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KDM6A alternative splicing induced by 25(OH)D inhibits breast cancer cell stemness through repressing TRAP1 transcription

Oncogene (2026)Cite this article

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Abstract

25-Hydroxyvitamin D (25(OH)D), a metabolite of vitamin D, has demonstrated anticancer properties; however, the role of alternative splicing in mediating these effects remains poorly understood. In this study, we reveal for the first time that 25(OH)D exerts antitumor effects by promoting exon 13 skipping of KDM6A (KDM6A Δexon13), which suppresses the proliferation and stemness of breast cancer cells and lacks H3K27 demethylase activity. Mechanistically, CUT&Tag and RNA-seq analyses demonstrated that KDM6A Δexon13 induces the accumulation of H3K27me3 at the promoter region of TRAP1, thereby inhibiting its transcription. Consequently, the downregulation of TRAP1 reduces Smad2/3 phosphorylation. Furthermore, KHDRBS3 was identified as the splicing factor of KDM6A Δexon13 and was regulated by 25(OH)D. Notably, 25(OH)D exhibited a synergistic effect with GSK-J4, a specific inhibitor of KDM6A, in suppressing breast cancer cell growth. Collectively, our findings uncover a novel anticancer mechanism of 25(OH)D, highlight the critical role of KDM6A Δexon13 in breast cancer progression, and provide further evidence supporting the correction of 25(OH)D deficiency in breast cancer patients.

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Fig. 1: 25-HydroxyvitaminD (25(OH)D) upregulates exon 13 skipping of KDM6A (KDM6A Δexon13) in breast cancer (BC).
Fig. 2: KDM6A Δexon13 inhibits proliferation and cell cycle progression of BC cells.
Fig. 3: RNA-seq analysis of KDM6A Δexon13 and its role in Smad2/3 phosphorylation (p-Smad2/3).
Fig. 4: KDM6A Δexon13 suppresses BC cell stemness and lacks H3K27 demethylase activity.
Fig. 5: KDM6A Δexon13 attenuates TRAP1 transcription via accumulation of H3K27me3 in the promoter region.
Fig. 6: TRAP1 promotes BC cell proliferation and stemness by activating the phosphorylation of Smad2/3.
Fig. 7: 25(OH)D promotes KDM6A Δexon13 by downregulating splicing factor KHDRBS3.
Fig. 8: KDM6A Δexon 13 suppresses tumor growth in vivo.

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Data availability

The RNA-seq and the CUT&Tag data have been deposited in the NCBI (https://www.ncbi.nlm.nih.gov/) Sequence Read Archive (SRA) database under accession number PRJNA1216307.

References

  1. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer J Clin. 2024;74:229–63.

    Google Scholar 

  2. Zheng W, Peng W, Qian F, Zhang M, Duan B, Fan Z, et al. Vitamin D suppresses CD133+/CD44 + cancer stem cell stemness by inhibiting NF-kappaB signaling and reducing NLRP3 expression in triple-negative breast cancer. Cancer Chemother Pharm. 2024;94:67–78.

    Article  CAS  Google Scholar 

  3. Kuznia S, Zhu A, Akutsu T, Buring JE, Camargo CA, Jr., Cook NR, et al. Efficacy of vitamin D(3) supplementation on cancer mortality: Systematic review and individual patient data meta-analysis of randomised controlled trials. Ageing Res Rev. 2023;87:101923.

  4. Kim Y, Chang Y, Cho Y, Chang J, Kim K, Park D-I, et al. Serum 25-hydroxyvitamin D levels and risk of colorectal cancer: an age-stratified analysis. Gastroenterology. 2023;165:920–31.

    Article  CAS  PubMed  Google Scholar 

  5. Zemlin C, Altmayer L, Stuhlert C, Schleicher JT, Wormann C, Lang M, et al. Prevalence and Relevance of Vitamin D Deficiency in Newly Diagnosed Breast Cancer Patients: A Pilot Study. Nutrients, 2023;15:1450.

  6. Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman BJ. The role of vitamin D in reducing cancer risk and progression. Nat Rev Cancer. 2014;14:342–57.

    Article  CAS  PubMed  Google Scholar 

  7. Townsend K, Banwell CM, Guy M, Colston KW, Mansi JL, Stewart PM, et al. Autocrine metabolism of vitamin D in normal and malignant breast tissu e. Clin Cancer Res. 2005;11:3579–86.

    Article  CAS  PubMed  Google Scholar 

  8. Aggarwal A, Feldman D, Feldman BJ. Identification of tumor-autonomous and indirect effects of vitamin D action that inhibit breast cancer growth and tumor progression. J Steroid Biochem Mol Biol. 2018;177:155–8.

