BACKGROUND Aberrant AKT activation contributes to cancer stem cell (CSC) traits in hepatocellular carcinoma (HCC). We previously reported that CD73 activated AKT signaling via the Rap1/P110β cascade. Here, we further explored the roles of CD73 in regulating CSC characteristics of HCC. METHODS CD73 expression modulations were conducted by lentiviral transfections. CD73+ fractions were purified by magnetic-based sorting, and fluorescent-activated cell sorting was used to assess differentiation potentials. A sphere-forming assay was performed to evaluate CSC traits in vitro, subcutaneous NOD/SCID mice models were generated to assess in vivo CSC features, and colony formation assays assessed drug resistance capacities. Stemness-associated gene expression was also determined, and underlying mechanisms were investigated by evaluating immunoprecipitation and ubiquitylation. RESULTS We found CD73 expression was positively associated with sphere-forming capacity and elevated in HCC spheroids. CD73 knockdown hindered sphere formation, Lenvatinib resistance, and stemness-associated gene expression, while CD73 overexpression achieved the opposite effects. Moreover, CD73 knockdown significantly inhibited the in vivo tumor propagation capacity. Notably, we found that CD73+ cells exhibited substantially stronger CSC traits than their CD73- counterparts. Mechanistically, CD73 exerted its pro-stemness activity through dual AKT-dependent mechanisms: activating SOX9 transcription via c-Myc, and preventing SOX9 degradation by inhibiting glycogen synthase kinase 3β. Clinically, the combined analysis of CD73 and SOX9 achieved a more accurate prediction of prognosis. CONCLUSIONS Collectively, CD73 plays a critical role in sustaining CSCs traits by upregulating SOX9 expression and enhancing its protein stability. Targeting CD73 might be a promising strategy to eradicate CSCs and reverse Lenvatinib resistance in HCC.

中文翻译：

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CD73通过促进肝细胞癌中SOX9的表达和稳定性来维持癌症干细胞特性。

背景技术异常的AKT激活有助于肝细胞癌（HCC）中的癌症干细胞（CSC）性状。我们先前曾报道CD73通过Rap1 /P110β级联激活AKT信号传导。在这里，我们进一步探讨了CD73在调节肝癌CSC特征中的作用。方法通过慢病毒转染进行CD73表达调节。通过磁性分选法纯化CD73 +馏分，并使用荧光激活的细胞分选法评估分化潜能。进行球形成测定以评估体外CSC特征，产生皮下NOD / SCID小鼠模型以评估体内CSC特征，并且菌落形成测定评估抗药性。还确定了与茎相关的基因表达，通过评估免疫沉淀和泛素化来研究其基本机制。结果我们发现CD73表达与成球能力正相关，并在HCC球体中升高。CD73击倒阻碍了球的形成，Lenvatinib耐药性和与干性相关的基因表达，而CD73的过表达则起到了相反的作用。而且，CD73敲低显着抑制了体内肿瘤的扩散能力。值得注意的是，我们发现CD73 +细胞比其CD73-细胞具有更强的CSC特性。在机制上，CD73通过双重AKT依赖性机制发挥其前茎活性：通过c-Myc激活SOX9转录，并通过抑制糖原合酶激酶3β防止SOX9降解。临床上 CD73和SOX9的组合分析可以更准确地预测预后。结论CD73通过上调SOX9表达并增强其蛋白质稳定性在维持CSCs性状中起着关键作用。靶向CD73可能是根除肝癌和逆转肝癌Lenvatinib耐药性的有前途的策略。