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ß-Tubulin Mutations Are Associated with Resistance to 2-Methoxyestradiol in MDA-MB-435 Cancer Cells

Departments of ¹ Medicine, ² Biochemistry and Molecular Biology, and ³ Pathology and Laboratory Medicine, Indiana University School of Medicine, and ⁴ Indiana Centers for Applied Protein Sciences, Indianapolis, Indiana; ⁵ Winship Cancer Institute, Emory University, Atlanta, Georgia; ⁶ Medical Oncology Center, Institut Català d'Oncologia, Hospital Universitari Germans Trias i Pujol, Barcelona, Spain; and ⁷ EntreMed, Inc., Rockville, Maryland

Requests for reprints: Yesim Gökmen-Polar, Indiana Cancer Research Institute, 1044 West Walnut Street, R4-202 Indianapolis, IN 46202-5254. Phone: 317-274-3605; Fax: 317-274-0396; E-mail: ypolar{at}iupui.edu.

2-Methoxyestradiol is an estradiol metabolite with significant antiproliferative and antiangiogenic activity independent of estrogen receptor status. To identify a molecular basis for acquired 2-methoxyestradiol resistance, we generated a stable 2-methoxyestradiol-resistant (2ME2R) MDA-MB-435 human cancer cell line by stepwise exposure to increasing 2-methoxyestradiol concentrations. 2ME2R cells maintained in the presence of the drug and W435 cells maintained in the absence of the drug showed 32.34- to 40.07-fold resistance to 2-methoxyestradiol. Cross-resistance was observed to Vinca alkaloids, including vincristine, vinorelbine, and vinblastine (4.29- to 6.40-fold), but minimal resistance was seen to colchicine-binding agents including colchicine, colcemid, and AVE8062A (1.72- to 2.86-fold). No resistance was observed to paclitaxel and epothilone B, polymerizing agents (0.89- to 1.14-fold). Genomic sequencing identified two different heterozygous point mutations in the class I (M40) isotype of ß-tubulin at amino acids 197 (Dß197N) and 350 (Kß350N) in 2ME2R cells. Tandem mass spectrometry confirmed the presence of both wild-type and the mutant ß-tubulin in 2ME2R cells at the protein level. Consistently, treatment of parental P435 cells with 2-methoxyestradiol resulted in a dose-dependent depolymerization of microtubules, whereas 2ME2R cells remained unaffected. In contrast, paclitaxel affected both cell lines. In the absence of 2-methoxyestradiol, 2ME2R cells were characterized by an elevated level of detyrosination. Upon 2-methoxyestradiol treatment, levels of acetylated and detyrosinated tubulins decreased in P435 cells, while remaining constant in 2ME2R cells. These results, together with our structure-based modeling, show a tight correlation between the antitubulin and antiproliferative effects of 2-methoxyestradiol, consistent with acquired tubulin mutations contributing to 2-methoxyestradiol resistance.

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