    Article  CAS  PubMed  Google Scholar 

  9. Shan NL, Wahler J, Lee HJ, Bak MJ, Gupta SD, Maehr H, et al. Vitamin D compounds inhibit cancer stem-like cells and induce differentiation in triple negative breast cancer. J Steroid Biochem Mol Biol. 2017;173:122–9.

    Article  CAS  PubMed  Google Scholar 

  10. Xie SP, Pirianov G, Colston KW. Vitamin D analogues suppress IGF-I signalling and promote apoptosis in breast cancer cells. Eur J Cancer. 1999;35:1717–23.

    Article  CAS  PubMed  Google Scholar 

  11. Carlberg C, Muñoz A. An update on vitamin D signaling and cancer. Semin Cancer Biol. 2022;79:217–30.

    Article  CAS  PubMed  Google Scholar 

  12. Marasco LE, Kornblihtt AR. The physiology of alternative splicing. Nat Rev Mol Cell Biol. 2022;24:242–54.

    Article  PubMed  Google Scholar 

  13. Bonnal SC, López-Oreja I, Valcárcel J. Roles and mechanisms of alternative splicing in cancer — implications for care. Nat Rev Clin Oncol. 2020;17:457–74.

    Article  PubMed  Google Scholar 

  14. Castagnoli L, Iezzi M, Ghedini GC, Ciravolo V, Marzano G, Lamolinara A, et al. Activated d16HER2 Homodimers and SRC Kinase Mediate Optimal Efficacy for Trastuzumab. Cancer Res. 2014;74:6248–59.

    Article  CAS  PubMed  Google Scholar 

  15. Castagnoli L, Ghedini GC, Koschorke A, Triulzi T, Dugo M, Gasparini P, et al. Pathobiological implications of the d16HER2 splice variant for stemnes s and aggressiveness of HER2-positive breast cancer. Oncogene. 2017;36:1721–32.

    Article  CAS  PubMed  Google Scholar 

  16. Lee MG, Villa R, Trojer P, Norman J, Yan K-P, Reinberg D, et al. Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquiti nation. Science. 2007;318:447–50.

    Article  CAS  PubMed  Google Scholar 

  17. Simon JA, Kingston RE. Occupying chromatin: polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell. 2013;49:808–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hosogane M, Funayama R, Shirota M, Nakayama K. Lack of transcription triggers H3K27me3 accumulation in the gene body. Cell Rep. 2016;16:696–706.

    Article  CAS  PubMed  Google Scholar 

  19. Shirai CL, Ley JN, White BS, Kim S, Tibbitts J, Shao J, et al. Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo. Cancer Cell. 2015;27:631–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wan X, Shi W, Ma L, Wang L, Zheng R, He J, et al. A 3′‐pre‐tRNA‐derived small RNA tRF‐1‐Ser regulated by 25(OH)D promotes proliferation and stemness by inhibiting the function of MBNL1 in breast cancer. Clin Transl Med. 2024;14:e1681.

  21. Mahmood N, Arakelian A, Muller WJ, Szyf M, Rabbani SA. An enhanced chemopreventive effect of methyl donor S-adenosylmethionine in combination with 25-hydroxyvitamin D in blocking mammary tumor growth and metastasis. Bone Res. 2020;8:28.

  22. Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2011;96:1911–30.

    Article  CAS  PubMed  Google Scholar 

  23. Seiler M, Peng S, Agrawal AA, Palacino J, Teng T, Zhu P, et al. Somatic mutational landscape of splicing factor genes and their functional consequences across 33 cancer types. Cell Rep. 2018;23:282–96.e4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Masgras I, Laquatra C, Cannino G, Serapian SA, Colombo G, Rasola A. The molecular chaperone TRAP1 in cancer: from the basics of biology to pharmacological targeting. Semin Cancer Biol. 2021;76:45–53.

    Article  CAS  PubMed  Google Scholar 

  25. Zhang B, Wang J, Huang Z, Wei P, Liu Y, Hao J, et al. Aberrantly upregulated TRAP1 is required for tumorigenesis of breast c ancer. Oncotarget. 2015;6:44495–508.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Sisinni L, Maddalena F, Lettini G, Condelli V, Matassa DS, Esposito F, et al. TRAP1 role in endoplasmic reticulum stress protection favors resistance to anthracyclins in breast carcinoma cells. Int J Oncol. 2014;44:573–82.

    Article  CAS  PubMed  Google Scholar 

  27. Wengert LA, Backe SJ, Bourboulia D, Mollapour M, Woodford MR. TRAP1 chaperones the metabolic switch in cancer. Biomolecules. 2022;12:786.

  28. Song C, Xu X, Wu Y, Ji B, Zhou X, Qin L. Study of the mechanism underlying hsa-miR338-3p downregulation to promote fibrosis of the synovial tissue in osteoarthritis patients. Mol Biol Rep. 2018;46:627–37.

