with ZA can prevent the acquisition of an invasive phenotype as well as the capacity to repopulate a tumor at a secondary site, thereby eliminating a cancer cell's ability to metastasize.