AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells

Guo,Peiyu; Lou,Zhenkai; Gong,Hongda; Hou,Xiaodong; Zhang,Chunqiang; Wang,Bing; Du,Kaili

doi:10.3892/mmr.2025.13613

AZD1080, a specific inhibitor of GSK‑3β, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells

Authors:
- Peiyu Guo
- Zhenkai Lou
- Hongda Gong
- Xiaodong Hou
- Chunqiang Zhang
- Bing Wang
- Kaili Du
View Affiliations

Affiliations: Department of Orthopedics, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China
Published online on: July 8, 2025 https://doi.org/10.3892/mmr.2025.13613
Article Number: 248
Copyright: © Guo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Osteosarcoma is the most common primary bone cancer, primarily affecting children and young adults. Cancer stem cells (CSCs), a subpopulation presenting stemness, critically influence prognosis and promote recurrence and metastasis. Due to the crucial role of glycogen synthase kinase‑3 beta (GSK‑3β) in maintaining stemness, it is considered as an important target for drug development. The aim of the present study was to evaluate the inhibitory effect of AZD1080, a GSK‑3β inhibitor, on osteosarcoma CSCs. AZD1080 treatment clearly inhibited sphere formation in U2OS and 143B cells and dissociated spheres in CSCs derived from U2OS and 143B; in both processes, stemness markers OCT4 and SOX2 were markedly decreased, without affecting cell proliferation or apoptosis. AZD1080 treatment inhibited phosphorylation of GSK‑3β and its downstream regulated genes, including HEY1, HES1, CyclinD1 and β‑catenin. It was also observed that GSK‑3β activity was critical for the inhibitory effects of AZD1080 treatment on sphere formation and stemness. Moreover, GSK‑3β knockdown inhibited sphere formation and invasion capacity, indicating that AZD1080 exerts inhibitory roles in a GSK‑3β‑dependent manner. Taken together, the results showed AZD1080 as a specific inhibitor of CSC stemness, without cytotoxicity, and indicated it was a promising therapeutic agent that targeted GSK‑3β signaling in osteosarcoma.