    Article  PubMed  Google Scholar 

  29. Jie P, Wu Y, Song C, Cheng Y, Liu Y, Chen K. Mechanism of Nrf2/miR338-3p/TRAP-1 pathway involved in hyperactivation of synovial fibroblasts in patients with osteoarthritis. Heliyon. 2023;9:e21412.

  30. Matsumoto Y, Itou J, Sato F, Toi M. SALL4 - KHDRBS3 network enhances stemness by modulating CD44 splicing in basal-like breast cancer. Cancer Med. 2018;7:454–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Dalpatraj N, Naik A, Thakur N. GSK-J4: an H3K27 histone demethylase inhibitor, as a potential anti-cancer agent. Int J Cancer. 2023;153:1130–8.

    Article  CAS  PubMed  Google Scholar 

  32. Ben-Eltriki M, Deb S, Guns EST. 1α,25-Dihydroxyvitamin D3 synergistically enhances anticancer effects of ginsenoside Rh2 in human prostate cancer cells. J Steroid Biochem Mol Biol. 2021;209:105828.

  33. Zheng C, Zhang B, Li Y, Liu K, Wei W, Liang S, et al. Donafenib and GSK-J4 synergistically induce ferroptosis in liver cancer by upregulating HMOX1 expression. Adv Sci. 2023;10:e2206798.

  34. Manson JE, Cook NR, Lee IM, Christen W, Bassuk SS, Mora S, et al. Vitamin D supplements and prevention of cancer and cardiovascular disease. N Engl J Med. 2019;380:33–44.

    Article  CAS  PubMed  Google Scholar 

  35. Rainville C, Khan Y, Tisman G. Triple negative breast cancer patients presenting with low serum vitamin D levels: a case series. Cases J. 2009;2:8390.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Lope V, Castello A, Mena-Bravo A, Amiano P, Aragones N, Fernandez-Villa T, et al. Serum 25-hydroxyvitamin D and breast cancer risk by pathological subtype (MCC-Spain). J Steroid Biochem Mol Biol. 2018;182:4–13.

    Article  CAS  PubMed  Google Scholar 

  37. Kim HJ, Lee YM, Ko BS, Lee JW, Yu JH, Son BH, et al. Vitamin D deficiency is correlated with poor outcomes in patients with luminal-type breast cancer. Ann Surg Oncol. 2011;18:1830–6.

    Article  PubMed  Google Scholar 

  38. Cristobo I, Larriba MJ, Ríos Vdl, García F, Muñoz A, Casal JI. Proteomic analysis of 1α,25-Dihydroxyvitamin D3 action on human colon cancer cells reveals a link to splicing regulation. J Proteom. 2011;75:384–97.

    Article  CAS  Google Scholar 

  39. Nurminen V, Neme A, Seuter S, Carlberg C. The impact of the vitamin D-modulated epigenome on VDR target gene regulation. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mech. 2018;1861:697–705.

    Article  CAS  Google Scholar 

  40. Yan N, Xu L, Wu X, Zhang L, Fei X, Cao Y, et al. GSKJ4, an H3K27me3 demethylase inhibitor, effectively suppresses the breast cancer stem cells. Exp Cell Res. 2017;359:405–14.

    Article  CAS  PubMed  Google Scholar 

  41. Lan J, Wei G, Liu J, Yang F, Sun R, Lu H. Chemotherapy-induced adenosine A2B receptor expression mediates epigenetic regulation of pluripotency factors and promotes breast cancer stemness. Theranostics. 2022;12:2598–612.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lu H, Xie Y, Tran L, Lan J, Yang Y, Murugan NL, et al. Chemotherapy-induced S100A10 recruits KDM6A to facilitate OCT4-mediated breast cancer stemness. J Clin Investig. 2020;130:4607–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yang Y, Chen C, Zuo Q, Lu H, Salman S, Lyu Y, et al. NARF is a hypoxia-induced coactivator for OCT4-mediated breast cancer stem cell specification. Sci Adv. 2022;8:eabo5000.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Fotouhi O, Nizamuddin S, Falk S, Schilling O, Knüchel-Clarke R, Biniossek ML, et al. Alternative mRNA Splicing Controls the Functions of the Histone H3K27 Demethylase UTX/KDM6A. Cancers. 2023;15:3117

  45. Lettini G, Sisinni L, Condelli V, Matassa DS, Simeon V, Maddalena F, et al. TRAP1 regulates stemness through Wnt/β-catenin pathway in human colorectal carcinoma. Cell Death Differ. 2016;23:1792–803.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bhreathnach U, Griffin B, Brennan E, Ewart L, Higgins D, Murphy M. Profibrotic IHG-1 complexes with renal disease associated HSPA5 and TRAP1 in mitochondria. Biochimica et Biophysica Acta (BBA) - Mol Basis Dis. 2017;1863:896–906.

