Annexin A3:潜在的索拉非尼疗效预测因子及肝癌治疗的新靶点

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. Sorafenib is the only small molecule inhibitor approved by the US Food and Drug Administration for HCC treatment to date. However, the benefits of sorafenib for HCC patients remain modest, likely due to acquired drug resistance. A better understanding on the mechanisms governing sorafenib resistance is much warranted. There is now clear evidence to show that tumor-initiating cells (T-ICs) represent an important subset responsible for HCC formation, therapy resistance and recurrence. We and others having previously shown that liver T-ICs, functionally defined and phenotypically marked by CD47, CD133, EpCAM and Side Population, are more resistant to sorafenib (Lo et al. Hepatology 2015). In line with these studies, we found that sorafenib resistant HCC cells are also endowed with enhanced T-IC properties. These results provide evidence that targeting regulatory mechanisms of liver T-ICs may be a possible way to evade sorafenib resistance in HCC. Recently, we have found endogenous and secretory annexin A3 (ANXA3) to play pivotal roles in promoting cancer and stem cell-like features in CD133+ liver T-ICs; while blockade of ANXA3 with a novel anti-ANXA3 monoclonal antibody produced in house resulted in a significant reduction in tumor growth and self-renewal (Tong et al. Stem Cell Reports 2015). Our current preliminary data suggests ANXA3 to play a critical role in mediating sorafenib resistance in HCC. ANXA3 expression was found to be significantly enriched in sorafenib resistant HepG2 cells in vitro as well as in two HCC sorafenib resistant patient derived xenografts in vivo. Knockdown of ANXA3 in Huh7 and sorafenib resistant HepG2 sensitized the cells to sorafenib treatment. Conversely, overexpression of ANXA3 attenuated the cells’ response. Transcriptome sequencing profiling comparing HepG2 sorafenib resistant, sorafenib resistant (non-target control) and sorafenib resistant (sh-ANXA3 knockdown) cells identified expression of both E1A binding protein p300 (p300) and CREB-binding protein (CBP), two closely related transcriptional co-activating proteins, to be repressed following stable ANXA3 knockdown in the resistant clone. Based on these findings, we hypothesize (i) that ANXA3 is critical in conferring sorafenib resistance in HCC through de-regulation of p300 and/or CBP; (ii) that the combinatorial use of anti-ANXA3 monoclonal antibody with sorafenib may possibly represent a novel treatment option for HCC patients; and (iii) that ANXA3 can be used as a novel predictive factor for the efficacy of sorafenib treatment in HCC. To test this hypothesis, functional / cell biological studies on HCC cell lines / non-tumor organoid cultures modulated by lentiviral based and genome-editing strategies, in vivo treatment modeling on HCC patient derived xenografts as well as analysis of clinical expression data will be performed. Findings from this study will further our understanding of how ANXA3 drives sorafenib resistance in HCC, and potentially lead to the establishment of a novel treatment option for HCC by circumventing sorafenib resistance, as well as development of a novel predictive marker for the efficacy of sorafenib in HCC patients.

肝癌是我国最常见的恶性肿瘤之一，且预后较差。目前，索拉非尼是唯一被美国FDA认可用于肝癌治疗的小分子抑制剂，但在治疗中存在获得性耐药因而收效甚微。有充分证据显示，肿瘤干细胞（T-ICs）在肝癌发生、耐药和复发中发挥重要作用。我们前期研究发现表达CD133等生物标记的肝癌干细胞具有更强的索拉非尼耐药性，针对干细胞在肝癌中调节机制的治疗可能会有效避免对索拉非尼产生耐药。体内外预实验发现ANXA3在索拉非尼耐药中发挥关键作用：ANXA3在耐药的肝癌细胞系和患者移植瘤中均有高表达；敲低ANXA3能增强细胞对索拉非尼的敏感性；转录组测序发现，敲低ANXA3能抑制转录共激活蛋白p300和CBP的表达。本研究将探索ANXA3通过调节p300和CBP对索拉非尼耐药机制的影响；研究ANXA3能否作为预测索拉非尼疗效的生物标记；明确ANXA3单克隆抗体与索拉非尼联合运用治疗肝癌的新方案。

晚期肝细胞癌是一种高致命性的恶性肿瘤，但其现行治疗手段相对匮乏。索拉菲尼是目前唯一得到FDA批准使用的用于治疗肝细胞癌的一线靶向药物，但是其对于提升患者生存率的效果依然有限，并且患者的肿瘤仍旧会继续发展。对索拉菲尼耐药性的进一步认知、提升索拉菲尼的疗效以及寻找可靠的预测性生物标记物对于有效控制肝细胞癌而言十分重要。此项课题中我们揭示了膜联蛋白ANXA3及其相关的信号通路在调节索拉菲尼治疗肝细胞癌过程中出现的耐药性。膜联蛋白ANXA3的存在使得肝细胞癌细胞获得了抵抗索拉菲尼药效的能力；同时在具有索拉菲尼抗性的肝细胞癌细胞系及人源化肿瘤异种移植组织中膜联蛋白ANXA3的表达也有所升高。在机理上，索拉菲尼耐药的肝细胞癌细胞通过过表达膜联蛋白ANXA3来抑制PKCδ/p38相关的细胞凋亡，同时激活细胞自噬从而使细胞得以存活。在临床上，肝细胞癌细胞中膜联蛋白ANXA3的高表达与细胞自噬标记物LC3B呈正相关，同时也与接受索拉菲尼治疗的病人的较差生存期有关，意味着膜联蛋白ANXA3可以作为肝细胞癌个体化治疗的标记物。抗膜联蛋白AXNA3单克隆抗体与索拉菲尼或雷戈菲尼的联合治疗无论在人源肝细胞癌细胞系、体外索拉菲尼抗性肝细胞癌细胞系、体外肝细胞癌类器官样本中，抑或在经过基因工程改造的具免疫功能的肝癌小鼠模型以及具有索拉菲尼抗性的肝癌细胞系或在免疫缺陷小鼠身上接种的人源化肿瘤异种移植组织中均被证明有效。我们的研究描述了目前为止针对抗ANXA3单克隆抗体最广泛的临床前表征，并且支持其与索拉菲尼/雷戈菲尼组合的临床试验开发。进一步的研究将有助于病人的针对性选择以及寻找最佳治疗组合方案。