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CHIP promotes CAD ubiquitination and degradation to suppress the proliferation and colony formation of glioblastoma cells

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Abstract

Purpose

Cancer cells are characterized as the uncontrolled proliferation, which demands high levels of nucleotides that are building blocks for DNA synthesis and replication. CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase) is a trifunctional enzyme that initiates the de novo pyrimidine synthesis, which is normally enhanced in cancer cells to preserve the pyrimidine pool for cell division. Glioma, representing most brain cancer, is highly addicted to nucleotides like pyrimidine to sustain the abnormal growth and proliferation of cells. CAD is previously reported to be dysregulated in glioma, but the underlying mechanism remains unclear.

Methods

The expression of CAD and CHIP (carboxyl terminus of Hsc70-interacting protein) protein in normal brain cells and three glioblastoma (GBM) cell lines were measured by immunoblots. Lentiviruses-mediated expression of target proteins or shRNAs were used to specifically overexpress or knock down CAD and CHIP. Cell counting, colony formation, apoptosis and cell cycle assays were used to assess the roles of CAD and CHIP in GBM cell proliferation and survival. Co-immunoprecipitation and ubiquitination assays were used to examine the interaction of CHIP with CAD and the ubiquitination of CAD. The correlation of CAD and CHIP expression with GBM patients’ survival was obtained by analyzing the GlioVis database.

Results

In this study, we showed that the expression of CAD was upregulated in glioma, which was positively correlated with the tumor grade and survival of glioma patients. Knockdown of CAD robustly inhibited the cell proliferation and colony formation of GBM cells, indicating the essential role of CAD in the pathogenesis of GBM. Mechanistically, we firstly identified that CAD was modified by the K29-linked polyubiquitination, which was mediated by the E3 ubiquitin ligase CHIP. By interacting with and ubiquitinating CAD, CHIP enhanced its proteasomal and lysosomal degradation, which accounted for the anti-proliferative role of CHIP in GBM cells. To sustain the expression of CAD, CHIP is significantly downregulated, which is correlated with the poor prognosis and survival of GBM patients. Notably, the low level of CHIP and high level of CAD overall predict the short survival of GBM patients.

Conclusion

Altogether, these results illustrated the essential role of CAD in GBM and revealed a novel therapeutic strategy for CAD-positive and CHIP-negative cancer.

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Data Availability

All data related to this study are available within the article.

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Acknowledgements

We thank all lab members for their helps and suggestions.

Funding

This work was supported by grants from the National Natural Science Foundation of China (8210082627), the Natural Science Foundation for Outstanding Young Scholars of Hunan Province (2022JJ20033) and the Key Project of Education Department of Hunan Province (22A0035) to T. Li to T. Li and Hunan Science and Technology Project (2017XK2020), the Key R&D Program of Hunan Province (2019NK2161) to S. He.

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Author notes

  1. Guanya Li and Kai Xiao contributed equally to this work.

Authors and Affiliations

  1. Hunan Provincial Key Laboratory of Animal Intestinal Function and Regulation, School of Life Sciences, Hunan Normal University, Changsha, Hunan, China

    Guanya Li, Yinan Li, Jianfang Gao, Shanping He & Tingting Li

  2. Department of Neurosurgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China

    Kai Xiao

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  1. Guanya Li
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Contributions

Experimental design, Guanya Li, Tingting Li; data acquisition and analysis, Guanya Li, Kai Xiao, Jianfang Gao, Yinan Li, Shanping He, Tingting Li; writing and revision, Guanya Li, Tingting Li; supervision, Shanping He, Tingting Li; funding source, Shanping He, Tingting Li. All authors reviewed the manuscript.

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Correspondence to Shanping He or Tingting Li.

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Li, G., Xiao, K., Li, Y. et al. CHIP promotes CAD ubiquitination and degradation to suppress the proliferation and colony formation of glioblastoma cells. Cell Oncol. (2023). https://doi.org/10.1007/s13402-023-00899-2

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