Integrated Analysis of the Anoikis-Related Signature Identifies Rac Family Small GTPase 3 as a Novel Tumor-Promoter Gene in Hepatocellular Carcinoma

Abstract

Anoikis resistance in hepatocellular carcinoma (HCC) cells boosts survival and metastasis. This study aimed to establish an anoikis-related genes (ARGs)-based model for predicting HCC patients’ outcomes and investigate the clinicopathological significance and function of crucial ARGs. The transcriptional expression patterns for HCC cohorts were compiled from TCGA, GEO and ICGC. Univariate and LASSO multivariate analyses were performed to screen for prognostic ARGs. Gain- and loss-of-function studies, RNA sequencing, and mass spectrometry were employed to elucidate the underlying mechanisms of ARGs in HCC. We established a five-gene ARGs risk model for HCC prognosis, with an AUC value of 0.812 for 1-year survival. Among the five genes, Rac family small GTPase 3 (RAC3) was upregulated in HCC relative to adjacent normal tissues and negatively correlated to overall survival and disease-free survival of patients with HCC. Silence of RAC3 in HCC cells resulted in an increased cell apoptosis and diminished cell proliferation and invasion. Mechanistically, we uncovered that RAC3 binding with SOX6 propelled the advancement of HCC cells through NNMT-mediated stimulation of the cAMP/MAPK/Rap1 signaling. In particular, EHop-016, a small molecule inhibitor targeting RAC3, significantly suppressed HCC progression.

Keywords

anoikis / hepatocellular carcinoma / prognostic signature / Rac family small GTPase 3 / risk model

References

[1] H. Sung, J. Ferlay, R. L. Siegel, et al., “Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries,” CA: A Cancer Journal for Clinicians 71, no. 3 (2021): 209-249.

[2] R. Zheng, C. Qu, S. Zhang, et al., “Liver Cancer Incidence and Mortality in China: Temporal Trends and Projections to 2030,” Chinese Journal of Cancer Research = Chung-kuo Yen Cheng Yen Chiu 30, no. 6 (2018): 571-579.

[3] X. Luo, X. He, X. Zhang, et al., “Hepatocellular Carcinoma: Signaling Pathways, Targeted Therapy, and Immunotherapy,” MedComm 5, no. 2 (2024): e474.

[4] A. Forner, M. Reig, and J. Bruix, “Hepatocellular Carcinoma,” Lancet 391, no. 10127 (2018): 1301-1314.

[5] S. Gerstberger, Q. Jiang, and K. Ganesh, “Metastasis,” Cell 186, no. 8 (2023): 1564-1579.

[6] S. U. Khan, K. Fatima, and F. Malik, “Understanding the Cell Survival Mechanism of Anoikis-Resistant Cancer Cells During Different Steps of Metastasis,” Clinical & Experimental Metastasis 39, no. 5 (2022): 715-726.

[7] J. Winkler, A. Abisoye-Ogunniyan, K. J. Metcalf, and Z. Werb, “Concepts of Extracellular Matrix Remodelling in Tumour Progression and Metastasis,” Nature Communications 11, no. 1 (2020): 5120.

[8] Y. Wang, S. Cheng, J. S. Fleishman, et al., “Targeting Anoikis Resistance as a Strategy for Cancer Therapy,” Drug Resistance Updates 75 (2024): 101099.

[9] J. Mei, X. Y. Jiang, H. X. Tian, et al., “Anoikis in Cell Fate, Physiopathology, and Therapeutic Interventions,” MedComm 5, no. 10 (2024): e718.

[10] Y. Chen, W. Huang, J. Ouyang, J. Wang, and Z. Xie, “Identification of Anoikis-Related Subgroups and Prognosis Model in Liver Hepatocellular Carcinoma,” International Journal of Molecular Sciences 24, no. 3 (2023): 2862.

[11] X. Hu, Y. Zhao, L. Wei, et al., “CCDC178 Promotes Hepatocellular Carcinoma Metastasis Through Modulation of Anoikis,” Oncogene 36, no. 28 (2017): 4047-4059.

[12] J. Song, Y. Liu, F. Liu, et al., “The 14-3-3σ Protein Promotes HCC Anoikis Resistance by Inhibiting EGFR Degradation and Thereby Activating the EGFR-Dependent ERK1/2 Signaling Pathway,” Theranostics 11, no. 3 (2021): 996-1015.

[13] S. K. Donnelly, R. Cabrera, S. P. H. Mao, et al., “Rac3 Regulates Breast Cancer Invasion and Metastasis by Controlling Adhesion and Matrix Degradation,” Journal of Cell Biology 216, no. 12 (2017): 4331-4349.

