丝氨酸蛋白酶抑制剂SERPINA12对肝癌干细胞特性的调控及机制研究

肿瘤干细胞可能是肿瘤复发、转移和耐药的根源。本实验室首先报道了CD133分子是一个重要的肝癌干细胞标志物。我们前期利用RNA-Seq测序分析发现丝氨酸蛋白酶抑制剂SERPINA12是肝癌CD133+细胞亚群的特异性基因，通过细胞功能学实验证实SERPINA12能够促进肝癌细胞的生长、转移、自我更新，临床样本及多个数据库显示SERPINA12的表达与患者预后密切相关，但它调控肝癌细胞干性特征的分子机制尚不清楚。本项目拟利用多种体内外实验模型和技术，从分子、细胞、动物模型以及临床肝癌标本等不同层次系统，研究内源性和分泌性SERPINA12基因对肝癌发生发展的作用；阐明调控分子机制；明确临床意义，评价AAV-8介导的SERPINA12基因靶向治疗肝癌的潜力。本项目的预期研究成果将为揭示SERPINA12在肝癌发生发展中的功能与作用分子机制，为CD133+肝癌干细胞的根源性治疗提供有效策略。

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in Southeast Asia and China. The prognosis of HCC is dismal and the disease remains a major public health concern in our locality. HCC is a particularly heterogenous disease, which contributes to inefficacy of current treatments. Intratumor molecular heterogeneity of HCC is partly attributed to the presence of cancer stem cells (CSCs), which we now know represents a critical root of tumor recurrence and therapy resistance. We and others have previously found CD133 to mark an important functional marker of liver CSCs. Yet unfortunately, CD133 is not specific to HCC, but is also expressed in the fetal and regenerating liver. Identifying critical factors expressed specifically in liver CSCs, but not in liver normal stem/progenitor cells may offer important therapeutic opportunities. Our current preliminary data by transcriptome sequencing profiling comparing sorted CD133+ and CD133- subsets of fetal liver, regenerating liver induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and HCC induced by either N-nitrosodiethylamine (DEN) + carbon tetrachloride (CCL4) or hydrodynamic tail vein injection of oncogenic plasmids identified serpin family A member 12 (SERPINA12) to be preferentially expressed in the HCC CD133+ subpopulation. SERPINA12 was also found to be upregulated in a non-alcoholic fatty liver disease (NAFLD)-HCC mouse model, generated by DEN and high-fat, high-cholesterol diet, suggesting that its up-regulation is observed across different etiology-driven HCC. Clinically, endogenous SERPINA12 is frequently overexpressed in HCC, where its overexpression correlates with poor prognosis. Pilot functional studies in vitro and in vivo found endogenous SERPINA12 overexpression to potentiate aggressive cancer features including proliferation, stemness, metastasis and drug resistance, possibly through a deregulated β-catenin signaling. Our initial data also suggest SERPINA12 up-regulation in HCC to be mediated by the transcriptional factor c-myb. We hypothesize c-myb to bind to the promoter of SERPINA12 and drive its transcriptional activity. Enhanced SERPINA12 expression will then result in enhanced cancer stemness properties via sustaining AKT – GSK-3β – β-catenin signaling. Here, we propose to study the clinical significance, functional role and underlying mechanism by which SERPINA12 contributes to cancer stemness in HCC. We also aim to examine whether SERPINA12, alone or in combination with CD133 can be used as novel prognostic biomarkers of HCC; and lastly explore, as a proof-of-concept, the potential of targeting SERPINA12 by AAV8-mediated liver-directed gene therapy, as mono- and combined-therapies with cisplatin or sorafenib, for HCC treatment.