EHMT2-mediated R-loop formation promotes the malignant progression of prostate cancer via activating Aurora B
Yuyang Zhang
Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Institute of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Anhui Province Key Laboratory of Urological and Andrological Diseases Research and Medical Transformation, Anhui Medical University, Hefei, Anhui, China
Search for more papers by this authorMingqin Su
Department of Pathology, The Second People's Hospital of Hefei, Hefei Hospital Affiliated to Anhui Medical University, Hefei, Anhui, China
Search for more papers by this authorYiming Chen
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Search for more papers by this authorLi Cui
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Search for more papers by this authorWei Xia
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Search for more papers by this authorRenfang Xu
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Search for more papers by this authorCorresponding Author
Dong Xue
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Correspondence
Xingliang Feng and Dong Xue, Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China.
Email: [email protected] and [email protected]
Xiansheng Zhang, Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
Email: [email protected]
Search for more papers by this authorCorresponding Author
Xiansheng Zhang
Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Institute of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Anhui Province Key Laboratory of Urological and Andrological Diseases Research and Medical Transformation, Anhui Medical University, Hefei, Anhui, China
Correspondence
Xingliang Feng and Dong Xue, Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China.
Email: [email protected] and [email protected]
Xiansheng Zhang, Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
Email: [email protected]
Search for more papers by this authorCorresponding Author
Xingliang Feng
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Correspondence
Xingliang Feng and Dong Xue, Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China.
Email: [email protected] and [email protected]
Xiansheng Zhang, Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
Email: [email protected]
Search for more papers by this authorYuyang Zhang
Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Institute of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Anhui Province Key Laboratory of Urological and Andrological Diseases Research and Medical Transformation, Anhui Medical University, Hefei, Anhui, China
Search for more papers by this authorMingqin Su
Department of Pathology, The Second People's Hospital of Hefei, Hefei Hospital Affiliated to Anhui Medical University, Hefei, Anhui, China
Search for more papers by this authorYiming Chen
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Search for more papers by this authorLi Cui
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Search for more papers by this authorWei Xia
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Search for more papers by this authorRenfang Xu
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Search for more papers by this authorCorresponding Author
Dong Xue
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Correspondence
Xingliang Feng and Dong Xue, Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China.
Email: [email protected] and [email protected]
Xiansheng Zhang, Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
Email: [email protected]
Search for more papers by this authorCorresponding Author
Xiansheng Zhang
Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Institute of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
Anhui Province Key Laboratory of Urological and Andrological Diseases Research and Medical Transformation, Anhui Medical University, Hefei, Anhui, China
Correspondence
Xingliang Feng and Dong Xue, Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China.
Email: [email protected] and [email protected]
Xiansheng Zhang, Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
Email: [email protected]
Search for more papers by this authorCorresponding Author
Xingliang Feng
Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China
Department of Urology, The First People's Hospital of Changzhou, Changzhou, Jiangsu, China
Correspondence
Xingliang Feng and Dong Xue, Department of Urology, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China.
Email: [email protected] and [email protected]
Xiansheng Zhang, Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.
Email: [email protected]
Search for more papers by this authorAbstract
Background
Chromosomal instability (CIN), a hallmark of cancer, is commonly linked to poor prognosis in high-grade prostate cancer (PCa). Paradoxically, excessively high levels of CIN may impair cancer cell viability. Consequently, understanding how tumours adapt to CIN is critical for identifying novel therapeutic targets.
Methods
Bioinformatic analyses were conducted to identify genes overexpressed in PCa tissues using The Cancer Genome Atlas (TCGA) and GEO datasets. Western blotting and immunohistochemistry assays were applied to determine the expression levels of euchromatic histone lysine methyltransferase 2 (EHMT2), pT232-Aurora B and Cullin 3 (CUL3). The proliferation of cells was measured through CCK-8 tests, clonogenesis and subcutaneous xenografts of human PCa cells in BALB/c nude mice. Live cell imaging, immunofluorescence (IF) and flow cytometry were used to confirm the role of EHMT2 in PCa cell mitosis. Co-immunoprecipitation, Western blotting and IF assays further elucidated the underlying molecular mechanisms.
