Del Bufalo Donatella, Desideri Marianna, De Luca Teresa, Di Martile Marta, Gabellini Chiara, Monica Valentina, Busso Simone, Eramo Adriana, De Maria Ruggero, Milella Michele, Trisciuoglio Daniela
Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy.
Mol Cancer. 2014 Oct 9;13:230. doi: 10.1186/1476-4598-13-230.
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed, a multi-target folate antagonist, has demonstrated efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS) expression. Among many other potential targets, histone deacetylase inhibitors (HDACi) modulate TS expression, potentially sensitizing to the cytotoxic action of anti-cancer drugs that target the folate pathway, such as pemetrexed. Since high levels of TS have been linked to clinical resistance to pemetrexed in NSCLC, herein we investigated the molecular and functional effects of combined pemetrexed and ITF2357, a pan-HDACi currently in clinical trials as an anti-cancer agent.
In NSCLC cell lines, HDAC inhibition by ITF2357 induced histone and tubulin acetylation and downregulated TS expression at the mRNA and protein level. In combination experiments in vitro ITF2357 and pemetrexed demonstrated sequence-dependent synergistic growth-inhibitory effects, with the sequence pemetrexed followed by ITF2357 inducing a strikingly synergistic reduction in cell viability and induction of both apoptosis and autophagy in all cell line models tested, encompassing both adenocarcinoma and squamous cell carcinoma. Conversely, simultaneous administration of both drugs achieved frankly antagonistic effects, while the sequence of ITF2357 followed by pemetrexed had additive to slightly synergistic growth-inhibitory effects only in certain cell lines. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells (LCSC) exposed to pemetrexed followed by ITF2357. In terms of molecular mechanisms of interaction, the synergistic growth-inhibitory effects observed were only partially related to TS modulation by ITF2357, as genetic silencing of TS expression potentiated growth inhibition by either pemetrexed or ITF2357 and, to a lesser extent, by their sequential combination. Genetic and pharmacological approaches provided an interesting link between the autophagic and apoptotic pathways, and showed that sequential pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival.
Overall, these data provide a strong rationale for the clinical development of sequential schedules employing pemetrexed followed by HDACi in NSCLC.
非小细胞肺癌（NSCLC）是全球癌症相关死亡的主要原因。培美曲塞是一种多靶点叶酸拮抗剂，已在胸苷酸合成酶（TS）表达较低的NSCLC组织学亚型中显示出疗效。在许多其他潜在靶点中，组蛋白去乙酰化酶抑制剂（HDACi）可调节TS表达，可能使细胞对靶向叶酸途径的抗癌药物（如培美曲塞）的细胞毒性作用敏感。由于高水平的TS与NSCLC患者对培美曲塞的临床耐药有关，因此我们研究了培美曲塞与ITF2357（一种目前正在进行抗癌药物临床试验的泛HDACi）联合使用的分子和功能效应。
在NSCLC细胞系中，ITF2357抑制HDAC可诱导组蛋白和微管蛋白乙酰化，并在mRNA和蛋白质水平下调TS表达。在体外联合实验中，ITF2357和培美曲塞显示出序列依赖性的协同生长抑制作用，培美曲塞后接ITF2357的序列在所有测试的细胞系模型（包括腺癌和鳞状细胞癌）中均显著协同降低细胞活力，并诱导凋亡和自噬。相反，同时给予两种药物产生明显的拮抗作用，而ITF2357后接培美曲塞的序列仅在某些细胞系中具有相加至轻微协同的生长抑制作用。同样，在接受培美曲塞后接ITF2357的患者来源的肺癌干细胞（LCSC）中也观察到高度协同的生长抑制。就相互作用的分子机制而言，观察到的协同生长抑制作用仅部分与ITF2357对TS的调节有关，因为TS表达的基因沉默增强了培美曲塞或ITF2357以及它们的序贯组合（程度较小）的生长抑制作用。遗传和药理学方法在自噬和凋亡途径之间建立了有趣的联系，并表明序贯培美曲塞/ITF2357会导致一种有毒形式的自噬，从而激活半胱天冬酶依赖性凋亡程序。NSCLC异种移植瘤的体内实验证实，序贯培美曲塞/ITF2357是可行的，并导致肿瘤生长抑制增加和小鼠生存期延长。
总体而言，这些数据为在NSCLC中采用培美曲塞后接HDACi的序贯方案进行临床开发提供了有力依据。