View Figures

View References

1	Longhi A, Errani C, De Paolis M, Mercuri M and Bacci G: Primary bone osteosarcoma in the pediatric age: State of the art. Cancer Treat Rev. 32:423–436. 2006. View Article : Google Scholar : PubMed/NCBI
2	Wittig JC, Bickels J, Priebat D, Jelinek J, Kellar-Graney K, Shmookler B and Malawer MM: Osteosarcoma: A multidisciplinary approach to diagnosis and treatment. Am Fam Physician. 65:1123–1132. 2002.PubMed/NCBI
3	Kansara M and Thomas DM: Molecular pathogenesis of osteosarcoma. DNA Cell Biol. 26:1–18. 2007. View Article : Google Scholar
4	Mohseny AB, Szuhai K, Romeo S, Buddingh EP, Briaire-de Bruijn I, de Jong D, van Pel M, Cleton-Jansen AM and Hogendoorn PC: Osteosarcoma originates from mesenchymal stem cells in consequence of aneuploidization and genomic loss of Cdkn2. J Pathol. 219:294–305. 2009. View Article : Google Scholar
5	Tang N, Song WX, Luo J, Haydon RC and He TC: Osteosarcoma development and stem cell differentiation. Clin Orthop Relat Res. 466:2114–2130. 2008. View Article : Google Scholar : PubMed/NCBI
6	Hogendoorn PC, Bove J, Karperien M and Cleton-Jansen AM: Skeletogenesis: Genetics. London: Nature Publishing Group; 2003
7	Adhikari AS, Agarwal N, Wood BM, Porretta C, Ruiz B, Pochampally RR and Iwakuma T: CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance. Cancer Res. 70:4602–1462. 2010. View Article : Google Scholar : PubMed/NCBI
8	Georgievska B, Sandin J, Doherty J, Mörtberg A, Neelissen J, Andersson A, Gruber S, Nilsson Y, Schött P, Arvidsson PI, et al: AZD1080, a novel GSK3 inhibitor, rescues synaptic plasticity deficits in rodent brain and exhibits peripheral target engagement in humans. J Neurochem. 125:446–456. 2013. View Article : Google Scholar : PubMed/NCBI
9	Chen S, Sun KX, Feng MX, Sang XB, Liu BL and Zhao Y: Role of glycogen synthase kinase-3β inhibitor AZD1080 in ovarian cancer. Drug Des Devel Ther. 10:1225–1232. 2016. View Article : Google Scholar : PubMed/NCBI
10	Chandra P, Sachan N and Pal D: Glycogen synthase kinase-3 (GSK-3) inhibitors as a new lead for treating breast and ovarian cancer. Current Drug Targets. 22:1548–1554. 2021. View Article : Google Scholar : PubMed/NCBI
11	Zhang X and Wang X, Hou L, Xu Z, Liu Y and Wang X: Nanoparticles overcome adaptive immune resistance and enhance immunotherapy via targeting tumor microenvironment in lung cancer. Front Pharmacol. 14:11309372023. View Article : Google Scholar
12	Watanabe M, Abe N, Oshikiri Y, Stanbridge EJ and Kitagawa T: Selective growth inhibition by glycogen synthase kinase-3 inhibitors in tumorigenic HeLa hybrid cells is mediated through NF-κB-dependent GLUT3 expression. Oncogenesis. 1:e212012. View Article : Google Scholar : PubMed/NCBI
13	Zhou W, Wang L, Gou SM, Wang TL, Zhang M, Liu T and Wang CY: ShRNA silencing glycogen synthase kinase-3 beta inhibits tumor growth and angiogenesis in pancreatic cancer. Cancer Lett. 316:178–186. 2012. View Article : Google Scholar
14	Augello G, Emma MR, Cusimano A, Azzolina A, Montalto G, McCubrey JA and Cervello M: The role of GSK-3 in cancer immunotherapy: GSK-3 inhibitors as a new frontier in cancer treatment. Cells. 9:14272020. View Article : Google Scholar : PubMed/NCBI
15	Nagini S, Sophia J and Mishra R: Glycogen synthase kinases: Moonlighting proteins with theranostic potential in cancer. Semin Cancer Biol. 56:25–36. 2019. View Article : Google Scholar
16	Fu Y, Hu D, Qiu J, Xie X, Ye F and Lu WG: Overexpression of glycogen synthase Kinase-3 in ovarian carcinoma cells with acquired paclitaxel resistance. Int J Gynecol Cancer. 21:439–444. 2011. View Article : Google Scholar : PubMed/NCBI
17	Kawazoe H, Bilim VN, Ugolkov AV, Yuuki K, Naito S, Nagaoka A, Kato T and Tomita Y: GSK-3 Inhibition in vitro and in vivo enhances antitumor effect of sorafenib in renal cell carcinoma (RCC). Biochem Biophys Res Commun. 423:490–495. 2012. View Article : Google Scholar : PubMed/NCBI
18	Kazi A, Xiang S, Yang H, Delitto D, Trevino J, Jiang RHY, Ayaz M, Lawrence HR, Kennedy P and Sebti SM: GSK3 Suppression Upregulates β-catenin and c-Myc to abrogate KRas-dependent tumors. Nat Commun. 9:51542018. View Article : Google Scholar : PubMed/NCBI
