Abstract: 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.

Carfilzomib通过上调Gadd45a表达抑制肺腺癌细胞生长

[1]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.

[2]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.

[3]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.

[4]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.

[5]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.

[6]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.

[7]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.

[8]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.

[9]Hollander MC, Sheikh MS, Bulavin DV, et al., 1999. Genomic instability in Gadd45a-deficient mice. Nat Genet, 23(2):176-184.

[10]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.

[11]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.

[12]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.

[13]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.

[14]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.

[15]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.

[16]Liebermann DA, Hoffman B, 2008. Gadd45 in stress signaling. J Mol Signal, 3:15.

[17]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.

[18]Liu Y, Ao X, Ding W, et al., 2018. Critical role of FOXO3a in carcinogenesis. Mol Cancer, 17:104.

[19]Manasanch EE, Orlowski RZ, 2017. Proteasome inhibitors in cancer therapy. Nat Rev Clin Oncol, 14(7):417-433.

[20]Miller KD, Siegel RL, Lin CC, et al., 2016. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin, 66(4):271-289.

[21]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.

[22]Orlowski RZ, Kuhn DJ, 2008. Proteasome inhibitors in cancer therapy: lessons from the first decade. Clin Cancer Res, 14(6):1649-1657.

[23]Park JE, Miller Z, Jun Y, et al., 2018. Next-generation proteasome inhibitors for cancer therapy. Transl Res, 198: 1-16.

[24]Plas DR, Thompson CB, 2003. Akt activation promotes degradation of tuberin and FOXO3a via the proteasome. J Biol Chem, 278(14):12361-12366.

[25]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://dx.doi.org/10.1631/jzus.B1800211

[26]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.

[27]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.

[28]Siegel RL, Miller KD, Jemal A, 2018. Cancer statistics, 2018. CA Cancer J Clin, 68(1):7-30.

[29]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.

[30]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.

[31]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.

[32]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.

[33]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.

[34]Yang C, Yang SH, Wood KB, et al., 2009. Multidrug resistant osteosarcoma cell lines exhibit deficiency of GADD45α expression. Apoptosis, 14(1):124-133.

[35]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.

[36]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.

[37]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.