BACKGROUND Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. METHODS CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110β and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. RESULTS In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110β to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. CONCLUSIONS CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.

中文翻译：

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CD73 通过诱导 Rap1 介导的 P110β 膜定位来激活 PI3K/AKT 信号，从而促进肝细胞癌进展和转移，并预测不良预后。


背景技术肝细胞癌（HCC）是世界范围内最常见的恶性肿瘤之一，因为其进展迅速且转移或复发的发生率高。越来越多的证据表明表达 CD73 的肿瘤细胞与多种癌症的发生有关。然而，CD73在HCC细胞中的作用尚未得到系统研究，其潜在机制仍不清楚。方法采用RT-PCR、Western blot、免疫组化等方法检测HCC细胞中CD73的表达。通过Cox回归分析评估CD73的临床意义。使用细胞计数试剂盒8和集落形成测定法进行增殖评估。 Transwell 测定用于运动性评估。应用免疫共沉淀、胞浆和质膜分级分离以及 ELISA 评估 P110β 的膜定位及其催化活性。采用NOD/SCID/γc(null) (NOG)小鼠模型研究CD73的体内功能。结果在本研究中，我们证明 CD73 对于 HCC 的上皮间质转化 (EMT)、进展和转移至关重要。 CD73 表达在 HCC 细胞中增加，并与侵袭性临床病理特征相关。临床上，CD73 被确定为复发时间和总生存期的独立不良预后指标。 CD73敲低在体外显着抑制HCC细胞增殖、迁移、侵袭和EMT，并在体内阻碍肿瘤生长和转移。当CD73过表达时可以观察到相反的结果。 从机制上讲，CD73 产生的腺苷与腺苷 A2A 受体 (A2AR) 结合并激活 Rap1，后者将 P110β 招募到质膜并触发 PIP3 产生，从而促进 HCC 细胞中的 AKT 磷酸化。值得注意的是，与单独使用单一药物相比，抗 CD73 和抗 A2AR 的组合对 HCC 生长和转移具有协同抑制作用。结论 CD73 通过激活 PI3K/AKT 信号传导促进进展和转移，表明 HCC 是一种新的预后生物标志物。我们的数据证明了 CD73 除了其免疫抑制功能之外在 HCC 中的重要性，并揭示了共同靶向 CD73 和 A2AR 策略可能是未来 HCC 管理的一种有前途的新型治疗策略。