Vorinostat Eliminates Multicellular Resistance of Mesothelioma 3D Spheroids via Restoration of Noxa Expression

When grown in 3D cultures as spheroids, mesothelioma cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found that resistance to the proteasome inhibitor bortezomib in 3D can be explained by a lack of upregulation of Noxa, the pro-apoptotic BH3 sensitizer that acts via displacement of the Bak/Bax-activator BH3-only protein, Bim. We hypothesized that the histone deacetylase inhibitor vorinostat might reverse this block to Noxa upregulation in 3D. Indeed, we found that vorinostat effectively restored upregulation of Noxa protein and message and abolished multicellular resistance to bortezomib in the 3D spheroids. The ability of vorinostat to reverse resistance was ablated by knockdown of Noxa or Bim, confirming the essential role of the Noxa/Bim axis in the response to vorinostat. Addition of vorinostat similarly increased the apoptotic response to bortezomib in another 3D model, the tumor fragment spheroid, which is grown from human mesothelioma ex vivo. In addition to its benefit when used with bortezomib, vorinostat also enhanced the response to cisplatin plus pemetrexed, as shown in both 3D models. Our results using clinically relevant 3D models show that the manipulation of the core apoptotic repertoire may improve the chemosensitivity of mesothelioma. Whereas neither vorinostat nor bortezomib alone has been clinically effective in mesothelioma, vorinostat may undermine chemoresistance to bortezomib and to other therapies thereby providing a rationale for combinatorial strategies.

当以3D球体（spheroids）培养体系培养时，间皮瘤（mesothelioma）细胞可获得与实体瘤相似的多细胞抗凋亡（apoptosis）特性。我们此前的研究发现，3D培养环境下肿瘤细胞对蛋白酶体抑制剂（proteasome inhibitor）硼替佐米（bortezomib）的耐药性，可归因于Noxa的表达上调缺失；Noxa是一种促凋亡BH3致敏因子，通过置换Bak/Bax激活型BH3-only蛋白（BH3-only protein）Bim发挥作用。我们提出假说，认为组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor）伏立诺他（vorinostat）可逆转3D培养体系中Noxa表达上调的受阻状态。实验结果证实，伏立诺他可有效恢复3D球体中Noxa蛋白与信使RNA的表达上调，并消除了其对硼替佐米的多细胞耐药性。通过敲低Noxa或Bim可消除伏立诺他逆转耐药性的能力，这证实了Noxa/Bim轴在伏立诺他应答过程中的关键作用。在另一项源自人间皮瘤组织离体（ex vivo）培养的3D模型——肿瘤碎片球体（tumor fragment spheroids）——中，加入伏立诺他同样增强了细胞对硼替佐米的凋亡应答反应。除与硼替佐米联用时的获益外，伏立诺他还可增强两种3D模型对顺铂（cisplatin）与培美曲塞（pemetrexed）联合疗法的应答反应。我们基于临床相关3D模型得到的研究结果表明，调控核心凋亡相关蛋白谱可提高间皮瘤的化疗敏感性。尽管单独使用伏立诺他或硼替佐米在间皮瘤的临床治疗中均未显现疗效，但伏立诺他可削弱肿瘤对硼替佐米及其他疗法的耐药性，从而为联合治疗策略提供了理论依据。