19	Mancinelli R, Carpino G, Petrungaro S, Mammola CL, Tomaipitinca L, Filippini A, Facchiano A, Ziparo E and Giampietri C: Multifaceted roles of GSK-3 in cancer and Autophagy-related diseases. Oxid Med Cell Longev. 2017:46294952017. View Article : Google Scholar : PubMed/NCBI
20	McCubrey JA, Steelman LS, Bertrand FE, Davis NM, Sokolosky M, Abrams SL, Montalto G, D'Assoro AB, Libra M, Nicoletti F, et al: GSK-3 as potential target for therapeutic irvention in cancer. Oncotarget. 5:2881–2911. 2014. View Article : Google Scholar
21	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
22	Lathia J, Liu H and Matei D: The clinical impact of cancer stem cells. Oncologist. 25:123–131. 2020. View Article : Google Scholar
23	Foltz DR, Santiago MC, Berechid BE and Nye JS: Glycogen synthase kinase-3beta modulates notch signaling and stability. Curr Biol. 12:1006–1011. 2002. View Article : Google Scholar
24	Guha S, Cullen JP, Morrow D, Colombo A, Lally C, Walls D, Redmond EM and Cahill PA: Glycogen synthase kinase 3 beta positively regulates Notch signaling in vascular smooth muscle cells: Role in cell proliferation and survival. Basic Res Cardiol. 106:773–785. 2011. View Article : Google Scholar
25	Zheng L and Conner SD: Glycogen synthase kinase 3β inhibition enhances Notch1 recycling. Mol Biol Cell. 29:389–395. 2018. View Article : Google Scholar
26	Saini MK and Sanyal SN: PTEN regulates apoptotic cell death through PI3-K/Akt/GSK3β signaling pathway in DMH induced early colon carcinogenesis in rat. Exp Mol Pathol. 93:135–146. 2012. View Article : Google Scholar
27	Mai W, Kong L, Yu H, Bao J, Song C and Qu G: Glycogen synthase kinase 3β promotes osteosarcoma invasion and migration via regulating PTEN and phosphorylation of focal adhesion kinase. Biosci Rep. 41:BSR201935142021. View Article : Google Scholar : PubMed/NCBI
28	Strang JE, Astridge DD, Nguyen VT and Reigan P: Small molecule modulators of AMP-activated protein kinase (AMPK) activity and their potential in cancer therapy. J Med Chem. 68:2238–2254. 2025. View Article : Google Scholar : PubMed/NCBI
29	Jiang H, Zhao X, Zang J, Wang R, Gao J, Chen J and Yu T: Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation. Front Pharmacol. 15:13996252024. View Article : Google Scholar
30	Shi Q, Xue C, Zeng Y, Yuan X, Chu Q, Jiang S, Wang J, Zhang Y, Zhu D and Li L: Notch signaling pathway in cancer: From mechanistic insights to targeted therapies. Signal Transduct Target Ther. 9:1282024. View Article : Google Scholar : PubMed/NCBI
31	Wei Y, Li Y, Chen Y, Liu P, Huang S, Zhang Y, Sun Y, Wu Z, Hu M, Wu Q, et al: ALDH1: A potential therapeutic target for cancer stem cells in solid tumors. Front Oncol. 12:10262782022. View Article : Google Scholar
32	Anderson ME: Update on survival in osteosarcoma. Orthop Clin North Am. 47:283–292. 2016. View Article : Google Scholar : PubMed/NCBI
33	Tsuru A, Setoguchi T, Matsunoshita Y, Nagao-Kitamoto H, Nagano S, Yokouchi M, Maeda S, Ishidou Y, Yamamoto T and Komiya S: Hairy/enhancer-of-split related with YRPW motif protein 1 promotes osteosarcoma metastasis via matrix metallopeptidase 9 expression. Br J Cancer. 112:1232–1240. 2015. View Article : Google Scholar : PubMed/NCBI
34	Kopan R and Turner DL: The Notch pathway: Democracy and aristocracy in the selection of cell fate. Curr Opin Neurobiol. 6:594–601. 1996. View Article : Google Scholar
35	Han X, Ju JH and Shin I: Glycogen synthase kinase 3-β phosphorylates novel S/T-P-S/T domains in Notch1 intracellular domain and induces its nuclear localization. Biochem Biophys Res Commun. 423:282–288. 2012. View Article : Google Scholar : PubMed/NCBI
36	Giovannini C, Baglioni M, Toaldo MB, Ventrucci C, D'Adamo S, Cipone M, Chieco P, Gramantieri L and Bolondi L: Notch3 inhibition enhances sorafenib cytotoxic efficacy by promoting GSK3b phosphorylation and p21 down-regulation in hepatocellular carcinoma. Oncotarget. 4:1618–1631. 2013. View Article : Google Scholar
37	Espinosa L, Inglés-Esteve J, Aguilera C and Bigas A: Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways. J Biol Chem. 278:32227–32235. 2003. View Article : Google Scholar : PubMed/NCBI