Regulation of THZ1, an inhibitor of cyclin-dependent kinase 7, on stemness of glioma stem cells and its mechanism

doi:10.12307/2025.528

Abstract

Abstract: BACKGROUND: THZ1, an inhibitor of cyclin-dependent kinase 7, has been shown to inhibit the proliferation of a variety of tumor cells, but whether THZ1 can affect the stemness of glioma stem cells through the Wnt/β-catenin signaling pathway remains unclear.
OBJECTIVE: To investigate the effect of THZ1 on stemness of glioma cell U87 and its mechanism.
METHODS: U87 adherent cells were cultured to form stem cell mammospheres. The expressions of stemness related proteins were verified by western blot assay. The effect of THZ1 on half maximal inhibitory concentration (IC50) of U87 cells was determined by Cell Counting Kit-8 (CCK-8) assays. The effects of THZ1 on proliferation and migration of U87 cells were determined by cell colony-formation assays, cell wound healing assays, and Transwell migration assays. The effect of THZ1 treatment on mammosphere forming rate and mammosphere size of U87 stem cells was analyzed. Stemness associated proteins CD133, ABCG2, Nanog, OCT4, SOX2, epithelial-mesenchymal transformation-related proteins E-cadherin, N-cadherin, Occludin, Snail, and Wnt/β-catenin pathway associated proteins Axin1, β-Catenin, WNT-5A, GSK3β, Cyclind-1, and C-myc were measured by western blot assay.
RESULTS AND CONCLUSION: (1) Compared with adherent cells, the expressions of stemness related proteins Nestin, CD133, ABCG2, Nanog, OCT4, and SOX2 were significantly increased. (2) Compared with the control group, THZ1 decreased the proliferation and migration of U87 cells. (3) THZ1 inhibited the mammosphere forming rate and mammosphere size of U87 stem cells. (4) After THZ1 treatment, the expression of N-cadherin and Snail decreased, while the protein expression of E-cadherin and Occludin increased. (5) THZ1 treatment decreased the expression of Wnt/β-catenin pathway related proteins Axin1, β-Catenin, Wnt-5A, GSK3β, Cyclind-1, and C-myc in U87 stem cells. It is concluded that THZ1 can suppress the proliferation and migration of U87 cells, and inhibit the mammosphere forming ability, stemness related protein expression, and epithelial-mesenchymal transformation ability of U87 stem cells by down-regulating the expression of Wnt/β-catenin signaling pathway related molecules.

Key words: THZ1, glioma, cancer stem cell, mammosphere culture, stemness marker, Wnt/β-catenin signaling pathway, epithelial-mesenchymal transformation, proliferation, migration

CLC Number:

Hu Enxi, He Wenying, Tao Xiang, Du Peijing, Wang Libin. Regulation of THZ1, an inhibitor of cyclin-dependent kinase 7, on stemness of glioma stem cells and its mechanism[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(25): 5374-5381.

