Department of Hepatobiliary Surgery, Liver Cancer Center, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China.
Department of Medicine II, University Hospital, University of Munich, Munich, 80333, Germany.
Cell Oncol (Dordr). 2021 Oct;44(5):1151-1166. doi: 10.1007/s13402-021-00625-w. Epub 2021 Aug 2.
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.
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.
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.
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.
肝细胞癌（HCC）已成为全球癌症相关死亡的主要原因，其中缺氧和激活的缺氧诱导因子（HIFs）起着重要作用。在缺氧肿瘤生长和进展中，HIF-1α 和 HIF-2α 之间的兄弟竞争仍有待解决，包括在 HCC 中。在这项研究中，我们旨在分析 HIF-1α 和 HIF-2α 如何平衡 HCC 细胞对缺氧的增殖反应。
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。通过共免疫沉淀、染色质免疫沉淀、原位邻近连接、 Annexin V-FITC/PI 染色凋亡和 MTT 测定，进一步评估了缺氧 HCC 细胞中 HIF-1α 和 HIF-2α 之间的关系及其各自的作用。此外，还应用针对 HIF-1α/2α 和 Rictor 的短发夹 RNA（shRNA）转染和 Western blot 在 HCC 细胞中研究潜在机制。
我们发现 HIF-2α 表达与肿瘤组织中 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 与 c-MYC 之间的相互作用增加。尽管轻度缺氧导致 HIF-1α、HIF-2α 和 c-MYC 上调，但我们发现 mTORC2 调节的 HIF-2α 与 HIF-1α 竞争与 c-MYC 结合。此外，我们发现 HIF-2α 敲低降低了下游 c-MYC 的表达，抑制了缺氧细胞的增殖，并诱导了 HCC 细胞凋亡，而 HIF-1α 敲低则没有。此外，我们发现 PI3K 抑制剂阿替利珠单抗拮抗了 HIF-2α 的作用，从而诱导 HCC 细胞凋亡。
我们的数据强调了 HIF-2α 在激活和与 c-MYC 结合从而诱导轻度慢性缺氧时 HCC 细胞增殖中的作用。PI3K/mTORC2/HIF-2α/c-MYC 轴可能在这个过程中起关键作用。PI3K 抑制剂阿替利珠单抗可能是患有 HCC 的患者的一种潜在治疗选择，特别是在轻度慢性缺氧条件下肿瘤快速生长的情况下。
