The expression level of SKP2 gene affects the radiosensitivity in hepatocellular carcinoma cells
Yang Weifang1,2,3, Wang Xuequan1,2,3, Xia Xinhang1,2, Ran Gang1, Wang Wei1,2, Zhou Jiahui1, Yang Haihua1,2,3, Pi Wenhu1,2
1Department of Radiation Oncology, Taizhou Hospital of Zhejiang Province, Linhai 317000, China; 2Key Laboratory of Radiation Oncology of Taizhou, Linhai 317000, China; 3Key Laboratory of Minimally Invasive Techniques & Rapid Rehabilitation of Digestive System Tumor of Zhejiang Province, Linhai 317000, China
Abstract：Objective To investigate the effect of S‐phase kinase‐associated protein 2 (SKP2) expression level on radiosensitivity of human hepatocellular carcinoma (HCC) cells and the correlation of SKP2 expression with clinical prognosis of patients with HCC. Methods The expression levels of SKP2 gene in liver cancer tissues and normal tissues were validated and its correlation with clinical prognosis of HCC patients was analyzed based on the TCGA database. Western blot was used to determine the SKP2 protein levels in HCC cell lines before and after radiation. CRISPR/Cas9 technology was employed to delete the promoter and first exon of SKP2 gene in PLC/PRF/5 (PLC) and Hep3B HCC cells for generating the SKP2 knockout cell lines. The difference of radiosensitivity and cell survival rate between normal (SKP2 +/ +) and SKP2 knockout (SKP2 ‐/ ‐) HCC cells was determined by using cell clonogenic assay and CCK8 kit. Results Compared with normal tissues, the expression levels of SKP2 gene in HCC were increased based on the results of TCGA database analysis. K‐M analysis showed that the HCC patients with high SKP2 expression had relatively poor prognosis. The 5‐year overall survival (OS) was 34.6% in high SKP2 expression HCC patients and 50.6% in low SKP2 expression HCC patients, respectively (HR=2.18, 95%CI=1.46‐3.27, P<0.001). In vitro experiment showed that the expression levels of SKP2 were significantly increased after radiation in HCC cells. Simultaneously, deletion of SKP2 significantly increased the radiosensitivity of HCC cells. Conclusion The expression level of SKP2 gene is increased in HCC patients, and patients with high SKP2 expression have worse prognosis than those with low expression. Radiation can upregulate the SKP2 expression levels in HCC cells, while the radiosensitivity of the cells is significantly increased after SKP2 deletion.
Yang Weifang,Wang Xuequan,Xia Xinhang et al. The expression level of SKP2 gene affects the radiosensitivity in hepatocellular carcinoma cells[J]. Chinese Journal of Radiation Oncology, 2022, 31(10): 922-927.
[1] Chen WQ, Zheng RS, Baade PD, et al. Cancer statistics in China, 2015[J]. Ca Cancer J Clin, 2016,66(2):115‐132. DOI: 10.3322/caac. 21338. [2] Villanueva A. Hepatocellular carcinoma[J]. N Engl J Med, 2019,380(15): 1450‐1462. DOI: 10. 1056 / NEJMra1713263. [3] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019[J]. CA Cancer J Clin, 2019,69(1): 7‐34. DOI: 10.3322/ caac. 21551. [4] Chen W, Chiang CL, Dawson LA.Efficacy and safety of radiotherapy for primary liver cancer[J]. Chin Clin Oncol, 2021,10(1):9. DOI: 10.21037/cco-20-89. [5] Chen B, Wu JX, Cheng SH, et al. Phase 2 study of adjuvant radiotherapy following narrow‐margin hepatectomy in patients with HCC[J]. Hepatology, 2021,74(5):2595‐2604. DOI: 10.1002/hep. 31993. [6] Asmamaw MD, Liu Y, Zheng YC, et al.Skp2 in the ubiquitin‐proteasome system: a comprehensive review[J]. Med Res Rev, 2020, 40(5):1920‐1949. DOI: 10.1002/med. 21675. [7] Barré B, Perkins ND.The Skp2 promoter integrates signaling through the NF-kappaB, p53, and Akt/GSK3beta pathways to regulate autophagy and apoptosis[J]. Mol Cell, 2010,38(4):524-538. DOI: 10.1016/j.molcel.2010.03.018. [8] Chan CH, Lee SW, Wang J, et al. Regulation of Skp2 expression and activity and its role in cancer progression[J]. Scientific World Journal, 2010,10:1001‐1015. DOI: 10.1100/tsw. 2010.89. [9] Wang S, Raven JF, Koromilas AE.STAT1 represses Skp2 gene transcription to promote p27Kip1 stabilization in Ras- transformed cells[J]. Mol Cancer Res, 2010,8(5):798-805. DOI: 10.1158/1541-7786.MCR-10-0027. [10] Liu SG, Wang BS, Jiang YY, et al.Atypical protein kinase Cι (PKCι) promotes metastasis of esophageal squamous cell carcinoma by enhancing resistance to Anoikis via PKCι-SKP2-AKT pathway[J]. Mol Cancer Res, 2011,9(4):390-402. DOI: 10.1158/1541-7786.MCR-10-0359. [11] Hung WC, Tseng WL, Shiea J, et al. Skp2 overexpression increases the expression of MMP‐2 and MMP‐9 and invasion of lung cancer cells[J]. Cancer Lett, 2010,288(2):156‐161. DOI: 10.1016/j. canlet. 