ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/β-Catenin Signaling Pathway

Abstract

Objective

Uterine corpus endometrial carcinoma (UCEC), a kind of gynecologic malignancy, poses a significant risk to women’s health. The precise mechanism underlying the development of UCEC remains elusive. Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein superfamily, was reported to be dysregulated in various illnesses, including malignant tumors. This study aimed to examine the involvement of ZNF554 in the development of UCEC.

Methods

The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay. Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection. CCK-8, wound healing, and Transwell invasion assays were employed to assess cell proliferation, migration, and invasion. Propidium iodide (PI) staining combined with fluorescence-activated cell sorting (FACS) flow cytometer was utilized to detect cell cycle distribution. qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels. Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5 (RBM5).

Results

The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines. Decreased expression of ZNF554 was associated with higher tumor stage, decreased overall survival, and reduced disease-free survival in UCEC. ZNF554 overexpression suppressed cell proliferation, migration, and invasion, while also inducing cell cycle arrest. In contrast, a decrease in ZNF554 expression resulted in the opposite effect. Mechanistically, ZNF554 transcriptionally regulated RBM5, leading to the deactivation of the Wingless (WNT)/β-catenin signaling pathway. Moreover, the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression on β-catenin and p-glycogen synthase kinase-3β (p-GSK-3β). Similarly, the deliberate activation of RBM5 reduced the increase in β-catenin and p-GSK-3β caused by the suppression of ZNF554. In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown. Additionally, when RBM5 was overexpressed, it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels.

Conclusion

ZNF554 functions as a tumor suppressor in UCEC. Furthermore, ZNF554 regulates UCEC progression through the RBM5/WNT/β-catenin signaling pathway. ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.

Keywords

zinc finger protein 554 / endometrial carcinoma / RNA binding motif 5 / Wingless/β-catenin signaling pathway