    Article  CAS  Google Scholar 

  47. Chen J-f, He Q, Dai M-h, Kong W. HSP75 inhibits TGF-β1-induced apoptosis by targeting mitochondria in human renal proximal tubular epithelial cells. Biochem Biophys Res Commun. 2019;515:64–71.

    Article  CAS  PubMed  Google Scholar 

  48. Takemoto K, Miyata S, Takamura H, Katayama T, Tohyama M. Mitochondrial TRAP1 regulates the unfolded protein response in the endoplasmic reticulum. Neurochem Int. 2011;58:880–7.

    Article  CAS  PubMed  Google Scholar 

  49. Anczuków O, Krainer AR. Splicing-factor alterations in cancers. Rna. 2016;22:1285–301.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Ukai S, Honma R, Sakamoto N, Yamamoto Y, Pham QT, Harada K, et al. Molecular biological analysis of 5-FU-resistant gastric cancer organoids; KHDRBS3 contributes to the attainment of features of cancer stem cell. Oncogene. 2020;39:7265–78.

    Article  PubMed  Google Scholar 

  51. Somrit K, Krobthong S, Yingchutrakul Y, Phueakphud N, Wongtrakoongate P, Komyod W. KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines. Life Sci. 2024;358:123132

  52. Zhao M, Zhang Y, Li L, Liu X, Zhou W, Wang C, et al. KHDRBS3 accelerates glycolysis and promotes malignancy of hepatocellular carcinoma via upregulating 14-3-3ζ. Cancer Cell Int. 2023;23:244

  53. Ukai S, Sakamoto N, Taniyama D, Harada K, Honma R, Maruyama R, et al. KHDRBS3 promotes multi-drug resistance and anchorage-independent growth in colorectal cancer. Cancer Sci. 2021;112:1196–208.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Bradley RK, Anczuków O. RNA splicing dysregulation and the hallmarks of cancer. Nat Rev Cancer. 2023;23:135–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Han T, Goralski M, Gaskill N, Capota E, Kim J, Ting TC, et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science. 2017;356:eaal3755.

    Article  PubMed  Google Scholar 

  56. Abu-Hanna J, Patel JA, Anastasakis E, Cohen R, Clapp LH, Loizidou M, et al. Therapeutic potential of inhibiting histone 3 lysine 27 demethylases: a review of the literature. Clin Epigenetics. 2022;14:98

  57. Kruidenier L, Chung C-w, Cheng Z, Liddle J, Che K, Joberty G, et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature. 2012;488:404–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This research was partially supported by the Chinese Society of Clinical Oncology Foundation (Y-HR2020MS-0421), Jiangsu Province Elderly Health Research Project (LKM2022033, LKM2023023), Wu Jieping Medical Foundation Special Fund for Clinical Research (320.6750.2023-11-29), China Breast Surgery Young Physician Research Award Advantage Support Program (2020-CHPASLP-01), National Natural Science Foundation of China (Grant No. 82503790).

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  1. These authors contributed equally: Lingjun Ma, Xingye Sheng, Lexin Wang, Xinyu Wan.

Authors and Affiliations

  1. Department of Breast Disease, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China

    Lingjun Ma, Xingye Sheng, Lexin Wang, Rui Chen, Xuan Li, Ran Zheng, Xiaoming Zha & Jue Wang

  2. Department of Gastrointestinal Surgery, Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University, The Third Affiliated Hospital of Nanjing Medical University, Changzhou Medical Center, Nanjing Medical University, Changzhou, China

    Lingjun Ma

  3. Department of Breast and Thyroid Surgery, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, China

    Xinyu Wan

  4. Department of Nutrition, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China

    Lu Xu

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Contributions

LJM, XYS, and LXW performed the experiments and analyzed the data. XYW provided technical support for in vitro experiments, and we would like to express our special gratitude for his significant contributions. XL and RZ accomplished clinical sample and data collection. LJM, LX, and JW conceived the study. LX, JW, and XMZ supervised the study.

Corresponding authors

Correspondence to Lu Xu, Xiaoming Zha or Jue Wang.

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The authors declare no competing interests.

Ethics approval and consent to participate

All BC tissue samples were obtained from the Department of Breast Disease of The First Affiliated Hospital of Nanjing Medical University. The collection of clinical samples was approved by the Ethics Committee of Jiangsu Province Hospital (ethics review number: 2023-SR-977). All participants provided written informed consent prior to sample collection. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (ethics review number: IACUC-2309011). All procedures were carried out in accordance with institutional and national ethical guidelines for animal research.

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Ma, L., Sheng, X., Wang, L. et al. KDM6A alternative splicing induced by 25(OH)D inhibits breast cancer cell stemness through repressing TRAP1 transcription. Oncogene (2026). https://doi.org/10.1038/s41388-026-03694-z

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