Purpose

Hepatocellular carcinoma (HCC) has emerged as a leading cause of cancer-related deaths globally, in which hypoxia and activated hypoxia-inducible factors (HIFs) play important roles. The sibling rivalry between HIF-1α and HIF-2α in hypoxic tumor growth and progression still remains to be resolved, including in HCC. In this study, we aimed to analyze the mechanism by which HIF-1α and HIF-2α balance the proliferative response of HCC cells to hypoxia.

Methods

The expression of HIF-1α, HIF-2α, c-MYC, Rictor and Raptor in corresponding tumor and non-tumor tissues from twenty-six patients with HCC was analyzed. The relationships between HIF-1α and HIF-2α and their respective effects were evaluated further in vitro in hypoxic HCC cells using co-immunoprecipitation, chromatin immunoprecipitation, in situ proximity ligation, annexin V-FITC/PI staining apoptosis and MTT assay. In addition, short hairpin RNA (shRNA) transfections targeting HIF-1α/2α and Rictor and Western blotting were applied in HCC cells to study the underlying mechanism.

Results

We found that HIF-2α expression showed a positive correlation with c-MYC expression in tumor tissues, whereas HIF-1α did not. In vitro, increased HCC cell proliferation and an increased interaction between HIF-2α and c-MYC were observed under mild chronic hypoxic conditions. Although mild hypoxia led to HIF-1α, HIF-2α and c-MYC up-regulation, we found that mTORC2-regulated HIF-2α competed with HIF-1α to bind to c-MYC. Moreover, we found that HIF-2α knockdown decreased the expression of downstream c-MYC, suppressed hypoxic cell proliferation, and induced HCC cell apoptosis, whereas HIF-1α knockdown did not. Additionally, we found that the PI3K inhibitor apitolisib counteracted the effect of HIF-2α, thereby inducing HCC cell apoptosis.

Conclusions

Our data highlight a role of HIF-2α in activating and binding c-MYC, thereby inducing HCC cell proliferation during mild chronic hypoxia. The PI3K/mTORC2/HIF-2α/c-MYC axis may play a key role in this process. The PI3K inhibitor apitolisib may serve as a potential treatment option for patients suffering from HCC, especially in cases with rapidly growing tumors under mild chronic hypoxic conditions.

中文翻译：

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轻度慢性缺氧诱导的 HIF-2α 通过与 HIF-1α 竞争与 c-MYC 相互作用诱导肝癌细胞增殖

目的

肝细胞癌（HCC）已成为全球癌症相关死亡的主要原因，其中缺氧和活化的缺氧诱导因子（HIF）发挥着重要作用。HIF-1α 和 HIF-2α 在低氧肿瘤生长和进展中的同胞竞争仍有待解决，包括在 HCC 中。在本研究中，我们旨在分析 HIF-1α 和 HIF-2α 平衡 HCC 细胞对缺氧的增殖反应的机制。

方法

分析了26例HCC患者相应肿瘤和非肿瘤组织中HIF-1α、HIF-2α、c-MYC、Rictor和Raptor的表达。使用免疫共沉淀、染色质免疫沉淀、原位邻近连接、膜联蛋白 V-FITC/PI 染色凋亡和 MTT 测定，在缺氧 HCC 细胞中进一步评估 HIF-1α 和 HIF-2α 之间的关系及其各自的作用。此外，将靶向 HIF-1α/2α 的短发夹 RNA (shRNA) 转染和 Rictor 和蛋白质印迹应用于 HCC 细胞以研究潜在机制。

结果

我们发现 HIF-2α 表达与肿瘤组织中的 c-MYC 表达呈正相关，而 HIF-1α 则没有。在体外，在轻度慢性缺氧条件下观察到 HCC 细胞增殖增加和 HIF-2α 和 c-MYC 之间的相互作用增加。虽然轻度缺氧导致 HIF-1α、HIF-2α 和 c-MYC 上调，但我们发现 mTORC2 调节的 HIF-2α 与 HIF-1α 竞争结合 c-MYC。此外，我们发现 HIF-2α 敲低降低了下游 c-MYC 的表达，抑制了缺氧细胞增殖，并诱导了 HCC 细胞凋亡，而 HIF-1α 敲低则没有。此外，我们发现 PI3K 抑制剂 apitolisib 抵消了 HIF-2α 的作用，从而诱导 HCC 细胞凋亡。

结论

我们的数据强调了 HIF-2α 在激活和结合 c-MYC 中的作用，从而在轻度慢性缺氧期间诱导 HCC 细胞增殖。PI3K/mTORC2/HIF-2α/c-MYC 轴可能在这个过程中起关键作用。PI3K 抑制剂 apitolisib 可作为 HCC 患者的潜在治疗选择，尤其是在轻度慢性缺氧条件下肿瘤快速生长的病例中。