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Mild chronic hypoxia-induced HIF-2α interacts with c-MYC through competition with HIF-1α to induce hepatocellular carcinoma cell proliferation

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Abstract

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.

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Data Availability

Data and material are available upon reasonable request.

Code Availability

Not applicable.

Abbreviations

HCC:

hepatocellular carcinoma

HIFs:

hypoxia-inducible factors

TAE/TACE:

transcatheter arterial (chemo) embolization

HK2:

glycolytic enzyme hexokinase 2

PDK1:

pyruvate dehydrogenase kinase 1

VEGFA:

vascular endothelial growth factor A

mTOR:

mammalian target of rapamycin

DAPI:

4′,6-diamidino-2-phenylindole

CoCl2:

Cobalt chloride

IC50:

50 % cell growth inhibitory concentrations

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PLA:

in situ Proximity Ligation Assay

Co-IP:

Co-Immunoprecipitation

shRNA:

Short hairpin RNA

ChIP:

Chromatin Immunoprecipitation

HREs:

Hypoxia-Response Element

UICC:

Union for International Cancer Control

PHD:

prolyl-4-hydroxylase

RCC:

renal cell carcinoma

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Acknowledgements

We appreciated the technical support from Mr. Andreas Schmitt and Ms. Weiwei Ma.

Funding

This work was supported by Tianjin Medical University Cancer Institute and Hospital, Tianjin, China (NO. TJ20170110).

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Authors and Affiliations

  1. 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

    Han Mu, Ge Yu, Huikai Li, Yunlong Cui, Ti Zhang & Tianqiang Song

  2. Department of Medicine II, University Hospital, University of Munich, Munich, 80333, Germany

    Mengmeng Wang

  3. Department of Interventional Treatment, 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

    Changfu Liu

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  1. Han Mu
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Contributions

HM and CFL conceived the project. HM and CFL performed all experiments and drafted the manuscript. MMW and TZ supported the experiments. HKL, GY and YLC collected clinical samples and information. CFL and TQS supervised all studies. All authors participated in preparing the manuscript and approved the submitted and published version.

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Correspondence to Changfu Liu.

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Conflict of interest

The authors declare that they have no conflicts of interests.

Ethics approval and consent to participate

All our experiments involving human participants were approved by The Ethics Committee of Tianjin Medical University and performed in accordance with the Declaration of Helsinki. We obtained human HCC tissue and adjacent normal tissue from HCC patients at the Tianjin Medical University Cancer Institute and Hospital with informed consent from all patients.

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Supplementary Information

Fig. S1

The expressions and correlations of Rictor and Raptor in HCC patients. A-B. The number of HCC cases with low or high expressions of Rictor and Raptor in different UICC stage (PNG 119 kb)

High Resolution Image (TIF 283 kb)

Fig. S2

HIF-1α or HIF-2α knockdown in mild chronic hypoxic hepatocellular carcinoma cell. (A) The expressions of HIF-1α and HIF-2α in shHIF-1α or shHIF-2α transfected HCC cells treated by Cocl2low for 72 h. (B) The relative fold changes of HIF-1α, HIF-2α and c-MYC in the QRT-PCR analysis and the relative fold changes of c-MYC in the HIF-2α binding ChiP assay from the HCC samples treated with/without Cocl2low for 72 h (PNG 208 kb)

High Resolution Image (TIF 340 kb)

Fig. S3

c-MYC knockdown suppressed cell growth in mild chronic hypoxic hepatocellular carcinoma cell. (A) The expressions of c-MYC in shRNA c-MYC transfected HCC cells treated by Cocl2low for 72 h. (B) Cell proliferation curve of c-MYC knockdown Huh7 and HepG2 in mild hypoxia for 6 days (PNG 219 kb)

High Resolution Image (TIF 319 kb)

Fig. S4

Rictor knockdown in mild chronic hypoxic hepatocellular carcinoma cell. A. The expressions of Rictor in shRictor transfected HCC cells treated by Cocl2low for 72 h (PNG 83 kb)

High Resolution Image (TIF 159 kb)

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Mu, H., Yu, G., Li, H. et al. Mild chronic hypoxia-induced HIF-2α interacts with c-MYC through competition with HIF-1α to induce hepatocellular carcinoma cell proliferation. Cell Oncol. 44, 1151–1166 (2021). https://doi.org/10.1007/s13402-021-00625-w

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  • DOI: https://doi.org/10.1007/s13402-021-00625-w

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