Solid tumors such as mesothelioma exhibit a resistance to chemotherapy that poses a challenge for curative therapy. We have attempted to model this chemoresistance in our 3D models, in which cells acquire multicellular resistance. Previously, we have reported that the acquired multicellular resistance is mediated by alterations in the Bcl-2 family of proteins, raising the hope that strategies directed to the Bcl-2 family could undermine resistance 
. In this study, we have found that a broadly active histone deacetylase inhibitor, vorinostat, is able to ablate the multicellular resistance to bortezomib by its ability to restore expression of a key pro-apoptotic Bcl-2 family member, Noxa.
In our previous work in 3D spheroids, resistance to bortezomib was shown to be due to a lack of upregulation of Noxa, a sensitizer BH3-only protein that acts via displacement of pro-apoptotic Bim from Mcl-1 and possibly also from Bcl-2 and Bcl-xl 
. Importantly, we found that spheroids expressed elevated Bim and were primed for apoptosis but, without Noxa to displace the Bim from anti-apoptotic buffers, the spheroids exhibited a resistance to apoptosis. Therefore, in this study, we sought means of restoring the upregulation of Noxa in the spheroids and turned to vorinostat, an HDAC inhibitor thought to act via enhanced transcription and expression of epigenetically-silenced genes, many of which are pro-apoptotic 
. Indeed, vorinostat effectively restored the pro-apoptotic Noxa upregulation seen in response to bortezomib. By RNA silencing, Noxa and Bim were each shown to be required for the ability of vorinostat to undermine multicellular resistance. Vorinostat appeared to act at least in part via the transcriptional upregulation of Noxa; in 3D, the increase in Noxa message in response to bortezomib was blunted and, with the addition of vorinostat, the Noxa message was restored to the same levels seen in monolayers. In addition to its beneficial effect on Noxa regulation, vorinostat increased baseline levels of Bim protein, in both multicellular spheroids and tumor fragment spheroids, suggesting that vorinostat could perhaps elevate Bim in tumors with low Bim, found to represent approximately 30% of mesotheliomas 
Our findings may have clinical relevance for the use of HDAC inhibitors in combinatorial therapy. Despite promising in vitro
and pre-clinical results, vorinostat and bortezomib have each been found to be largely ineffective as single agents in clinical trials of patients with relapsed mesothelioma [unpublished
. Our results in 3D spheroids are similar: neither agent was effective in inducing apoptosis in 3D spheroids when given alone. In particular, vorinostat alone had almost no apparent effect against mesothelioma cells in 3D spheroids; there was no evident impact of vorinostat alone on cellular morphology, Noxa transcription, Noxa protein levels or apoptosis. In our models, the benefit of vorinostat was only seen when it was used with other agents. Indeed, in clinical trials, whereas vorinostat as a single agent has yielded disappointing results, its use in combination with chemotherapy has been reported to have positive results in a variety of solid and hematological malignancies 
. It is of interest then that, despite the lack of benefit reported for vorinostat as a single agent in mesothelioma, another HDAC inhibitor, valproic acid, was recently found to have benefit when used with doxorubicin in mesothelioma patients 
. Our findings in mesothelioma lead us to believe that the key to clinical efficacy of HDAC inhibition lies in its ability to potentiate responses to chemotherapy 
, a potentially valuable strategy against chemoresistant solid tumors. Recently, it has been reported that resistance to manipulation of Noxa can develop over a period of 10 days 
. Although we did not study our spheroids for that duration, we saw no resistance to vorinostat over 2 days (data not shown). Issues including drug resistance as well as drug penetrance and toxicity will be important considerations in the use of this combination in the clinic 
In summary, we have used 3D models to show the value of histone deacetylase inhibition in undermining multicellular resistance to chemotherapy. This raises the possibility that the resistance of the tumor cells in 3D, which can be localized to the Bcl-2 family, is mediated via epigenetic mechanisms. In the 3D resistant setting, chemoresponsiveness can be enhanced by either targeting the Bcl-2 family directly, as we have previously done by using small molecules such as ABT-737 
or indirectly, by restoring transcription of silenced pro-apoptotic genes, as we show here. Possibly both strategies could be used together, especially if vorinostat enhances Bim expression in tumors with low baseline expression. These studies may provide the basis for the use of these agents in combination in future clinical trials.