Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.
最近有研究表明卡非佐米 (Carfilzominb, CFZ) 能有效抑制肺腺癌细胞生长, 但是其中的内在机制仍然需要进一步研究. 本文针对 CFZ 抑制肺腺癌生长机制进行了系统研究.
揭示了蛋白酶体抑制剂抗实体肿瘤的新机制, 为这类药物用于实体肿瘤治疗提供了有利依据. 同时 Gadd45a 可做为候选指标用于蛋白酶体抑制剂抗肿瘤疗效的预测.
应用流式细胞术检测 CFZ 对肺腺癌细胞周期和凋亡的影响; 通过 MTS 比色法及平板克隆形成实验分析 CFZ 对肺腺癌细胞生长的抑制作用; 使用蛋白质印迹法 (western blot) 和定量聚合酶链 反应 (qPCR) 检测相关基因表达水平的改变.
CFZ 通过 AKT/FOXO3a 通路上调 Gadd45a 基因的表达, 诱导肺腺癌细胞周期阻滞和凋亡, 从而发挥抗肿瘤效应.
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Ao L, Reichel D, Hu D, et al., 2015. Polymer micelle formulations of proteasome inhibitor carfilzomib for improved metabolic stability and anticancer efficacy in human multiple myeloma and lung cancer cell lines. J Pharmacol Exp Ther, 355(2):168–173. https://doi.org/10.1124/jpet.115.226993
Baker AF, Hanke NT, Sands BJ, et al., 2014. Carfilzomib demonstrates broad anti-tumor activity in pre-clinical non-small cell and small cell lung cancer models. J Exp Clin Cancer Res, 33:111. https://doi.org/10.1186/s13046-014-0111-8
Bouchard C, Marquardt J, Brás A, et al., 2004. Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins. EMBO J, 23(14): 2830–2840. https://doi.org/10.1038/sj.emboj.7600279
Chung HK, Yi YW, Jung NC, et al., 2003. CR6-interacting factor 1 interacts with Gadd45 family proteins and modulates the cell cycle. J Biol Chem, 278(30):28079–28088. https://doi.org/10.1074/jbc.M212835200
da Silva GN, Filoni LT, Salvadori MC, et al., 2018. Gemcitabine/cisplatin treatment induces concomitant SERTAD1, CDKN2B and GADD45A modulation and cellular changes in bladder cancer cells regardless of the site of TP53 mutation. Pathol Oncol Res, 24(2):407–417. https://doi.org/10.1007/s12253-017-0255-x
Gao M, Li XG, Dong W, et al., 2013. Ribosomal protein S7 regulates arsenite-induced GADD45α expression by attenuating MDM2-mediated GADD45α ubiquitination and degradation. Nucleic Acids Res, 41(10):5210–5222. https://doi.org/10.1093/nar/gkt223
Hanke NT, Garland LL, Baker AF, 2016. Carfilzomib combined with suberanilohydroxamic acid (SAHA) synergistically promotes endoplasmic reticulum stress in non-small cell lung cancer cell lines. J Cancer Res Clin Oncol, 142(3):549–560. https://doi.org/10.1007/s00432-015-2047-6
Hildesheim J, Belova GI, Tyner SD, et al., 2004. Gadd45a regulates matrix metalloproteinases by suppressing ΔNp63α and β-catenin via p38 MAP kinase and APC complex activation. Oncogene, 23(10):1829–1837. https://doi.org/10.1038/sj.onc.1207301
Hollander MC, Sheikh MS, Bulavin DV, et al., 1999. Genomic instability in Gadd45a-deficient mice. Nat Genet, 23(2): 176–184. https://doi.org/10.1038/13802
Hollander MC, Kovalsky O, Salvador JM, et al., 2001. Dimethylbenzanthracene carcinogenesis in Gadd45a-null mice is associated with decreased DNA repair and increased mutation frequency. Cancer Res, 61(6):2487–2491.