Results
EHMT2 was highly expressed in metastatic PCa tissues exhibiting elevated CIN and was strongly associated with adverse clinical outcomes in patients with PCa. Silencing EHMT2 impaired cell division, inducing G2/M-phase arrest and mitotic catastrophe in PCa cells. Mechanistically, EHMT2 is indispensable to ensure the full activation of Aurora B through centromeric R-loop-driven ATR–CHK1 pathway, with EHMT2 protein expression peaking during the G2/M-phase. Moreover, CUL3 was identified as a binding partner of EHMT2, mediating its polyubiquitination and destabilising its protein levels.
Conclusions
This study reveals a CUL3–EHMT2–Aurora B regulatory axis that safeguards accurate chromosome segregation in PCa cells, supporting the potential therapeutic application of EHMT2 inhibitors.
Key points
- Euchromatic histone lysine methyltransferase 2 (EHMT2) is overexpressed in advanced prostate cancer, restraining catastrophic chromosomal instability (CIN) and enhancing cell fitness.
- EHMT2 functions via the centromeric R-loop-driven ATR–CHK1–Aurora B pathway to promote chromosomal stability.
- EHMT2 confers enzalutamide resistance via activating Aurora B.
- Cullin 3 (CUL3) promotes EHMT2 destabilisation via deubiquitination.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
Open Research
DATA AVAILABILITY STATEMENT
Data are available from the corresponding author upon reasonable request.
Supporting Information
| Filename | Description |
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| ctm270164-sup-0001-Figures.pdf6.4 MB | Supporting Information |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
REFERENCES
- 1Chen Y, Zhou Z, Zhou Y, et al. Updated prevalence of latent prostate cancer in Chinese population and comparison of biopsy results: an autopsy-based study. Innovation (Camb). 2024; 5:100558.
- 2Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024; 74: 12-49.
- 3Carceles-Cordon M, Orme JJ, Domingo-Domenech J, Rodriguez-Bravo V. The yin and yang of chromosomal instability in prostate cancer. Nat Rev Urol. 2024; 21: 357-372.
- 4Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat Rev Cancer. 2015; 15: 701-711.
- 5Dhital B, Santasusagna S, Kirthika P, et al. Harnessing transcriptionally driven chromosomal instability adaptation to target therapy-refractory lethal prostate cancer. Cell Rep Med. 2023; 4:100937.
- 6Fraser M, Sabelnykova VY, Yamaguchi TN, et al. Genomic hallmarks of localized, non-indolent prostate cancer. Nature. 2017; 541: 359-364.
- 7Hieronymus H, Murali R, Tin A, et al. Tumor copy number alteration burden is a pan-cancer prognostic factor associated with recurrence and death. Elife. 2018; 7:e37294.
- 8Grasso CS, Wu Y-M, Robinson DR, et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature. 2012; 487: 239-243.
- 9Janssen A, Kops GJ, Medema RH. Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells. Proc Natl Acad Sci U S A. 2009; 106: 19108-19113.
- 10Taylor BS, Schultz N, Hieronymus H, et al. Integrative genomic profiling of human prostate cancer. Cancer Cell. 2010; 18: 11-22.
- 11Carter SL, Eklund AC, Kohane IS, Harris LN, Szallasi Z. A signature of chromosomal instability inferred from gene expression profiles predicts clinical outcome in multiple human cancers. Nat Genet. 2006; 38: 1043-1048.
- 12Krenn V, Musacchio A. The Aurora B kinase in chromosome bi-orientation and spindle checkpoint signaling. Front Oncol. 2015; 5: 225.
- 13Welburn JPI, Vleugel M, Liu D, et al. Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface. Mol Cell. 2010; 38: 383-392.
- 14Den Hollander J, Rimpi S, Doherty JR, et al. Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state. Blood. 2010; 116: 1498-1505.
- 15Huang D, Huang Y, Huang Z, Weng J, Zhang S, Gu W. Relation of AURKB over-expression to low survival rate in BCRA and reversine-modulated aurora B kinase in breast cancer cell lines. Cancer Cell Int. 2019; 19: 166.
- 16Failes TW, Mitic G, Abdel-Halim H, et al. Evolution of resistance to Aurora kinase B inhibitors in leukaemia cells. PLoS One. 2012; 7:e30734.
- 17Zhao H, Wang Y, Yang Z, Wei W, Cong Z, Xie Y. High expression of aurora kinase B predicts poor prognosis in hepatocellular carcinoma after curative surgery and its effects on the tumor microenvironment. Ann Transl Med. 2022; 10: 1168.
