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CRHBP is degraded via autophagy and exerts anti-hepatocellular carcinoma effects by reducing cyclin B2 expression and dissociating cyclin B2-CDK1 complex

Cancer Gene Therapy volume 29, pages 1217–1227 (2022)Cite this article

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Abstract

Autophagy is the predominant self-eating catabolic pathway activated in response to nutrient starvation and hypoxia within the microenvironment of varied malignancies, including hepatocellular carcinoma (HCC). SQSTM1/p62 links its cargos to autophagosomes for degradation, and reportedly acts as a contributor for hepatocarcinogenesis. Five GEO gene microarrays identified corticotropin releasing hormone (CRH) binding protein (CRHBP) as a significantly downregulated gene in HCC (log2 Fold change < −3 and p < 0.001), and an earlier human interactome study indicated that CRHBP may interact with p62. This study aimed to explore (1) the role of CRHBP in HCC development, and (2) whether p62-mediated autophagy was responsible for low CRHBP expression within HCC tissue. Following functional experiments first revealed an anti-proliferative, anti-metastatic, and anti-angiogenic role of CRHBP in HCC cells (Huh-7, Li-7 and HCCLM3) and xenografts. CRHBP negatively regulated cyclin B2 expression, and dissociated cyclin B2-CDK1 complex in HCC cells, thereby leading to cell cycle arrest at G2 phase. To simulate HCC microenvironment in vitro, Huh-7 cells were incubated in Earle’s Balanced Salt Solution (nutrient starvation) or exposed to 1% O2 (hypoxic exposure). In addition to activating autophagy, nutrient starvation and hypoxic exposure also induced CRHBP degradation. Interestingly, CRHBP was demonstrated as a novel cargo targeted by p62 for degradation in autophagosomes. Blocking autophagy with 3-MA, chloroquine or siSQSTM1 prevented CRHBP degradation in HCC cells. Collectively, our study uncovers a role for CRHBP in retarding HCC development, reducing cyclin B2 expression and impairing cyclin B2-CDK1 interaction. CRHBP downregulation in HCC may attribute to p62-mediated autophagy.

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Fig. 1: CRHBP: a downregulated gene that predicts worse overall survival in HCC patients.
Fig. 2: Anti-proliferative and anti-metastatic role of CRHBP in HCC cells.
Fig. 3: Anti-angiogenic role of CRHBP identified in HCC cells.
Fig. 4: CRHBP overexpression impairs tumor formation of HCC cells in nude mice.
Fig. 5: CRHBP induces cell cycle arrest at G2 stage, reduces cyclin B2 expression, and disrupts cyclin B2-CDK1 complex.
Fig. 6: Tumor microenvironment-associated autophagy is responsible for CRHBP degradation in HCC cells.

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All data generated or analyzed during this study are included in this published article.

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Acknowledgements

We acknowledge assistance with the access of analytic instruments from Translational Medical Center at The FirstAffiliated Hospital of Zhengzhou University.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82000453), and theJoint Construction Project of Henan Medical Science and Technology Research Plan (LHGJ20190134).

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  1. These authors contributed equally: Tao Bai and Zhiwei Wang.

Authors and Affiliations

  1. Department of Vascular and Endovascular Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, People’s Republic of China

    Zhiwei Wang, Mingxing Li, Yuanfeng Liu, Zhentao Qiao & Tao Bai

  2. Department of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, People’s Republic of China

    Zhiwei Wang, Mingxing Li, Zhentao Qiao, Ling Yang, Bo Liu & Tao Bai

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Contributions

ZW and TB were responsible for designing designed the study, conducting the experiments, extracting and analyzing data. TB was responsible for drafting the manuscript, ZW was responsible for revising the manuscript. ML, YL and ZQ, were responsible for conducting the experiments. LY and BL were responsible for performed the experiments and interpreting results. All authors read and approved the final manuscript.

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Correspondence to Zhiwei Wang or Tao Bai.

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All institutional and national guidelines for the care and use of laboratory animals were followed. The study was approved by ethics committee of Zhengzhou University.

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Wang, Z., Li, M., Liu, Y. et al. CRHBP is degraded via autophagy and exerts anti-hepatocellular carcinoma effects by reducing cyclin B2 expression and dissociating cyclin B2-CDK1 complex. Cancer Gene Ther 29, 1217–1227 (2022). https://doi.org/10.1038/s41417-021-00423-4

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