[14] C. Zhang, T. Liu, G. Wang, et al., “Rac3 Regulates Cell Invasion, Migration and EMT in Lung Adenocarcinoma Through p38 MAPK Pathway,” Journal of Cancer 8, no. 13 (2017): 2511-2522.

[15] L. Wang, J. Shi, S. Liu, et al., “RAC3 Inhibition Induces Autophagy to Impair Metastasis in Bladder Cancer Cells via the PI3K/AKT/mTOR Pathway,” Frontiers in Oncology 12 (2022): 915240.

[16] T. Wang, K. Guo, S. Yang, et al., “Identification and Validation of SLCO4C1 as a Biological Marker in Hepatocellular Carcinoma Based on Anoikis Classification Features,” Aging (Albany NY) 16, no. 2 (2024): 1440-1462.

[17] M. P. Walker, M. Zhang, T. P. Le, P. Wu, M. Lainé, and G. L. Greene, “RAC3 is a Pro-Migratory Co-Activator of ERα,” Oncogene 30, no. 17 (2011): 1984-1994.

[18] R. Donne and A. Lujambio, “The Liver Cancer Immune Microenvironment: Therapeutic Implications for Hepatocellular Carcinoma,” Hepatology 77, no. 5 (2023): 1773-1796.

[19] J. Kim, S. J. Hong, E. K. Lim, et al., “Expression of Nicotinamide N-Methyltransferase in Hepatocellular Carcinoma Is Associated With Poor Prognosis,” Journal of Experimental & Clinical Cancer Research 28, no. 1 (2009): 20.

[20] G. Li, B. Kong, Q. Tong, et al., “Vanillin Downregulates NNMT and Attenuates NNMT‑Related Resistance to 5‑Fluorouracil via ROS‑Induced Cell Apoptosis in Colorectal Cancer Cells,” Oncology Reports 45, no. 6 (2021): 110.

[21] P. N. Fernandez Larrosa, C. V. Alvarado, M. F. Rubio, et al., “Nuclear Receptor Coactivator RAC3 Inhibits Autophagy,” Cancer Science 103, no. 12 (2012): 2064-2071.

[22] S. Dharmawardhane, E. Hernandez, and C. Vlaar, “Development of EHop-016: A Small Molecule Inhibitor of Rac,” Enzymes 33 (2013): 117-146.

[23] K. L. Schaefer, K. Wada, H. Takahashi, et al., “Peroxisome Proliferator-Activated Receptor Gamma Inhibition Prevents Adhesion to the Extracellular Matrix and Induces Anoikis in Hepatocellular Carcinoma Cells,” Cancer Research 65, no. 6 (2005): 2251-2259.

[24] Y. Chen, Q. X. Lin, Y. T. Xu, et al., “An Anoikis-Related Gene Signature Predicts Prognosis and Reveals Immune Infiltration in Hepatocellular Carcinoma,” Frontiers in Oncology 13 (2023): 1158605.

[25] X. Diao, C. Guo, and S. Li, “Identification of a Novel Anoikis-Related Gene Signature to Predict Prognosis and Tumor Microenvironment in Lung Adenocarcinoma,” Thoracic Cancer 14, no. 3 (2023): 320-330.

[26] Z. Cai and F. Zhou, “A Novel Anoikis and Immune-Related Genes Marked Prognostic Signature for Colorectal Cancer,” Medicine 101, no. 46 (2022): e31127.

[27] Z. Zhong, F. Xie, J. Yin, et al., “Development of a Prognostic Model for Anoikis and Identifies Hub Genes in Hepatocellular Carcinoma,” Scientific Reports 13, no. 1 (2023): 14723.

[28] M. D. Asmamaw, Y. Liu, Y. C. Zheng, X. J. Shi, and H. M. Liu, “Skp2 in the Ubiquitin-Proteasome System: A Comprehensive Review,” Medicinal Research Reviews 40, no. 5 (2020): 1920-1949.

[29] D. F. Calvisi, S. Ladu, F. Pinna, et al., “SKP2 and CKS1 Promote Degradation of Cell Cycle Regulators and Are Associated With Hepatocellular Carcinoma Prognosis,” Gastroenterology 137, no. 5 (2009): 1816-1826.

[30] J. Yao, S. Tang, C. Shi, et al., “Isoginkgetin, a Potential CDK6 Inhibitor, Suppresses SLC2A1/GLUT1 Enhancer Activity to Induce AMPK-ULK1-Mediated Cytotoxic Autophagy in Hepatocellular Carcinoma,” Autophagy 19, no. 4 (2023): 1221-1238.

