title: beta-Tubulin mutations are associated with resistance to 2-methoxyestradiol in MDA-MB-435 cancer cells.
abstract: 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 beta-tubulin at amino acids 197 (Dbeta197N) and 350 (Kbeta350N) in 2ME2R cells. Tandem mass spectrometry confirmed the presence of both wild-type and the mutant beta-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.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, and Indiana Centers for Applied Protein Sciences, Indianapolis, IN 46202-5254, USA. ypolar@iupui.edu
authors.forename: Yesim
authors.initials: Y
authors.lastname: Gökmen-Polar
authors.forename: Daniel
authors.initials: D
authors.lastname: Escuin
authors.forename: Chad D
authors.initials: CD
authors.lastname: Walls
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Soule
authors.forename: Yuefang
authors.initials: Y
authors.lastname: Wang
authors.forename: Kerry L
authors.initials: KL
authors.lastname: Sanders
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Lavallee
authors.forename: Mu
authors.initials: M
authors.lastname: Wang
authors.forename: Brian D
authors.initials: BD
authors.lastname: Guenther
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Giannakakou
authors.forename: George W
authors.initials: GW
authors.lastname: Sledge
mesh_terms: D000077584:2-Methoxyestradiol; D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D020033:Protein Isoforms; D013329:Structure-Activity Relationship; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*
chemical_list: D004273:DNA, Neoplasm; D020033:Protein Isoforms; D014404:Tubulin; D050257:Tubulin Modulators; D004958:Estradiol; D000077584:2-Methoxyestradiol