β-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 B-tubulin at amino acids 197 (DB197N) and 350 (KB350N) in 2ME2R cells. Tandem mass spectrometry confirmed the presence of both wild-type and the mutant B-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. (Cancer Res 2005; 65(20): 9406-14)

Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

“…A previous study of breast cancer cells resistant to 2ME2 identified mutations in class I h-tubulin that resulted in amino acid substitutions, D197N and K350N mutations (15). Of interest, the 2ME2-resistant leukemia cells selected in the current study acquired four heterozygous mutations in class I h-tubulin including, D197N and K350N.…”

“…5), the 2ME2-resistant cells were hypersensitive to colchicine. In contrast, 2ME2-resistant breast cancer cells that also acquired the D197N mutation were cross-resistant to colchicine and Vinca alkaloids (15). The differences therefore could arise from the carboxamide of asparagine in the form of additional hydrogen-bond interactions.…”

“…Additionally, the change caused by the S25N mutation is subtler, as both serine and asparagine are polar and uncharged residues. Gokmen-Polar et al (15) suggested that the amino acid substitution at position 197 in h-tubulin might alter the salt bond with Arg 156 , which anchors the COOH-terminal at the end of helix 4, and results in propagation of a conformational change within h-tubulin. Other than that, little is known about this mutation, except that the D197N mutation site is not within any known drug-binding site on h-tubulin.…”

“…Mutations in the human class I h-tubulin gene, HM40, have been reported previously in cells resistant to microtubule-destabilizing agents that exhibited increases in tubulin polymer levels (15,18). Gene sequencing clearly identified four distinct heterozygous class I h-tubulin gene (HM40) point mutations in 2ME2-resistant cells that were not present in parental CCRF-CEM cells ( Supplementary Fig.…”

“…13,14). Breast cancer cells selected for resistance to 2ME2 acquired mutations in the human class I (HM40) h-tubulin gene, yet they were only minimally resistant to colchicine and related agents (15). To date, this has been the only study of 2ME2-resistant cancer cells.…”