Background Paclitaxel (PTX), a first-line therapy for triple negative breast cancers (TNBC) induces anti-tumor activity by microtubule stabilization and inhibition of cell division. Its dose-limiting toxicity and short half-life, however, pose clinical challenges underscoring the need for strategies that increase its efficiency. RAD6, a E2 ubiquitin conjugating enzyme, is associated with centrosomes at all phases of cell cycle. Constitutive overexpression of the RAD6B homolog in normal breast cells induces centrosome amplification and multipolar spindle formation, indicating its importance in centrosome regulation. Methods TNBC centrosome numbers were scored by pericentrin immunostaining. PTX sensitivities and interactions with SMI#9, a RAD6-selective small molecule inhibitor, on TNBC cell survival were analyzed by MTT and colony forming assays and an isogenic MDA-MB-468 TNBC model of PTX resistance. The molecular mechanisms underlying PTX and SMI#9 induced cytotoxicity were determined by flow cytometry, immunoblot analysis of cyclin B1 and microtubule associated protein TAU, and dual immunofluorescence staining of TAU and α-tubulin. Results Our data show aberrant centrosome numbers and that PTX sensitivities are not correlated with TNBC BRCA1 status. Combining PTX with SMI#9 synergistically enhances PTX sensitivities of BRCA1 wild-type and mutant TNBC cells. Whereas SMI#9/PTX combination treatment increased cyclin B1 levels in MDA-MB-468 cells, it induced cyclin B1 loss in HCC1937 cells with accumulation of reproductively dead giant cells, a characteristic of mitotic catastrophe. Cell cycle analysis revealed drug-induced accumulation of tetraploid cells in S and G2/M phases, and robust increases in cells with 4 N DNA content in HCC1937 cells. TAU overexpression is associated with reduced PTX efficacy. Among the six TAU isoforms, both SMI#9 and PTX downregulated 1N3R TAU in MDA-MB-468 and HCC1937 cells, suggesting a common mechanism of 1N3R regulation. Dual TAU and α-tubulin immunostaining showed that SMI#9 induces monopolar mitotic spindles. Using the isogenic model of PTX resistance, we show that SMI#9 treatment restores PTX sensitivity. Conclusions These data support a common mechanism of microtubule regulation by SMI#9 and PTX and suggest that combining PTX with RAD6 inhibitor may be beneficial for increasing TNBC sensitivities to PTX and alleviating toxicity. This study demonstrates a new role for RAD6 in regulating microtubule dynamics.
Triple negative breast cancers (TNBC) lack estrogen receptors, and progesterone receptors, and HER2/neu receptor amplifications, making targeted therapies unsuitable. The taxane based drug paclitaxel (PTX) is used as a first line chemotherapy. PTX is a microtubule stabilizer that induces G2/M arrest and mitotic catastrophe. Loss of BRCA1 often seen in TNBC patients is associated with PTX resistance. Rad6 is an E2 ubiquitin conjugating enzyme that has two human homologues, Rad6A (UBE2A) and Rad6B (UBE2B), and high Rad6B expression rather than Rad6A is associated with poor survival of TNBC patients. Rad6 is associated with centrosomes at all phases of the cell cycle, and constitutive overexpression of Rad6B in nontransformed MCF10A breast cells leads to centrosome amplification and aneuploidy. Inhibition of Rad6 enzymatic activity, with our novel Rad6-selective small molecule inhibitor (SMI#9) induces G2/M arrest, much like PTX. We hypothesize that the overexpression of Rad6B commonly found in BRCA1 wt and BRCA1 mut TNBCs contributes to taxane resistance by promoting centrosome amplification and enhancing microtubule dynamics. Thus, targeting Rad6 will confer taxane sensitivity by preventing centrosome reduplication and/or microtubule hypernucleation, strengthening G2/M arrest, and ensuring terminal mitotic catastrophe. Immunostaining shows Rad6 is overexpressed in BRCA1 wt and BRCA1 mut breast cancer tissues and TNBC cell lines. Immunostaining of the centrosomal protein pericentrin shows centrosome amplification in both BRCA1 wt and BRCA1 mut TNBC cells. MTT data show SMI#9 synergistically sensitizes both BRCA1 wt (MDA-MB-468) and BRCA1 mut (HCC1937) TNBC cell lines to PTX. Colony forming assays corroborate these data and show that addition of SMI#9 diminishes colony forming efficiency of PTX pretreated MDA-MB-468 cells. SMI#9 treatment of HCC1937 PTX resistant colonies results in increases in cells with enlarged and multiple nuclei (characteristic of mitotic catastrophe). Western blot analysis of Tau, a marker of PTX sensitivity, indicates PTX, SMI#9, and PTX+SMI#9 treatments decrease the steady state levels of the 1N3R and 0N4R, or 1N3R Tau isoforms in MDA-MB-468 and HCC1937 cells, respectively. Analysis of cyclin B1, a marker of G2/M arrest, shows increases in cyclin B1 levels in PTX, SMI#9, and PTX+SMI#9 treated MDA-MB-468 cells, whereas cyclin B1 is degraded in HCC1937 cells treated with these agents (indicative of mitotic catastrophe). Immunofluorescence staining shows that Tau localization to the mitotic spindles is unaffected by PTX, SMI#9, or PTX+SMI#9. However, treatments including SMI#9 resulted in mitotic cells with defective or monopolar mitotic spindles. These data implicate a role for Rad6 in centrosome duplication/separation and provide mechanistic support for inhibiting Rad6 to enhance PTX sensitivity. Supported by NCI R21 CA178117 and T32-CA009531. Citation Format: Brittany Haynes, Kristen Cunningham, Malathy Shekhar. Rad6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by inducing mitotic spindle defects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3786.
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