溶瘤病毒通过PI3K/Akt信号通路抑制前列腺癌干细胞增殖的分子机制研究

Prostate cancer stem cells have the characteristics of self-renew and proliferation, and they are resistant to the chemo-radiotherapy. It is the root of tumor recurrence. Oncolytic herpes simplex type 1 viruses (oHSVs) are genetically-engineered replication-competent viruses that can selectively replicate in and kill cancer cells, amplifying themselves and spreading within cancer, but sparing normal tissue. oHSVs have already been safely used in human subjects with a variety of cancers including prostate cancer, and it’s a novel antitumor drug. We established PCSC-enriched spheres (DU145 sphere and TRAMP-C2 sphere) by culturing human and murine PCa cells in growth factor-defined serum-free medium. Our results showed Sphere-culture-enriched PCSCs express stem cell markers, resistant to the chemo-radiotherapy and drive the high sphere formation ratio in vitro, and they are highly tumorigenic in vivo. Human and murine PCSCs were sensitive to oHSVs. But oHSVs also induced the p-AKT increased apparently in two spheres. This feature can reduce the antitumor effect of oHSVs. My previous data showed oHSVs actived PI3K/Akt pathway when working on PCSCs instead of acting other stem cell pathways such as Notch and Wnt. We hypothesis that oncolytic herpes simplex viruses suppressing the proliferation of prostate cancer stem-like cells by targeting the PI3K/Akt signal pathway. Our project illuminate the mechanism of the oHSVs enhancing the ability of killing PCSC by inhibiting the PI3K/Akt/c-Myc signal axis, which will be helpful and useful to promote the oHSVs using in the clinical for the treatment of prostate cancer in future and prolong the overall survival of PCa patients.

前列腺癌干细胞具有自我更新及增殖能力，对放化疗抵抗，是肿瘤复发的根源。溶瘤病毒通过特异性感染肿瘤细胞并完成感染复制周期，从而特异性地杀伤裂解肿瘤细胞，成为目前最具研究前景的抗肿瘤生物治疗药物之一。申请人的前期研究表明，前列腺癌细胞系经富集得到的前列腺癌干细胞样细胞（PCSCs）具有自我更新、放化疗抵抗及极强的致瘤能力。溶瘤病毒对PCSCs有高效的杀伤作用，但同时其磷酸化Akt表达却明显升高，影响溶瘤病毒对其杀伤作用。通过对多个干细胞通路的检测，发现溶瘤病毒作用于PCSCs时伴有PI3K/Akt信号通路的活化。由此我们提出假设，靶向PI3K/Akt信号通路将增强溶瘤病毒对PCSCs的抑制作用。本项目拟通过体外实验和动物实验，阐明溶瘤病毒通过PI3K/Akt信号通路抑制PCSCs增殖的分子机制，为溶瘤病毒在前列腺癌治疗的临床应用提供新的科学依据。

前列腺癌（PCa）是男性最常见的恶性肿瘤之一，欧美国家高发。随着人口的老龄化、生活方式的改变及诊断技术进步，PCa在我国的发病率逐年上升。随着疾病的进展，绝大部分PCa患者进展为去势抵抗性前列腺癌（CRPC）而导致肿瘤的复发、转移进而危及生命。前列腺癌干细胞被认为是肿瘤复发及转移的根源，具有自我更新及增殖能力且对放化疗抵抗。新型的溶瘤性单纯疱疹病毒（oHSV）能利用靶细胞中抑癌基因的失活或缺陷从而特异性地感染肿瘤细胞，在其内大量复制并最终摧毁肿瘤细胞，且对正常组织和细胞无损伤，成为最具有应用前景的肿瘤生物治疗手段之一。PCa近年来的研究热点聚焦于精准的分子靶向治疗。多种靶向PI3K/Akt信号通路的新型药物进入PCa的临床试验，但总体的客观有效率及总生存期尚不令人满意。本项目使用的G47Δ是第三代基因工程溶瘤病毒，具有对肿瘤细胞杀伤效率高、靶向性好、安全性高的特点。.研究结果显示，新型溶瘤病毒G47Δ在体外能高效杀伤前列腺癌干细胞，对DU145 sphere及TRAMP-C2 sphere的IC50分别为：0.006MOI和0.565MOI。在裸鼠体内实验中，G47Δ能明显抑制DU145 sphere皮下移植瘤的生长，有良好的生存获益，差异有统计学意义。而经过溶瘤病毒感染的前列腺癌干细胞其磷酸化Akt表达却明显升高，影响了溶瘤病毒对其杀伤作用。因此我们通过新型的PI3K/Akt信号通路的抑制剂BKM120与溶瘤病毒联合，在前列腺癌干细胞DU145 sphere及TRAMP-C2 sphere上均显示出协同效应。进一步的分子水平研究探讨了抑制PI3K/Akt信号通路增强溶瘤病毒对前列腺癌干细胞增殖的具体机制。该研究有望为临床上复发难治的前列腺癌带来新的治疗策略及新思路，为溶瘤病毒在实体肿瘤中临床研究的推广带来新的理论依据，为多药耐药的晚期前列腺癌患者带来新的希望。