Mitotic Arrest Deficiency Protein 2 (MAD2) and Histone Deacetylase 6 (HDAC6) Present a Complex Relationship in Their Regulation and Expression and Subsequent Impact on Chemoresponsiveness

Introduction:<br/>Ovarian cancer patients presenting with advanced stage (III/IV)<br/>canceraretreatedwithcarboplatinumincombinationwithpaclitaxel.Despitea<br/>significant initial response rate, fewer than 20% of patients become long-term<br/>survivors. We have published that low MAD2 expression levels associate with<br/>reduced progression free survival (PFS) in patients with high-grade serous<br/>epithelial ovarian cancer (EOC). Moreover, we have demonstrated that MAD2<br/>expressionisdown-regulatedbythemicroRNAmiR-433(<br/>Furlong et al., 2011<br/>).<br/>Interestingly, miR-433 also down-regulates HDAC6 (<br/>Simon et al., 2010<br/>), which<br/>uniquely deacetylates<br/>a<br/>-tubulin prior to HDAC6s binding to<br/>b<br/>-tubulin.<br/>In vitro<br/>studies have shown that HDAC6 inhibition in combination with paclitaxel<br/>treatment enhances chemoresistant cancer cell death. To date, an interaction<br/>between MAD2 and HDAC6 has not been reported.<br/>Experimental design:<br/>MAD2 and HDAC6 immunohistochemistry (IHC) and<br/>Western blot analyses were performed to investigate the role of HDAC6 and<br/>MAD2 in chemoresistance to paclitaxel in high-grade serous EOC.<br/>Results and Discussion:<br/>In vitro<br/>experiments demonstrated that overex-<br/>pression of pre-miR-433, which targets MAD2, resulted in down-regulation<br/>of HDAC6 in EOC cell lines. High levels of HDAC6 are co-expressed with<br/>MAD2 in the paclitaxel resistant UPN251 and OVCAR7 cell lines. While, all<br/>4 paclitaxel resistant EOC cell lines express higher levels of miR-433 than<br/>the paclitaxel sensitive A2780 cells, only ovca432 and ovca433 demonstrated<br/>down-regulation of both HDAC6 and MAD2. Paclitaxel binds to<br/>b<br/>-tubulin and<br/>causesmicrotubulepolymerizationinpaclitaxelsensitivecellsasdemonstrated<br/>by tubulin acetylation in A2780 cells. However, paclitaxel failed to cause a<br/>significant acetylation of<br/>a<br/>-tubulin and microtubule stabilisation in the resistant<br/>UPN251 cells. Therefore resistance in this cell line may be mediated by<br/>aberrantly high HDAC6 activity. We have previously shown that MAD2 knock-<br/>down cells are resistant to paclitaxel (<br/>Furlong F., et al., 2011; Prencipe M.,<br/>et al., 2009<br/>). We measured HDAC6 protein expression in MAD2 knockdown<br/>cells and showed that MAD2 knockdown is associated with concomitant<br/>up-regulation of HDAC6. We hypothesise that the up-regulation of HDAC6<br/>by MAD2 knockdown renders cancer cells more resistant to paclitaxel and<br/>increases the invasive potential of these cells. On-going experiments will test<br/>this hypothesis. Lastly we have observed differential MAD2 and HDAC6 IHC<br/>staining intensity in formalin fixed paraffin embedded EOC samples.<br/>In conclusion<br/>, we have reported on a novel interaction between MAD2 and<br/>HDAC6 which may have important consequences for paclitaxel resistant EOC.<br/>Moreover, understanding chemo-responsiveness in ovarian tumours will lead<br/>to improved patient management and treatment options for women diagnosed<br/>with this disease