基于小分子诱导人ES细胞分化肝细胞的肝癌诱导分化策略及其机制研究

Hepatocellular carcinoma (HCC), is the sixth most common malignant disease worldwide and the third greatest cause of cancer-related death. The increasing incidence, the high heterogeneity of HCC, the limited therapeutic options and its resistance to traditional anticancer therapies such as chemotherapy and radiotherapy, and the recurrence and metastasis result in the poor prognosis of HCC. Therefore, development novel therapeutic strategies for HCC treatment are urgent. The observation of cancer stem cell (CSC) led to establish a model in which tumour would arise from rare to moderately frequent cells able to initiate a hierarchy of proliferative and progressively differentiating non-tumourigenic cells. This non-tumourigenic population of cells would then form the bulk of the tumour. In this model, CSC shares similar properties to normal adult stem cells or embryonic stem cell (ESC). These properties include the capacity of self-renewal, the use of key regulatory pathways and the establishment of dynamic epigenetic profiles. Hence, the tumour hierarchical organization would be some level comparable to that found in normal tissue, where genetic and epigenetic changes trigger both cell differentiation and cell fate. Recently, we have developed a novel approach using small molecule combination to efficiently different human ESC to homogenous mature functional hepatocytes. Due to some level similarity of cancer cell and CSC with ESC, we postulated that our protocol for hESC differentiation might also be suitable for HCC differentiation induction. We took several HCC lines to do the same experiments and the primilary data showed that based on the HCC lines,10-60% cell apoptosis in 3-9 days treatment and the left cells differentiate to be matured cell which re-gained its normal morphology and behavior such as reduced proliferation rate, increased ALB and HNF4a expression, non-colonies growth pattern, no longer aerobic glycolysis metabolism. Further analysis revealed that our protocol to induce HCC apoptosis and differentiation was p53 independent and compared to CD133- population, CD133+ HCC (CSC) was much easier to be induced apoptosis and differentiation. These results suggested that hESC differentiation approach also facilitates HCC differentiation. However, what signaling pathway(s) and key regulation factor(s) involve in and work together to induce HCC apoptosis and differentiation? What is the endogenous difference between apoptotic and differentiated HCC? Whether our observation can be confirmed in clinic HCC samples, and whether this small molecule combination can be used for in vitro fresh isolated HCC from clinic and in vivo HCC animal models for apoptosis and differentiation induction? We plan to perform systematic investigation to answer these questions and elucidate the regulation mechanism. Our research results would help to develop a promising novel approach for HCC diagnosis and treatment.

肝细胞癌（HCC）有发病率高、病情隐匿、预后凶险等特征，目前缺乏有效的治疗方案。肿瘤诱导分化治疗在血液系统肿瘤治疗上有成功先例，但针对HCC分化信号调控系统的特异性分化诱导剂及方法却未见任何报道。根据HCC（干）细胞与ES细胞具同源性和相似性特点，申请者拟研究基于"小分子定向诱导ES细胞分化为高纯度的功能性肝细胞"的方法和策略，应用于HCC的诱导分化。前期实验结果表明：小分子组合处理多株HCC细胞，3－9天内10－60％凋亡，其余则向成熟细胞方向分化，不再具有肿瘤细胞的生物学行为。HCC干细胞则更易于被诱导凋亡和分化，且该方法对正常肝细胞无损伤。本研究拟完善此方法，深入研究其信号网络通路和关键调控因子及相关调控机理；采用临床肝癌样本和肝癌实验动物模型验证其临床相关性、有效性和普适性。本研究有助于明确肝癌诱导分化机理，为临床诊治肝癌提供理论依据，并可能为临床治疗肝癌提供一种新方法、新药物。

中国是肝病大国，肝癌患者占全球的一半以上(55%)。肝癌具发病原因及发病机理复杂、异质性强、病情隐匿、预后凶险等临床特征。目前，治疗肝癌的有效方法除外科手术应用于早期肝癌效果较好外，其他治疗手段均存在疗效有限、毒副作用大、复发率高和五年存活率低的特点。研究开发有效治疗肝癌及其他恶性肿瘤的新方法和新手段是刻不容缓的任务。 .细胞重编程(Cell Reprogramming)指不改变基因序列的情况下，通过调控细胞信号通路及表观遗传(Epigenetics)的变化来改变细胞命运的过程。随着干细胞科学发展，细胞重编程技术也取得极大进展：1、不仅通过外源导入转录因子实现细胞命运的转换，也可通过化学小分子实现细胞重编程；2、不仅可将终分化细胞转化为多能干细胞，也可将一种分化细胞直接转化为另一种分化细胞（Direct Reprogramming）；3、甚至可将异常分化细胞（肿瘤细胞）重编程为功能细胞。.基于细胞重编程机理，我们筛选出一组小分子组合，可将不同来源的人HCC细胞，包括各种不同的HCC细胞系、肝癌干细胞（CSC）、耐索拉菲尼的HCC细胞以及从病人肿瘤组织分离的原代肝癌细胞直接转分化为非致瘤肝样细胞，同时伴随不同程度的肝癌细胞凋亡。体内实验验证了体外实验的结果。GSK3β信号通路在HCC细胞转分化过程中发挥重要作用。其机理之一是GSK3β通过AMPK/mTOR信号通路调控HCC细胞的代谢和蛋白合成发挥调控HCC发生发展的作用。研究还发现了一个有效抑制HCC的靶向治疗方法。通过双靶点抑制，对HCC的抑癌效率可高达90%。 .我们的小分子转分化方法不需要导入外源基因，避免了体内应用可能的致癌风险；该方法对正常体细胞没有杀伤和转化作用，有极大的安全性；而且小分子具价格低廉，易于调控，安全可靠等优势，易于临床转化。该方法对致病原因及发病机理复杂，异质性强且缺乏有效治疗手段的肝癌的控制或有效治疗具有重要意义。研究结果将为临床治疗肝癌提供新思想、新方法和新药物，为其他肿瘤治疗研究提供借鉴，开辟了直接转分化肿瘤细胞研究新领域。研究具重大的社会意义、极大的市场价值及应用前景，也满足国家人口健康重大需求。