[31] I. Cosi, A. Pellecchia, E. De Lorenzo, et al., “ETV4 Promotes Late Development of Prostatic Intraepithelial Neoplasia and Cell Proliferation Through Direct and p53-Mediated Downregulation of p21,” Journal of Hematology & Oncology 13, no. 1 (2020): 112.

[32] X. Xu, B. Wang, Y. Liu, et al., “ETV4 Potentiates Nuclear YAP Retention and Activities to Enhance the Progression of Hepatocellular Carcinoma,” Cancer Letters 537 (2022): 215640.

[33] D. Manna, S. C. Reghupaty, M. D. C. Camarena, et al., “Melanoma Differentiation Associated Gene-9/Syndecan Binding Protein Promotes Hepatocellular Carcinoma,” Hepatology 78, no. 6 (2023): 1727-1741.

[34] M. Nishikawa, M. Scala, M. Umair, et al., “Gain-of-Function p.F28S Variant in RAC3 Disrupts Neuronal Differentiation, Migration and Axonogenesis During Cortical Development, Leading to Neurodevelopmental Disorder,” Journal of Medical Genetics 60, no. 3 (2023): 223-232.

[35] C. Cheng, D. Song, Y. Wu, and B. Liu, “RAC3 Promotes Proliferation, Migration and Invasion via PYCR1/JAK/STAT Signaling in Bladder Cancer,” Frontiers in Molecular Biosciences 7 (2020): 218.

[36] M. D. M. Maldonado and S. Dharmawardhane, “Targeting Rac and Cdc42 GTPases in Cancer,” Cancer Research 78, no. 12 (2018): 3101-3111.

[37] H. Song, Y. Ge, J. Xu, et al., “Identification and Validation of Novel Signature Associated With Hepatocellular Carcinoma Prognosis Using Single-Cell and WGCNA Analysis,” International Journal of Medical Sciences 20, no. 7 (2023): 870-887.

[38] D. L. Gu, Y. H. Chen, J. H. Shih, C. H. Lin, Y. S. Jou, and C. F. Chen, “Target Genes Discovery Through Copy Number Alteration Analysis in Human Hepatocellular Carcinoma,” World Journal of Gastroenterology 19, no. 47 (2013): 8873-8879.

[39] C. Meijuan, L. Fang, F. Min, and W. Qian, “Hypomethylated Gene RAC3 Induces Cell Proliferation and Invasion by Increasing FASN Expression in Endometrial Cancer,” International Journal of Biochemistry & Cell Biology 150 (2022): 106274.

[40] M. F. Rubio, M. C. Lira, F. D. Rosa, A. D. Sambresqui, M. C. Salazar Güemes, and M. A. Costas, “RAC3 Influences the Chemoresistance of Colon Cancer Cells Through Autophagy and Apoptosis Inhibition,” Cancer Cell International 17 (2017): 111.

[41] H. Zhang, Q. Kong, J. Wang, Y. Jiang, and H. Hua, “Complex Roles of cAMP-PKA-CREB Signaling in Cancer,” Experimental Hematology & Oncology 9, no. 1 (2020): 32.

[42] Q. Huang, H. Chen, D. Yin, et al., “Multi-Omics Analysis Reveals NNMT as a Master Metabolic Regulator of Metastasis in Esophageal Squamous Cell Carcinoma,” NPJ Precision Oncology 8, no. 1 (2024): 24.

[43] D. Dixit, B. C. Prager, R. C. Gimple, et al., “Glioblastoma Stem Cells Reprogram Chromatin in Vivo to Generate Selective Therapeutic Dependencies on DPY30 and Phosphodiesterases,” Science Translational Medicine 14, no. 626 (2022): eabf3917.

[44] H. Huang, Y. Wang, M. Kandpal, et al., “FTO-Dependent N6-Methyladenosine Modifications Inhibit Ovarian Cancer Stem Cell Self-Renewal by Blocking cAMP Signaling,” Cancer Research 80, no. 16 (2020): 3200-3214.

[45] Z. Huang, L. Zhou, J. Duan, et al., “Oxidative Stress Promotes Liver Cancer Metastasis via RNF25-Mediated E-Cadherin Protein Degradation,” Advanced Science 11, no. 13 (2024): e2306929.

[46] G. Shan, G. Bi, G. Zhao, et al., “Inhibition of PKA/CREB1 Pathway Confers Sensitivity to Ferroptosis in Non-Small Cell Lung Cancer,” Respiratory Research 24, no. 1 (2023): 277.

RIGHTS & PERMISSIONS

2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.