- 18Kabeche L, Nguyen HD, Buisson R, Zou L. A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation. Science. 2018; 359: 108-114.
- 19Tanaka K, Yu HA, Yang S, et al. Targeting Aurora B kinase prevents and overcomes resistance to EGFR inhibitors in lung cancer by enhancing BIM- and PUMA-mediated apoptosis. Cancer Cell. 2021; 39: 1245-1261 e1246.
- 20Bertran-Alamillo J, Cattan V, Schoumacher M, et al. AURKB as a target in non-small cell lung cancer with acquired resistance to anti-EGFR therapy. Nat Commun. 2019; 10: 1812.
- 21Hole S, Pedersen AM, Lykkesfeldt AE, Yde CW. Aurora kinase A and B as new treatment targets in aromatase inhibitor-resistant breast cancer cells. Breast Cancer Res Treat. 2015; 149: 715-726.
- 22Al-Khafaji AS, Davies MP, Risk JM, et al. Aurora B expression modulates paclitaxel response in non-small cell lung cancer. Br J Cancer. 2017; 116: 592-599.
- 23Phadke MS, Sini P, Smalley KS. The novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 overcomes acquired BRAF inhibitor resistance through suppression of Mcl-1 and MEK expression. Mol Cancer Ther. 2015; 14: 1354-1364.
- 24Padeken J, Methot SP, Gasser SM. Establishment of H3K9-methylated heterochromatin and its functions in tissue differentiation and maintenance. Nat Rev Mol Cell Biol. 2022; 23: 623-640.
- 25Shankar SR, Bahirvani AG, Rao VK, Bharathy N, Ow JR, Taneja R. G9a, a multipotent regulator of gene expression. Epigenetics. 2013; 8: 16-22.
- 26Tachibana M, Sugimoto K, Nozaki M, et al. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 2002; 16: 1779-1791.
- 27Ding J, Li T, Wang X, et al. The histone H3 methyltransferase G9A epigenetically activates the serine-glycine synthesis pathway to sustain cancer cell survival and proliferation. Cell Metab. 2013; 18: 896-907.
- 28Rowbotham SP, Li F, Dost AFM, et al. H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression. Nat Commun. 2018; 9: 4559.
- 29Ni Y, Shi M, Liu L, et al. G9a in cancer: mechanisms, therapeutic advancements, and clinical implications. Cancers (Basel). 2024; 16: 2175.
- 30Yang C, Ma S, Zhang J, et al. EHMT2-mediated transcriptional reprogramming drives neuroendocrine transformation in non-small cell lung cancer. Proc Natl Acad Sci U S A. 2024; 121:e2317790121.
- 31Yang Q, Zhu Q, Lu X, et al. G9a coordinates with the RPA complex to promote DNA damage repair and cell survival. Proc Natl Acad Sci U S A. 2017; 114: E6054-E6063.
- 32Fan HT, Shi YY, Lin Y, Yang XP. EHMT2 promotes the development of prostate cancer by inhibiting PI3K/AKT/mTOR pathway. Eur Rev Med Pharmacol Sci. 2019; 23: 7808-7815.
- 33Rajagopalan H, Lengauer C. Aneuploidy and cancer. Nature. 2004; 432: 338-341.
- 34Carmena M, Wheelock M, Funabiki H, Earnshaw WC. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nat Rev Mol Cell Biol. 2012; 13: 789-803.
- 35Yasui Y, Urano T, Kawajiri A, et al. Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis. J Biol Chem. 2004; 279: 12997-13003.
- 36Kapoor TM, Mayer TU, Coughlin ML, Mitchison TJ. Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5. J Cell Biol. 2000; 150: 975-988.
- 37Muneer A, Wang Li, Xie L, et al. Non-canonical function of histone methyltransferase G9a in the translational regulation of chronic inflammation. Cell Chem Biol. 2023; 30: 1525-1541 e1527.
- 38Hussain M, Fizazi K, Saad F, et al. Enzalutamide in men with nonmetastatic, castration-resistant prostate cancer. N Engl J Med. 2018; 378: 2465-2474.
- 39Scher HI, Fizazi K, Saad F, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012; 367: 1187-1197.
- 40Armstrong AJ, Eisenberger MA, Halabi S, et al. Biomarkers in the management and treatment of men with metastatic castration-resistant prostate cancer. Eur Urol. 2012; 61: 549-559.