References

[1]	Makker V, MacKay H, Ray-Coquard I, et al. Endometrial cancer. Nat Rev Dis Primers, 2021,7(1):88
[2]	Crosbie EJ, Kitson SJ, McAlpine JN, et al. Endometrial cancer. Lancet, 2022,399(10333):1412–1428
[3]	Sun X, Hou L, Qiu C, et al. MiR-501 promotes tumor proliferation and metastasis by targeting HOXD10 in endometrial cancer. Cell Mol Biol Lett, 2021,26(1):20
[4]	Miller KD, Nogueira L, Devasia T, et al. Cancer treatment and survivorship statistics, 2022. CA Cancer J Clin, 2022,72(5):409–436
[5]	Huvila J, Pors J, Thompson EF, et al. Endometrial carcinoma: molecular subtypes, precursors and the role of pathology in early diagnosis. J Pathol, 2021,253(4):355–365
[6]	Bogani G, Ray-Coquard I, Concin N, et al. Endometrial carcinosarcoma. Int J Gynecol Cancer, 2023,33(2):147–174
[7]	Nees LK, Heublein S, Steinmacher S, et al. Endometrial hyperplasia as a risk factor of endometrial cancer. Arch Gynecol Obstet, 2022,306(2):407–421
[8]	MacKintosh ML, Crosbie EJ. Prevention Strategies in Endometrial Carcinoma. Curr Oncol Rep, 2018,20(12):101
[9]	Hamilton SN, Tinker AV, Kwon J, et al. Treatment and outcomes in undifferentiated and dedifferentiated endometrial carcinoma. J Gynecol Oncol, 2022,33(3): e25
[10]	Chen H, Strickland AL, Castrillon DH. Histopathologic diagnosis of endometrial precancers: Updates and future directions. Semin Diagn Pathol, 2022,39(3):137–147
[11]	Jamieson A, Thompson EF, Huvila J, et al. Endometrial carcinoma molecular subtype correlates with the presence of lymph node metastases. Gynecol Oncol, 2022,165(2):376–384
[12]	Karpel HC, Slomovitz B, Coleman RL, et al. Treatment options for molecular subtypes of endometrial cancer in 2023. Curr Opin Obstet Gynecol, 2023,35(3):270–278
[13]	Concin N, Matias-Guiu X, Vergote I, et al. ESGO/ESTRO/ESP guidelines for the management of patients with endometrial carcinoma. Int J Gynecol Cancer, 2021,31(1):12–39
[14]	Guo Y, Huang C, Xu C, et al. Dysfunction of ZNF554 promotes ROS-induced apoptosis and autophagy in Fetal Growth Restriction via the p62-Keap1-Nrf2 pathway. Placenta, 2023,143:34–44
[15]	Ecco G, Imbeault M, Trono D. KRAB zinc finger proteins. Development, 2017,144(15):2719–2729
[16]	Zhang W, Zhangyuan G, Wang F, et al. The zinc finger protein Miz1 suppresses liver tumorigenesis by restricting hepatocyte-driven macrophage activation and inflammation. Immunity, 2021,54(6):1168–1185
[17]	Ito J, Kimura I, Soper A, et al. Endogenous retroviruses drive KRAB zinc-finger protein family expression for tumor suppression. Sci Adv, 2020,6(43):eabc3020
[18]	Jen J, Wang YC. Zinc finger proteins in cancer progression. J Biomed Sci, 2016,23(1):53
[19]	Xie G, Peng Z, Liang J, et al. Zinc finger protein 277 is an intestinal transit-amplifying cell marker and colon cancer oncogene. JCI Insight, 2022,7(4):e150894
[20]	Iyer AS, Shaik MR, Raufman JP, et al. The Roles of Zinc Finger Proteins in Colorectal Cancer. Int J Mol Sci, 2023,24(12):10249
[21]	Balogh A, Reiniger L, Hetey S, et al. Decreased Expression of ZNF554 in Gliomas is Associated with the Activation of Tumor Pathways and Shorter Patient Survival. Int J Mol Sci, 2020,21(16):5762
[22]	Berek JS, Matias-Guiu X, Creutzberg C, et al. FIGO staging of endometrial cancer: 2023. Int J Gynaecol Obstet, 2023,162(2):383–394
[23]	Yang D, Ma X, Xu J, et al. Zfx-induced upregulation of UBE2J1 facilitates endometrial cancer progression via PI3K/AKT pathway. Cancer Biol Ther, 2021,22(3):238–247
[24]	Zhang Y, Wang X. Targeting the Wnt/β-catenin signaling pathway in cancer. J Hematol Oncol, 2020,13(1):165
[25]	Sun M, Ju J, Ding Y, et al. The signaling pathways regulated by KRAB zinc-finger proteins in cancer. Biochim Biophys Acta Rev Cancer, 2022,1877(3): 188731
[26]	Zhang X, Zheng Q, Yue X, et al. ZNF498 promotes hepatocellular carcinogenesis by suppressing p53-mediated apoptosis and ferroptosis via the attenuation of p53 Ser46 phosphorylation. J Exp Clin Cancer Res, 2022,41(1):79
[27]	Ying Y, Wang M, Chen Y, et al. Zinc finger protein 280C contributes to colorectal tumorigenesis by maintaining epigenetic repression at H3K27me3-marked loci. Proc Natl Acad Sci U S A, 2022,119(22):e2120633119
[28]	Geng R, Zheng Y, Zhou D, et al. ZBTB7A, a potential biomarker for prognosis and immune infiltrates, inhibits progression of endometrial cancer based on bioinformatics analysis and experiments. Cancer Cell Int, 2020,20(1):542
[29]	Wang L, Dong L, Xu J, et al. Hypermethylated CDO1 and ZNF454 in Cytological Specimens as Screening Biomarkers for Endometrial Cancer. Front Oncol, 2022,12:714663
[30]	Zhang J, Zheng Z, Zheng J, et al. Epigenetic-Mediated Downregulation of Zinc Finger Protein 671 (ZNF671) Predicts Poor Prognosis in Multiple Solid Tumors. Front Oncol, 2019,9:342
[31]	Albrecht LV, Tejeda-Mu?oz N, De Robertis EM. Cell Biology of Canonical Wnt Signaling. Annu Rev Cell Dev Biol, 2021,37:369–389
[32]	Katoh M, Katoh M. WNT signaling and cancer stemness. Essays Biochem, 2022,66(4): 319–331
[33]	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
[34]	Nusse R, Clevers H. Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell, 2017,169(6):985–999
[35]	Parsons MJ, Tammela T, Dow LE. WNT as a Driver and Dependency in Cancer. Cancer Discov, 2021,11(10):2413–2429
[36]	Krishnamurthy N, Kurzrock R. Targeting the Wnt/beta-catenin pathway in cancer: Update on effectors and inhibitors. Cancer Treat Rev, 2018,62:50–60
[37]	Neiheisel A, Kaur M, Ma N, et al. Wnt pathway modulators in cancer therapeutics: An update on completed and ongoing clinical trials. Int J Cancer, 2022,150(5):727–740
[38]	Chatterjee A, Paul S, Bisht B, et al. Advances in targeting the WNT/β-catenin signaling pathway in cancer. Drug Discov Today, 2022,27(1):82–101
[39]	Sutherland LC, Wang K, Robinson AG. RBM5 as a putative tumor suppressor gene for lung cancer. J Thorac Oncol, 2010,5(3):294–298
[40]	Yu J, Ji G, Shi W, et al. RBM5 Acts as Tumor Suppressor in Medulloblastoma through Regulating Wnt/β-Catenin Signaling. Eur Neurol, 2020,83(3):242–250