PKA 激活剂对天然溶瘤病毒M1 的增效机制与临床意义

M1 is a kind of naturally oncolytic virus. It can reproduce to a great mount of copies within the tumor tissue and thus destroy tumor cells without any harm to the normal tissue cells. However, some kind of cancer cells are not sensitive to M1, it is imperative to enhance the sensitivity through screening synergist. We found that the PKA activator can effectively and safely promote M1 duplication in the tumor and amplify its oncolytic effect, but the mechanism is unknown. Experiment within cell lines and in the nude mice indicated that PKA activator could undermine the action of interferon and exacerbate a type of apoptosis derived from endoplasmic reticulum stress in M1-infected tumor cells. GADD34 is a critical gene with the functions to maintain the active effect of interferon, and inhibit the endoplasmic reticulum stress in a negative feedback way. Studies have shown that PKA can inhibit the activity of GADD34 via I-1. Therefore, PKA activator is likely to play its synergistic activity by inhibiting GADD34. In this study, we will use different methods as gene silencing, gene over-expression and others to confirm the role played by the interferon response, endoplasmic reticulum stress in PKA activator synergistic mechanism. We further integrate results of the reporter gene assay and mutant rescue experiments to determine how GADD34 regulated these two aspects mentioned above. In vivo experiments of animals will be combined with the clinical correlation analysis to elaborate the key role of GADD34 in synergistic mechanisms of PKA activator. This study will open up a broader prospects for the clinical application of M1, and provide a new model for the study of oncolytic viruses synergist.

天然溶瘤病毒M1能在瘤内大量复制，破坏癌细胞，而无害于正常细胞。但部分癌细胞对M1敏感性不足，当务之急是筛选增效剂以增强其敏感性。细胞及裸鼠实验发现，PKA激活剂可安全高效地发挥增效剂作用，但机制不明。研究发现，PKA激活剂既能通过破坏干扰素合成而促进M1复制，又能通过加剧内质网应激性凋亡而促进M1溶瘤。已知GADD34促进干扰素合成，且可抑制内质网应激性凋亡。研究表明PKA可抑制GADD34。因此，提出假说“PKA激活剂通过抑制GADD34而破坏干扰素合成并加剧内质网应激性凋亡，最终促进M1复制与溶瘤”。我们拟采用过表达、RNAi等手段确认干扰素与内质网应激在此增效机制中的意义；并结合报告基因和突变体拯救等实验确定GADD34如何对这两方面进行调控；最终通过动物实验结合临床相关性分析，确证GADD34在PKA激活剂增效机制中的关键作用，为M1临床应用奠定基础，为溶瘤增效剂研究提供新思路。

溶瘤病毒疗法是一种新兴的使用可复制的溶瘤病毒用于抗癌的治疗方法。虽然不同类型的癌细胞对溶瘤病毒敏感，但溶瘤病毒治疗的主要挑战之一是不同类型的癌细胞对溶瘤病毒的敏感程度不同。溶瘤病毒治疗的潜在机制尚不完全清楚。在这里，我们报道了在体外、体内和体外培养的病人标本中，激活环磷酸腺苷（cAMP）信号显著地使低敏感的癌细胞对甲病毒M1敏感。我们发现，cAMP信号通路的激活抑制了M1诱导的低敏感的癌细胞表达抗病毒因子，进而导致内质网应激延长和加重，并最终导致细胞凋亡。我们还证明，这种可被cAMP信号所增强的M1病毒的溶瘤作用，涉及了“cAMP 1直接激活的交换蛋白（Epac1）”，而不是经典的cAMP依赖性蛋白激酶A。在此，我们报道了一种经典的蛋白激酶AD的抑制剂H89可通过激活Epac1（cAMP 1直接激活的交换蛋白）与溶瘤病毒M1在多种癌细胞中协同作用。H89显著增加病毒在低敏感的癌细胞的复制，导致不可缓解的内质网应激和细胞凋亡。微阵列分析表明，H89通过抑制NF-kB的核移位而减弱低敏感的癌细胞发生抗病毒应激反应。重要的是，体内研究表明M1/H89联合治疗具有显著的抗肿瘤作用。综上所述，我们的研究发现cAMP/Epac1信号通路的激活抑制了抗病毒因子，促进了低敏感的癌细胞对M1介导的病毒治疗的反应性。