Tumor cells resistant to a microtubule-depolymerizing hemiasterlin analogue, HTI-286, have mutations in alpha- or beta-tubulin and increased microtubule stability

摘要

Hemiasterlins are sponge-derived tripeptides that inhibit cell growth by depolymerizing existing microtubules and inhibiting microtubule assembly. Since hemiasterlins are poor substrates for P-glycoprotein, they are attractive candidates for cancer therapy and have been undergoing clinical trials. The basis of resistance to a synthetic analogue of hemiasterlin, HTI-286 (HTI), was examined in cell populations derived from ovarian carcinoma (A2780/1A9) cells selected in HTI-286. 1A9-HTI-resistant cells (1A9-HTIR series) were 57-89-fold resistant to HTI. Cross-resistance (3-186-fold) was observed to other tubulin depolymerizing drugs, with collateral sensitivity (2-14-fold) to tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble tubulin in 1A9 parental and 1A9-HTIR Cells corroborated the HTI cytotoxicity data. At 22 degreesC or 37 degreesC, in the absence of any drug, the percentage of polymerized microtubules for each of the 1A9-HTIR populations was greater than that in the 1A9 parental cells, consistent with more stable microtubules. Furthermore, microtubules in the 1A9-HTIR populations were also more resistant to depolymerization at 4 degreesC and had more acetylated and detyrosinated (Glu-tubulin) alpha-tubulin, all characteristic of more stable microtubules. The 1A9-HTIR cell populations exhibited either a single nucleotide change in the M40 beta-tubulin isotype, S172A, or in two cell populations where no beta-tubulin mutation was detected, mutations in the Kalpha-1 alpha-tubulin isotype, S165P and R221H in one resistant cell population and 1384V in another. Unlike reports of mutations resulting in reduced drug affinity, the experimental data and location of mutations are consistent with resistance to HTI-286 mediated by microtubule-stabilizing mutations in beta- or alpha-tubulin.