P1-02-02: Zoledronic Acid Reverses the Epithelial-Mesenchymal Transition While Inhibiting the Tumor Initiating Cell Population of Highly Tumorigenic Breast Cancer Cell Lines.
Authors: AJ Schech, RA Gilani, AA Kazi, AH Brodie
Journal: Cancer Research (2011)

Abstract Breast cancer remains the second leading cause of cancer related death amongst women in the United States. This is largely due to metastasis of cancer cells from the primary tumor to other parts of the body, and to the putative cancer stem cell population. Both are driven by the epithelial-mesenchymal transition (EMT), a cellular process whereby cancer cells of epithelial origin lose their epithelial characteristics and acquire a mesenchymal phenotype. Cells which undergo EMT tend to be motile and invasive, and therefore can metastasize to other parts of the body. EMT has also been implicated in the generation of cells expressing the cancer stem cell phenotype. As metastatic disease and the cancer stem cell are difficult to eliminate, more effective therapy is required. Zoledronic acid, originally indicated for use in the treatment of osteoporosis, has been reported to inhibit the growth of breast cancer cells. The mechanism of this effect however has yet to be determined. In preliminary data studies, treatment with zoledronic acid was found to reduce activation of nuclear factor kappa-b, an established regulator of EMT. These findings suggest that treatment with zoledronic acid may reverse EMT in breast cancer cells, driving them to express a more epithelial phenotype. To test this hypothesis, the highly metastatic, triple negative breast cancer cell lines MDA-MB-231 and Hs578t, which largely express mesenchymal proteins, were used to measure cell viability and changes in protein and mRNA expression following treatment with zoledronic acid. Dose response analysis for inhibition of cell viability showed an IC50 of approximately 2μM in the Hs578t and 6 μM in the MDA-MB-231 cell line. Zoledronic acid treated cells displayed a decreased mesenchymal phenotype, as evidenced by reduced expression of mesenchymal markers N-cadherin (75% reduction, p&amp;lt;0.076; 65% reduction, p&amp;lt;0.005) and TWIST (67% reduction, p&amp;lt;0.08; 64% reduction, p&amp;lt;0.009) in MDA-MB-231 and Hs578t cells, respectively. This was accompanied by a subsequent increase in epithelial phenotype as evidenced by increased expression of epithelial marker E-cadherin (223% increase, p&amp;lt;0.006) in MDA-MB-231 cells. To further elucidate the effects on the mesenchymal and epithelial phenotypes of these cells, surface expression of CD24 and CD44 was measured by flow cytometry. While vehicle treated samples of both cell lines stained positive for CD44 and negative for CD24, zoledronic acid treatment decreased CD44 expression. As both increased the ratio of surface expression of CD44high/CD24low and mammosphere formation are characteristics of the breast cancer stem cell, effects of zoledronic acid on this subpopulation in Hs578t cells were determined. Cells pretreated with zoledronic acid were seeded under mammosphere conditions and allowed to propagate for 7 days. Zoledronic acid treated cells formed significantly fewer mammospheres (86% reduction, p&amp;lt;0.041), while the ones that formed were smaller in size. These findings suggest that zoledronic acid is able to reverse the epithelial mesenchymal transition, which may reduce the tumor initiating capacity of highly metastatic cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-02.