Gastroenterology

Gastroenterology

Volume 165, Issue 3, September 2023, Pages 629-646
Gastroenterology

Original Research
Full Report: GI Cancer
Nucleolar HEAT Repeat Containing 1 Up-regulated by the Mechanistic Target of Rapamycin Complex 1 Signaling Promotes Hepatocellular Carcinoma Growth by Dominating Ribosome Biogenesis and Proteome Homeostasis

https://doi.org/10.1053/j.gastro.2023.05.029Get rights and content

Background & Aims

Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression.

Methods

HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells.

Results

HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth in vitro and in vivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1–mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis.

Conclusions

Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1–mammalian target of rapamycin complex 1–specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.

Section snippets

Reagents and Treatments

Cells starved for 24 hours were supplemented with serum-free medium containing recombinant human insulin-like growth factor 1 (IGFI; 20 ng/mL; R&D System) or insulin (200 nmol/L; HY-P0035, MedChemExpress) for 3 hours and then were analyzed for protein or messenger (m)RNA expression. To block, mechanistic target of rapamycin complex 1 (mTORC1) signaling or SP1, cells were incubated with rapamycin (10 nmol/L; Cell Signaling Technology) or tolfenamic acid (15 μg/mL; Selleck) for 24 hours. To block

HEAT Repeat Containing 1 Is Up-regulated in Hepatocellular Carcinoma and Correlates With Hepatocellular Carcinoma Progression and Prognosis

Given the importance of ribosome biogenesis in HCC carcinogenesis,5,25 we vertically compared the expression profiles of 307 ribosome biogenesis-related genes (Gene Ontology [GO]: 0042254) (Supplementary Table 4) in the Gene Expression Omnibus Series (GSE) 14520 data set (the largest data set, containing 247 HCC samples and 242 normal tissues). We found that HEATR1 was the most overexpressed gene with the lowest P value (fold change = 2.837, false discovery rate–adjusted P = 7.46E−85); its

Discussion

Ribosome biogenesis, which is regulated by growth signaling that converges in the nucleolus, has been revealed as a druggable pathway for cancer therapeutics.9,44 In this study, we found that HEATR1 is up-regulated in HCC via the IGF1–mTORC1–SP1 axis and contributes to HCC growth by promoting ribosome biogenesis. Disruption of HEATR1 impaired ribosome biogenesis and global protein synthesis, leading to decreased cytoplasmic proteasome activity and IκB/NF-κB signaling. Simultaneously, nucleolar

Acknowledgments

Xiao-Mei Yang, Xiao-Qi Wang, and Li-Peng Hu contributed equally to this article. The authors thank Xiao Li, Shan Zhang, Hui-Ling Wang, Jun-Ping Ao, and Juan Yang for their technical support.

CRediT Authorship Contributions

Zhi-Gang Zhang, PhD (Conceptualization: Lead; Data curation: Lead; Funding acquisition: Lead; Project administration: Lead; Supervision: Lead; Writing – review & editing: Lead).

Xiao-Mei Yang, PhD (Conceptualization: Lead; Data curation: Lead; Funding acquisition: Equal; Investigation: Lead; Methodology:

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    Conflicts of interest The authors disclose no conflicts.

    Funding This study was supported by the National Natural Science Foundation of China (82230087 to Zhi-Gang Zhang and 81972582 to Xiao-Mei Yang), the Medicine and Engineering Interdisciplinary Research Fund of Shanghai Jiao Tong University (YG2021ZD08 to Zhi-Gang Zhang), the Shanghai Pilot Program for Basic Research—Shanghai Jiao Tong University (21TQ1400225 to Shu-Heng Jiang), the Natural Science Foundation of Shanghai (22ZR1460000 to Xue-Li Zhang, 21ZR1461300 to Li-Peng Hu), the Shanghai Municipal Health Commission (No. 202040092 to Xue-Li Zhang), Shanghai Sailing Program (21YF1445200 to Li-Peng Hu), and the Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20210802).

    Author names in bold designate shared co-first authorship.

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