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Title:Investigating the role of zoledronate for inhibition of osteosarcoma metastasis
Author(s):Shor, Sharon
Advisor(s):Fan, Timothy M.
Department / Program:Vet Clinical Medicine
Discipline:VMS-Veterinary Clinical Medcne
Degree Granting Institution:University of Illinois at Urbana-Champaign
Rho GTPases
Abstract:Osteosarcoma (OS) is the most common primary bone tumor in dogs and it accounts for nearly 10% of all diagnosed canine cancers. Despite aggressive multimodality therapy, the mortality rate for canine OS exceeds 90% from the development of distant metastases to lungs and other visceral organs. Given the high metastatic rate and associated mortality, there is a compelling rationale to explore novel therapeutic approaches that may slow the progression or inhibit the development of distant metastases. Successful metastasis requires cytoskeletal morphogenic changes of tumor cells, allowing for efficient and directional cell migration. Cell motility requires coordinated cytoskeletal rearrangement largely controlled by Rho GTPase proteins (RhoA, RhoC, Rac-1 and Cdc42) and effector pathways (Rho-kinase; ROCK). Given their pivotal role in directional cell migration, perturbing the function of Rho GTPases is considered a viable anti-metastasis strategy. Disruption of Rho GTPase functions can be achieved by interfering with protein prenylation, a pivotal post-translational modification necessary for Rho GTPase subcellular localization and protein-protein interactions. Zoledronate (ZOL) is a potent antiresorptive agent that definitively reduces the frequency and severity of malignant skeletal complications in people. As an antiresorptive agent, ZOL exerts its biologic effects on osteoclasts by inhibiting the mevalonate pathway which is necessary for protein prenylation. Although a large body of scientific information exists characterizing its anticancer effects within the local bone tumor microenvironment, ZOL’s potential antimetastatic properties remain less well defined. Despite several different murine tumor models describing ZOL’s capacity to reduce visceral metastases, a unifying molecular mechanism for these observations has yet to be reported. We hypothesized that ZOL will impair Rho GTPase prenylation and activity, with subsequent perturbation of the cell cytoskeleton and motility in OS cells, and serve as the molecular basis for ZOL’s observed antimetastatic effects. The effects of ZOL on K7M2 murine OS cells were investigated by characterizing perturbations in 1) membrane and cytosolic Rho GTPases and phosphorylated myosin light chain 2 (p-MLC2) by western blot analysis; 2) cytoskeletal structure with confocal fluorescence microscopy; 3) migration by qualitative and quantitative analyses; and 4) metastases development ex vivo and in vivo using an experimental lung metastasis model. The results of this study were that exposure to ZOL decreased membrane-bound Rho GTPases with concurrent accumulation of unprenylated Rho GTPases within the cytosol, reduced expression of p-MLC2, attenuated the expression of focal adhesions and filamentous actin, and inhibited in vitro cell migration. Ex vivo single cell videomicroscopy also showed that ZOL can significantly decrease early pulmonary nodule formation. Lastly, ZOL treatment induced a significant increase in survival of mice from OS lung nodules; however, this improvement was modest and was achieved only with pre-treatment of tumor cells in vitro. Findings from this investigation document the prenylation-dependent effects of ZOL on Rho GTPases and their impact on OS cell cytoskeletal organization, migration, survival, proliferation, and metastases formation. These observations provide a foundational basis for evaluating adjuvant ZOL for managing micrometastatic disease in dogs with spontaneously-arising OS.
Issue Date:2013-05-24
Rights Information:Copyright 2013 Sharon Shor
Date Available in IDEALS:2013-05-24
Date Deposited:2013-05

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