- 41Geigl JB, Obenauf AC, Schwarzbraun T, Speicher MR. Defining ‘chromosomal instability’. Trends Genet. 2008; 24: 64-69.
- 42Broad AJ, DeLuca KF, DeLuca JG. Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells. J Cell Biol. 2020; 219:e201905144.
- 43Cimini D, Wan X, Hirel CB, Salmon ED. Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors. Curr Biol. 2006; 16: 1711-1718.
- 44Adams RR, Carmena M, Earnshaw WC. Chromosomal passengers and the (aurora) ABCs of mitosis. Trends Cell Biol. 2001; 11: 49-54.
- 45Chen B, Dragomir MP, Fabris L, et al. The long noncoding RNA CCAT2 induces chromosomal instability through BOP1-AURKB signaling. Gastroenterology. 2020; 159: 2146-2162 e2133.
- 46Ruchaud S, Carmena M, Earnshaw WC. The chromosomal passenger complex: one for all and all for one. Cell. 2007; 131: 230-231.
- 47Ruchaud S, Carmena M, Earnshaw WC. Chromosomal passengers: conducting cell division. Nat Rev Mol Cell Biol. 2007; 8: 798-812.
- 48Wang F, Ulyanova NP, Daum JR, et al. Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation. J Cell Biol. 2012; 199: 251-268.
- 49Wang F, Dai J, Daum JR, et al. Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis. Science. 2010; 330: 231-235.
- 50Santaguida S, Tighe A, D'Alise AM, Taylor SS, Musacchio A. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J Cell Biol. 2010; 190: 73-87.
- 51Li J, Zhao J, Gan X, et al. The RPA-RNF20-SNF2H cascade promotes proper chromosome segregation and homologous recombination repair. Proc Natl Acad Sci U S A. 2023; 120:e2303479120.
- 52Wang W, Wang J, Liu S, et al. An EHMT2/NFYA-ALDH2 signaling axis modulates the RAF pathway to regulate paclitaxel resistance in lung cancer. Mol Cancer. 2022; 21: 106.
- 53Nachiyappan A, Gupta N, Taneja R. EHMT1/EHMT2 in EMT, cancer stemness and drug resistance: emerging evidence and mechanisms. FEBS J. 2022; 289: 1329-1351.
- 54Lee RA, Chang M, Yiv N, et al. Transcriptional coactivation by EHMT2 restricts glucocorticoid-induced insulin resistance in a study with male mice. Nat Commun. 2023; 14: 3143.
- 55Wang L, Dong X, Ren Y, et al. Targeting EHMT2 reverses EGFR-TKI resistance in NSCLC by epigenetically regulating the PTEN/AKT signaling pathway. Cell Death Dis. 2018; 9: 129.
- 56Li W, Wang H-Y, Zhao X, et al. A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair. Sci Adv. 2019; 5:eaau7566.
- 57Metzger T, Kleiss C, Sumara I. CUL3 and protein kinases: insights from PLK1/KLHL22 interaction. Cell Cycle. 2013; 12: 2291-2296.
- 58Zhou Z, Qin J, Song C, et al. circROBO1 promotes prostate cancer growth and enzalutamide resistance via accelerating glycolysis. J Cancer. 2023; 14: 2574-2584.
- 59Wang H, Li Ni, Liu Q, et al. Antiandrogen treatment induces stromal cell reprogramming to promote castration resistance in prostate cancer. Cancer Cell. 2023; 41: 1345-1362 e1349.
- 60Ye Z, Deng X, Zhang J, et al. Causal relationship between immune cells and prostate cancer: a Mendelian randomization study. Front Cell Dev Biol. 2024; 12:1381920.
- 61Comstock CE, Knudsen KE. The complex role of AR signaling after cytotoxic insult: implications for cell-cycle-based chemotherapeutics. Cell Cycle. 2007; 6: 1307-1313.
- 62Wang Q, Li W, Zhang Y, et al. Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer. Cell. 2009; 138: 245-256.
- 63Maximum androgen blockade in advanced prostate cancer: an overview of the randomised trials. Prostate Cancer Trialists' Collaborative Group. Lancet. 2000; 355: 1491-1498.
- 64Petrylak DP, Tangen CM, Hussain MHA, et al. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med. 2004; 351: 1513-1520.