Ji J, Liu R, Tong T, et al., 2007. Gadd45a regulates β-catenin distribution and maintains cell-cell adhesion/contact. Oncogene, 26(44):6396–6405. https://doi.org/10.1038/sj.onc.1210469
Jin SQ, Antinore MJ, Lung FDT, et al., 2000. The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression. J Biol Chem, 275(22):16602–16608. https://doi.org/10.1074/jbc.M000284200
Jin SQ, Tong T, Fan WH, et al., 2002. GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene, 21(57):8696–8704. https://doi.org/10.1038/sj.onc.1206034
Li Q, Wei X, Zhou ZW, et al., 2018. GADD45α sensitizes cervical cancer cells to radiotherapy via increasing cytoplasmic ape1 level. Cell Death Dis, 9(5):524. https://doi.org/10.1038/s41419-018-0452-x
Li TH, Ho L, Piperdi B, et al., 2010. Phase II study of the proteasome inhibitor bortezomib (PS-341, Velcade®) in chemotherapy-naive patients with advanced stage non-small cell lung cancer (NSCLC). Lung Cancer, 68(1): 89–93. https://doi.org/10.1016/j.lungcan.2009.05.009
Liebermann DA, Hoffman B, 2008. Gadd45 in stress signaling. J Mol Signal, 3:15. https://doi.org/10.1186/1750-2187-3-15
Liu LQ, Tian FJ, Xiong Y, et al., 2018. Gadd45a gene silencing by RNAi promotes cell proliferation and inhibits apoptosis and senescence in skin squamous cell carcinoma through the p53 signaling pathway. J Cell Physiol, 233(9):7424–7434. https://doi.org/10.1002/jcp.26588
Liu Y, Ao X, Ding W, et al., 2018. Critical role of FOXO3a in carcinogenesis. Mol Cancer, 17:104. https://doi.org/10.1186/s12943-018-0856-3
Manasanch EE, Orlowski RZ, 2017. Proteasome inhibitors in cancer therapy. Nat Rev Clin Oncol, 14(7):417–433. https://doi.org/10.1038/nrclinonc.2016.206
Miller KD, Siegel RL, Lin CC, et al., 2016. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin, 66(4): 271–289. https://doi.org/10.3322/caac.21349
Morgillo F, D’Aiuto E, Troiani T, et al., 2011. Antitumor activity of bortezomib in human cancer cells with acquired resistance to anti-epidermal growth factor receptor tyrosine kinase inhibitors. Lung Cancer, 71(3):283–290. https://doi.org/10.1016/j.lungcan.2010.06.005
Orlowski RZ, Kuhn DJ, 2008. Proteasome inhibitors in cancer therapy: lessons from the first decade. Clin Cancer Res, 14(6):1649–1657. https://doi.org/10.1158/1078-0432.CCR-07-2218
Park JE, Miller Z, Jun Y, et al., 2018. Next-generation proteasome inhibitors for cancer therapy. Transl Res, 198: 1–16. https://doi.org/10.1016/j.trsl.2018.03.002
Plas DR, Thompson CB, 2003. Akt activation promotes degradation of tuberin and FOXO3a via the proteasome. J Biol Chem, 278(14):12361–12366. https://doi.org/10.1074/jbc.M213069200
Ren WB, Xia XJ, Huang J, et al., 2019. Interferon-γ regulates cell malignant growth via the c-Abl/HDAC2 signaling pathway in mammary epithelial cells. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 20(1):39–48. https://doi.org/10.1631/jzus.B1800211
Salvador JM, Brown-Clay JD, Fornace AJ Jr, 2013. Gadd45 in stress signaling, cell cycle control, and apoptosis. In: Liebermann DA, Hoffman B (Eds.), Gadd45 Stress Sensor Genes. Springer, New York, p. 1–19. https://doi.org/10.1007/978-1-4614-8289-5_1
Siegel DS, Martin T, Wang M, et al., 2012. A phase 2 study of single-agent carfilzomib (PX-171-003-A1) in patients with relapsed and refractory multiple myeloma. Blood, 120(14):2817–2825. https://doi.org/10.1182/blood-2012-05-425934
Siegel RL, Miller KD, Jemal A, 2018. Cancer statistics, 2018. CA Cancer J Clin, 68(1):7–30. https://doi.org/10.3322/caac.21442
Smith ML, Chen IT, Zhan Q, et al., 1994. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science, 266(5189):1376–1380. https://doi.org/10.1126/science.7973727
Takekawa M, Saito H, 1998. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell, 95(4):521–530. https://doi.org/10.1016/S0092-8674(00)81619-0
Tong T, Ji JF, Jin SQ, et al., 2005. Gadd45a expression induces Bim dissociation from the cytoskeleton and translocation to mitochondria. Mol Cell Biol, 25(11):4488–4500. https://doi.org/10.1128/MCB.25.11.4488-4500.2005
Tran H, Brunet A, Grenier JM, et al., 2002. DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science, 296(5567): 530–534. https://doi.org/10.1126/science.1068712
van der Wekken AJ, Saber A, Hiltermann TJN, et al., 2016. Resistance mechanisms after tyrosine kinase inhibitors afatinib and crizotinib in non-small cell lung cancer, a review of the literature. Crit Rev Oncol Hematol, 100: 107–116. https://doi.org/10.1016/j.critrevonc.2016.01.024
Yang C, Yang SH, Wood KB, et al., 2009. Multidrug resistant osteosarcoma cell lines exhibit deficiency of GADD45α expression. Apoptosis, 14(1):124–133. https://doi.org/10.1007/s10495-008-0282-x
Yang F, Zhang WM, Li D, et al., 2013. Gadd45a suppresses tumor angiogenesis via inhibition of the mTOR/STAT3 protein pathway. J Biol Chem, 288(9):6552–6560. https://doi.org/10.1074/jbc.M112.418335
Yao K, Fu XF, Du X, et al., 2018. PGC-1α coordinates with Bcl-2 to control the cell cycle in U251 cells through reducing ROS. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 19(6):415–424. https://doi.org/10.1631/jzus.B1700148
Zhan QM, Antinore MJ, Wang XW, et al., 1999. Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene, 18(18):2892–2900. https://doi.org/10.1038/sj.onc.1202667
We thank Dr. Li-feng FENG (Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China) for modifying the article.
Fang YANG, Wang-wang LIU, Hui CHEN, Jia ZHU, Ai-hua HUANG, Fei ZHOU, Yi GAN, Yan-hua ZHANG, and Li MA declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
Project supported by the National Natural Science Foundation of China (Nos. 81601992, 81802986, and 81601029), the Natural Science Foundation of Zhejiang Province (No. LQ16H160008), and the Medical and Health Program of Zhejiang Province (No. 2019338991), China
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Yang, F., Liu, W., Chen, H. et al. Carfilzomib inhibits the growth of lung adenocarcinoma via upregulation of Gadd45a expression. J. Zhejiang Univ. Sci. B 21, 64–76 (2020) doi:10.1631/jzus.B1900551
- Lung adenocarcinoma
- Cell cycle arrest
- 卡非佐米 (Carfilzomib)