2009.06.032. [12] Frescas D, Pagano M.Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer[J]. Nat Rev Cancer, 2008,8(6):438-449. DOI: 10.1038/nrc2396. [13] Jia J, Yan DM, Wang YZ, et al.Skp2 regulates DNA damage repair and apoptosis via interaction with Ku70[J]. Exp Cell Res, 2020,397(1):112335. DOI: 10.1016/j. yexcr. 2020.112335. [14] Yan W, Yang Y, Yang W. Inhibition of SKP2 activity impaired ATM‐mediated DNA repair and enhanced sensitivity of cisplatin‐resistant mantle cell lymphoma cells[J]. Cancer Biother Radiopharm, 2019,34(7):451‐458. DOI: 10.1089/cbr. 2019.2787. [15] Panaccione A, Chang MT, Carbone BE, et al.NOTCH1 and SOX10 are essential for proliferation and radiation resistance of cancer stem-like cells in adenoid cystic carcinoma[J]. Clin Cancer Res, 2016,22(8):2083-2095. DOI: 10.1158/1078-0432.CCR-15-2208. [16] Wang XC, Tian LL, Tian J, et al.Overexpression of SKP2 promotes the radiation resistance of esophageal squamous cell carcinoma[J]. Radiat Res, 2012,177(1):52-58. DOI: 10.1667/rr2679.1. [17] Li C, Du L, Ren Y, et al.SKP2 promotes breast cancer tumorigenesis and radiation tolerance through PDCD4 ubiquitination[J]. J Exp Clin Cancer Res, 2019,38(1):76. DOI: 10.1186/s13046-019-1069-3. [18] Chen J, Xuan Z, Song W, et al.EAG1 enhances hepatocellular carcinoma proliferation by modulating SKP2 and metastasis through pseudopod formation[J]. Oncogene, 2021,40(1):163-176. DOI: 10.1038/s41388-020-01522-6. [19] Lu M, Ma J, Xue W, et al.The expression and prognosis of FOXO3a and Skp2 in human hepatocellular carcinoma[J]. Pathol Oncol Res, 2009,15(4):679-687. DOI: 10.1007/s12253-009-9171-z. [20] Calvisi DF, Ladu S, Pinna F, et al. SKP2 and CKS1 promote degradation of cell cycle regulators and are associated with hepatocellular carcinoma prognosis[J]. Gastroenterology, 2009, 137(5):1816‐26.e1‐10. DOI: 10.1053/j. gastro. 2009.08.005. [21] Anwanwan D, Singh SK, Singhet S, et al.Challenges in liver cancer and possible treatment approaches[J]. Biochim Biophys Acta Rev Cancer, 2020, 1873(1): 188314. DOI: 10.1016/j. bbcan. 2019.188314. [22] Rim CH, Kim HJ, Seong J.Clinical feasibility and efficacy of stereotactic body radiotherapy for hepatocellular carcinoma: a systematic review and meta-analysis of observational studies[J]. Radiother Oncol, 2019,131:135-144. DOI: 10.1016/j.radonc.2018.12.005. [23] Wang J, Han F, Wu J, et al.The role of Skp2 in hematopoietic stem cell quiescence, pool size, and self-renewal[J]. Blood, 2011,118(20):5429-5438. DOI: 10.1182/blood-2010-10- 312785. [24] Cai Z, Moten A, Peng DN, et al. The Skp2 pathway: a critical target for cancer therapy[J]. Semin Cancer Biol, 2020, 67(Pt 2): 16‐33. DOI: 10.1016/j. semcancer. 2020.01.013. [25] Tang X, Li G, Shi L, et al.Combined intermittent fasting and ERK inhibition enhance the anti-tumor effects of chemotherapy via the GSK3β-SIRT7 axis[J]. Nat Commun, 2021,12(1):5058. DOI: 10.1038/s41467-021-25274-3. [26] Kumarasamy V, Vail P, Nambiar R, et al.Functional determinants of cell cycle plasticity and sensitivity to CDK4/6 inhibition[J]. Cancer Res, 2021,81(5):1347-1360. DOI: 10.1158/0008-5472.CAN-20-2275. [27] Hume S, Grou CP, Lascaux P, et al.The NUCKS1-SKP2-p21/p27 axis controls S phase entry[J]. Nat Commun, 2021,12(1):6959. DOI: 10.1038/s41467-021-27124-8. [28] Wu J, Zhang X, Zhang L, et al. Skp2 E3 ligase integrates ATM activation and homologous recombination repair by ubiquitinating NBS1[J]. Mol Cell, 2012, 46(3): 351‐361. DOI: 10.1016/j. molcel. 2012.02.018. [29] Li F, Mladenov E, Mortoga S, et al.SCFSKP2 regulates APC/CCDH1-mediated degradation of CTIP to adjust DNA-end resection in G2-phase[J]. Cell Death Dis, 2020,11(7):548. DOI: 10.1038/s41419-020-02755-9. [30] Ye C, Xiong W, Lei CT, et al. MAD2B contributes to parietal epithelial cell activation and crescentic glomerulonephritis via Skp2[J]. Am J Physiol Renal Physiol, 2020, 319(4):F636‐F646. DOI: 10.1152/ajprenal. 00216.2020. [31] Vignard J, Mirey G, Salles B.Ionizing-radiation induced DNA double-strand breaks: a direct and indirect lighting up[J]. Radiother Oncol, 2013,108(3):362-369. DOI: 10.1016/j.radonc.2013.06.013.
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