tubacin and Cell-Transformation--Neoplastic

Enhanced microtubule acetylation has been identified as a negative prognostic indicator in breast cancer. We reported previously that primary cultured human mammary epithelial cells manifest breast cancer-related aneuploidization via the activation of severing protein katanin-like (KL)1 when tau is deficient. To address in this current study whether microtubule hyperacetylation is involved in breast carcinogenesis through mitosis, the effects of tubacin on human mammary epithelial cells were tested using immunofluorescence techniques. Tau-knockdown cells showed enhancement of KL1-dependent events, chromosome-bridging and micronucleation in response to tubacin. These enhancements were suppressed by further expression of an acetylation-deficient tubulin mutant. Consistently, using a rat fibroblast-based microtubule sensitivity test, it was confirmed that KL1 also shows enhanced activity in response to microtubule hyperacetylation as well as katanin. It was further observed in rat fibroblasts that exogenously expressed KL1 results in more micronucleation under microtubule hyperacetylation conditions. These data suggest that microtubule acetylation upregulates KL1 and induces more aneuploidy if tau is deficient. It is thus plausible that microtubule hyperacetylation promotes tumor progression by enhancing microtubule sensitivity to KL1, thereby disrupting spindle microtubules and this process could be reversed by the microtubule-binding and microtubule protective octapeptide NAPVSIPQ (NAP) which recruits tau to the microtubules.

Topics: Acetylation; Anilides; Animals; Cell Line; Cell Transformation, Neoplastic; Epithelial Cells; Fibroblasts; Gene Expression Regulation, Neoplastic; Histone Deacetylase 6; Humans; Hydroxamic Acids; Katanin; Mammary Glands, Human; Microtubules; Mitosis; Plasmids; Rats; RNA, Small Interfering; Signal Transduction; Spindle Apparatus; tau Proteins; Transfection

Histone deacetylase 6 (HDAC6) is structurally and functionally unique among the 11 human zinc-dependent histone deacetylases. Here we show that chemical inhibition with the HDAC6-selective inhibitor tubacin significantly enhances cell death induced by the topoisomerase II inhibitors etoposide and doxorubicin and the pan-HDAC inhibitor SAHA (vorinostat) in transformed cells (LNCaP, MCF-7), an effect not observed in normal cells (human foreskin fibroblast cells). The inactive analogue of tubacin, nil-tubacin, does not sensitize transformed cells to these anticancer agents. Further, we show that down-regulation of HDAC6 expression by shRNA in LNCaP cells enhances cell death induced by etoposide, doxorubicin, and SAHA. Tubacin in combination with SAHA or etoposide is more potent than either drug alone in activating the intrinsic apoptotic pathway in transformed cells, as evidenced by an increase in PARP cleavage and partial inhibition of this effect by the pan-caspase inhibitor Z-VAD-fmk. HDAC6 inhibition with tubacin induces the accumulation of γH2AX, an early marker of DNA double-strand breaks. Tubacin enhances DNA damage induced by etoposide or SAHA as indicated by increased accumulation of γH2AX and activation of the checkpoint kinase Chk2. Tubacin induces the expression of DDIT3 (CHOP/GADD153), a transcription factor up-regulated in response to cellular stress. DDIT3 induction is further increased when tubacin is combined with SAHA. These findings point to mechanisms by which HDAC6-selective inhibition can enhance the efficacy of certain anti-cancer agents in transformed cells.

Topics: Anilides; Antineoplastic Agents; Caspases; Cell Death; Cell Line, Tumor; Cell Transformation, Neoplastic; Checkpoint Kinase 2; DNA Damage; DNA Replication; Down-Regulation; Doxorubicin; Drug Screening Assays, Antitumor; Drug Synergism; Etoposide; G1 Phase; Gene Expression Regulation, Neoplastic; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Neoplasm Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Topoisomerase II Inhibitors; Up-Regulation

Histone deacetylase (HDAC) 6 is a subtype of the HDAC family; it deacetylates alpha-tubulin and increases cell motility. Here, we investigate the impact of an alteration of HDAC6 expression in estrogen receptor alpha (ER)-positive breast cancer MCF-7 cells, as we identified that HDAC6 is a novel estrogen-regulated gene. MCF-7 treated with estradiol showed increased expression of HDAC6 mRNA and protein and a four-fold increase in cell motility in a migration assay. Cell motility was increased to the same degree by stably transfecting the HDAC6 expression vector into MCF-7 cells. In both cases, the cells changed in appearance from their original round shape to an axon-extended shape, like a neuronal cell. This HDAC6 accumulation caused the deacetylation of alpha-tubulin. Either the selective estrogen receptor modulator tamoxifen (TAM) or the pure antiestrogen ICI 182,780 prevented estradiol-induced HDAC6 accumulation and deacetylation of alpha-tubulin, leading to reduced cell motility. Tubacin, an inhibitory molecule that binds to the tubulin deacetylation domain of HDAC6, also prevented estradiol-stimulated cell migration. Finally, we evaluated HDAC6 protein expression in 139 consecutively archived human breast cancer tissues by immunohistochemical staining. The prognostic analyses for these patients revealed no significant differences based on HDAC6 expression. However, subset analysis of ER-positive patients who received adjuvant treatment with TAM (n = 67) showed a statistically significant difference in relapse-free survival and overall survival in favor of the HDAC6-positive group (P < 0.02 and P < 0.05, respectively). HDAC6 expression was an independent prognostic indicator by multivariate analysis (odds ratio = 2.82, P = 0.047). These results indicate the biological significance of HDAC6 regulation via estrogen signaling.

Topics: Anilides; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Multivariate Analysis; Prognosis; Selective Estrogen Receptor Modulators; Tamoxifen; Tubulin