Figures/Tables 2

References

[1] KLEMM F, MAAS RR, BOWMAN RL, et al. Interrogation of the Microenvironmental Landscape in Brain Tumors Reveals Disease-Specific Alterations of Immune Cells. Cell. 2020;181(7):1643-1660.e17.
[2] TAN AC, ASHLEY DM, LÓPEZ GY, et al. Management of glioblastoma: State of the art and future directions. CA Cancer J Clin. 2020;70(4): 299-312.
[3] KARLSSON J, LULY KM, TZENG SY, et al. Nanoparticle designs for delivery of nucleic acid therapeutics as brain cancer therapies. Adv Drug Deliv Rev. 2021;179:113999.
[4] PRASETYANTI PR, MEDEMA JP. Intra-tumor heterogeneity from a cancer stem cell perspective. Mol Cancer. 2017;16(1):41.
[5] LI D, WANG L, JIANG B, et al. Improving cancer immunotherapy by preventing cancer stem cell and immune cell linking in the tumor microenvironment. Biomed Pharmacother. 2024;170:116043.
[6] SHEN Q, HILL T, CAI X, et al. Physical confinement during cancer cell migration triggers therapeutic resistance and cancer stem cell-like behavior. Cancer Lett. 2021;506:142-151.
[7] TSAI YT, WU AC, YANG WB, et al. ANGPTL4 Induces TMZ Resistance of Glioblastoma by Promoting Cancer Stemness Enrichment via the EGFR/AKT/4E-BP1 Cascade. Int J Mol Sci. 2019;20(22):5625.
[8] LAMBERT AW, WEINBERG RA. Linking EMT programmes to normal and neoplastic epithelial stem cells. Nat Rev Cancer. 2021;21(5):325-338.
[9] HE P, DAI Q, WU X. New insight in urological cancer therapy: From epithelial-mesenchymal transition (EMT) to application of nano-biomaterials. Environ Res. 2023;229:115672.
[10] RAMESH V, BRABLETZ T, CEPPI P. Targeting EMT in Cancer with Repurposed Metabolic Inhibitors. Trends Cancer. 2020;6(11):942-950.
[11] NUSSE R, CLEVERS H. Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell. 2017;169(6):985-999.
[12] NAJAFI M, FARHOOD B, MORTEZAEE K. Cancer stem cells (CSCs) in cancer progression and therapy. J Cell Physiol. 2019;234(6):8381-8395.
[13] LI Q, LAI Q, HE C, et al. RUNX1 promotes tumour metastasis by activating the Wnt/β-catenin signalling pathway and EMT in colorectal cancer. J Exp Clin Cancer Res. 2019;38(1):334.
[14] SUN J, ZHANG Q, SUN X, et al. THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours. Cell Commun Signal. 2022;20(1):138.
[15] CHOW PM, CHANG YW, KUO KL, et al. CDK7 inhibition by THZ1 suppresses cancer stemness in both chemonaïve and chemoresistant urothelial carcinoma via the hedgehog signaling pathway. Cancer Lett. 2021;507:70-79.
[16] ATTIA YM, SALAMA SA, SHOUMAN SA, et al. Targeting CDK7 reverses tamoxifen resistance through regulating stemness in ER+ breast cancer. Pharmacol Rep. 2022;74(2):366-378.
[17] HORBINSKI C, BERGER T, PACKER RJ, et al. Clinical implications of the 2021 edition of the WHO classification of central nervous system tumours. Nat Rev Neurol. 2022;18(9):515-529.
[18] BARTHEL L, HADAMITZKY M, DAMMANN P, et al. Glioma: molecular signature and crossroads with tumor microenvironment. Cancer Metastasis Rev. 2022;41(1):53-75.
[19] CAYROL F, PRADITSUKTAVORN P, FERNANDO TM, et al. THZ1 targeting CDK7 suppresses STAT transcriptional activity and sensitizes T-cell lymphomas to BCL2 inhibitors. Nat Commun. 2017;8:14290.
[20] CHEN HD, HUANG CS, XU QC, et al. Therapeutic Targeting of CDK7 Suppresses Tumor Progression in Intrahepatic Cholangiocarcinoma. Int J Biol Sci. 2020;16(7):1207-1217.
[21] TANG L, JIN J, XU K, et al. SOX9 interacts with FOXC1 to activate MYC and regulate CDK7 inhibitor sensitivity in triple-negative breast cancer. Oncogenesis. 2020;9(5):47.
[22] CHENG ZJ, MIAO DL, SU QY, et al. THZ1 suppresses human non-small-cell lung cancer cells in vitro through interference with cancer metabolism. Acta Pharmacol Sin. 2019;40(6):814-822.
[23] ABUDUREHEMAN T, XIA J, LI MH, et al. CDK7 Inhibitor THZ1 Induces the Cell Apoptosis of B-Cell Acute Lymphocytic Leukemia by Perturbing Cellular Metabolism. Front Oncol. 2021;11:663360.
[24] HUANG T, DING X, XU G, et al. CDK7 inhibitor THZ1 inhibits MCL1 synthesis and drives cholangiocarcinoma apoptosis in combination with BCL2/BCL-XL inhibitor ABT-263. Cell Death Dis. 2019;10(8):602.
[25] ZHANG T, LI J, YANG M, et al. CDK7/GRP78 signaling axis contributes to tumor growth and metastasis in osteosarcoma. Oncogene. 2022; 41(40):4524-4536.
[26] XIA L, ZHENG Z, LIU JY, et al. Targeting Triple-Negative Breast Cancer with Combination Therapy of EGFR CAR T Cells and CDK7 Inhibition. Cancer Immunol Res. 2021;9(6):707-722.
[27] HUANG T, SONG X, XU D, et al. Stem cell programs in cancer initiation, progression, and therapy resistance. Theranostics. 2020;10(19): 8721-8743.
[28] SUVÀ ML, TIROSH I. The Glioma Stem Cell Model in the Era of Single-Cell Genomics. Cancer Cell. 2020;37(5):630-636.
[29] BOYD NH, TRAN AN, BERNSTOCK JD, et al. Glioma stem cells and their roles within the hypoxic tumor microenvironment. Theranostics. 2021;11(2):665-683.
[30] CHEN X, NIU W, FAN X, et al. Oct4A palmitoylation modulates tumorigenicity and stemness in human glioblastoma cells. Neuro Oncol. 2023;25(1):82-96.
[31] AKHMETKALIYEV A, ALIBRAHIM N, SHAFIEE D, et al. EMT/MET plasticity in cancer and Go-or-Grow decisions in quiescence: the two sides of the same coin? Mol Cancer. 2023;22(1):90.
[32] LAMBERT AW, FIORE C, CHUTAKE Y, et al. ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells. Dev Cell. 2022;57(24): 2714-2730.e8.
[33] BAI X, NI J, BERETOV J, et al. Cancer stem cell in breast cancer therapeutic resistance. Cancer Treat Rev. 2018;69:152-163.
[34] MCCABE EM, RASMUSSEN TP. lncRNA involvement in cancer stem cell function and epithelial-mesenchymal transitions. Semin Cancer Biol. 2021;75:38-48.
[35] BABU D, MUDIRAJ A, YADAV N, et al. Rabeprazole has efficacy per se and reduces resistance to temozolomide in glioma via EMT inhibition. Cell Oncol (Dordr). 2021;44(4):889-905.
[36] ZHANG J, CAI H, SUN L, et al. LGR5, a novel functional glioma stem cell marker, promotes EMT by activating the Wnt/β-catenin pathway and predicts poor survival of glioma patients. J Exp Clin Cancer Res. 2018;37(1):225.
[37] LIU J, XIAO Q, XIAO J, et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther. 2022;7(1):3.
[38] YU F, YU C, LI F, et al. Wnt/β-catenin signaling in cancers and targeted therapies. Signal Transduct Target Ther. 2021;6(1):307.
[39] WEI B, CAO J, TIAN JH, et al. Mortalin maintains breast cancer stem cells stemness via activation of Wnt/GSK3β/β-catenin signaling pathway. Am J Cancer Res. 2021;11(6):2696-2716.
[40] GRINAT J, HEUBERGER J, VIDAL RO, et al. The epigenetic regulator Mll1 is required for Wnt-driven intestinal tumorigenesis and cancer stemness. Nat Commun. 2020;11(1):6422.
[41] YIN J, DING F, CHENG Z, et al. METTL3-mediated m6A modification of LINC00839 maintains glioma stem cells and radiation resistance by activating Wnt/β-catenin signaling. Cell Death Dis. 2023;14(7):417.