组织蛋白酶S (CTSS)在CD47+肿瘤干细胞驱动肝细胞癌发生中的地位及其靶向治疗的效应

A growing body of evidence has suggested that tumor growth is driven by stem-like cells within the tumor called tumor-initiating cells (T-ICs) that sustain tumor growth and confer resistance to conventional therapies. Thus, isolation and identification of T-ICs and their signaling pathways will lead to development of new targeted therapy against HCC. In our pilot study, we have enriched liver T-ICs derived from PLC/PRF/5 by combining serial passages of hepatospheres with chemotherapeutic regimens. Using cDNA microarray, we compared the expression profiles between the enriched T-IC population and their differentiated progeny, which differ in their capacities for self-renewal and tumorigenicity. This analysis showed that the cell surface marker, CD47 is up-regulated in the enriched liver T-IC population compared to its differentiated progeny. Subsequent analysis by cell sorting showed that CD47+ HCC cells possess T-IC properties including tumorigenicity, self-renewal, metastasis and chemoresistance. This data, together with the suppressive effect of CD47 knockdown on liver T-IC phenotype, suggests that these CD47+ HCC cells represent the source of tumor recurrence after chemotherapy. By cDNA microarray and subsequent analysis, we found cathepsin S (CTSS) was the potential downstream effector of CD47 signaling. We have preliminary data showing the role of CTSS in CD47+ HCC cells plays part in enhancing self-renewal, inducing tumor growth and promoting angiogensis in endothelial cells. Based on our current data, we hypothesize that elevated CD47 expression in HCCs may contribute to T-IC maintenance and growth via preferential production of CTSS. In this proposal, we aim to (1) examine and correlate both endogenous and secretory forms of CTSS in sorted CD47+ T-ICs and CD47- cells from fresh human HCC clinical tissue and serum specimens; (2) to elucidate the role of CTSS in CD47+ T-IC-driven HCC; and (3) to examine the use of CTSS as a potential therapeutic target against CD47+ T-ICs, when alone or in combination with conventional chemotherapeutic agents in mouse tumor model. Our study will further our understanding of how CD47-expressing HCC cells drives hepatocarcinogenesis and progression, and may shed light on the development of more specific targeted therapies against this deadly disease.

证据显示肝肿瘤是由一群肿瘤组织内的干细胞样的细胞。运用cDNA 微阵列技术，我们发现富集的肝癌干细胞表面分子CD47显著上调。CD47+肝癌细胞拥有肿瘤干细胞特性。进一步的研究发现组织蛋白酶S (CTSS)是CD47信号通路潜在的下游效应分子。我们推测肝细胞癌CD47表达的升高通过优先产生CTSS可能有助于肝癌干细胞维持和生长。本研究旨在：（1）研究肝癌患者标本中CD47+细胞和CTSS的关系及患者血清中的分泌性CTSS的临床意义；（2）阐明CTSS在CD47+肝癌干细胞驱动的肝细胞癌中的地位；（3）研究CTSS在老鼠肿瘤模型中作为靶向CD47+肝癌干细胞的治疗作用。我们的研究将为研发更特异的抗肝癌靶向治疗药物铺平道路。

本课题组前期研究发现并确认以CD47为标记的癌症干细胞（tumor initiating cells, T-ICs）能诱导肝癌的发生、自我更新、化疗耐药和转移。为靶向治疗肝T-ICs，鉴定新的T-ICs标记分子并阐明其与肿瘤发生、自我更新和转移相关的关键信号通路是很重要的。鉴于此，我们将深入研究和认识CD47+HCC细胞调控肝癌干细胞不同特性的分子机制。针对这一目的，本研究在前期工作基础上，通过cDNA 基因微阵列技术和进一步的研究，发现组织蛋白酶S (CTSS)是CD47信号通路潜在的下游效应分子。本项目旨在研究CTSS对CD47＋T-IC驱动肝细胞癌的效应。为达此目的，我们用qPCR分析CD47和细胞内CTSS表达。在42例HCC患者标本中，CD47与CTSS表达显著相关。肿瘤组织CTSS高表达的患者具有更短的总体生存期和无病生存期(P<0.001 and P=0.008) 。患者的血清标本，以用于定量ELISA(R&DSystems)检测循环CTSS水平。根据患者CTSS表达水平（低，中和高）进行分组。我们总共检测了60个患者和31个正常对照者的血清CTSS水平，发现血清CTSS水平从正常对照者、早期肝癌患者到进展期肝癌患者逐步升高。高血清CTSS水平的患者，肿瘤状态为进展期(P=0.002, 2 test)，肿瘤体积较大(P= 0.008, 2 test) 。体外细胞培养模型证实CD47与CTSS的相关性。 通过抑制CD47表达，我们发现肝癌细胞 CTSS下调。通过细胞迁移, NOD/SCID小鼠致瘤和浸润实验, 证实了CD47通过上调CTSS来调节肿瘤发生、自我更新和侵袭性。进一步分析发现CD47是以自分泌的方式，通过分泌CTSS来调节肝癌干细胞。单用抗CD47抗体能有效的诱导人巨噬细胞吞噬肝癌细胞。我们在体外与体内小鼠研究中检测了纯化的抗CD47抗体（B6H12克隆）不仅对高表达CD47的肿瘤发挥抗肿瘤的作用, 但也增加对索拉非尼和化疗药物的敏感性。总而言之，本项目研究表明CTSS和CD47在HCC癌症特性的调控中起重要作用。 这一数据为治疗肝癌开辟了一条新的治疗途径。向治疗药物铺平道路。