TGFβ3-SMAD2/ETV4/CARM1 axis drives metastatic progression in Muscle-invasive Bladder Cancer via succinate metabolic rewiring

Abstract

The mechanistic connection between intramuscular invasion and distant metastasis in muscle-invasive bladder cancer (MIBC) remains unclear. In this study, a TGFβ3 signaling pathway that drives metastatic progression by altering succinate metabolism was identified. Mechanistically, a SMAD3/4-independent TGFβ3 signaling cascade that promotes the formation of a SMAD2/ETV4/CARM1 transcriptional complex was discovered. This complex epigenetically activates sulfide quinone oxidoreductase (SQOR), leading to mitochondrial metabolic reprogramming and succinate accumulation. The accumulated succinate then acts as a powerful paracrine signal, activating succinate receptor (SUCNR1) on smooth muscle cells (SMCs) to coordinate stromal remodeling and vascular niche formation that supports metastasis. Additionally, structure-based virtual screening reveals L-chicoric acid as a specific inhibitor that disrupts the critical SMAD2-ETV4 interaction, thereby blocking the metabolic loop and inhibiting metastasis. These findings establish TGFβ3 as an essential mediator of metabolic flexibility, and highlight the TGFβ3/SMAD2/ETV4/succinate axis as a promising therapeutic target.

Keywords

TGF-β3 / Succinate / SQOR / Smooth muscle cells / Metastasis / Muscle-invasive bladder cancer

Ethical statements

This study was approved by the Institutional Medical Research Ethics Committee of the Sun Yat-sen Memorial Hospital and Sun Yat-sen University Cancer Center of Sun Yat-sen University, complying with all relevant ethical regulations involving human participants (Approval number: SYSKY-2023-034-01). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University and the approval number was SYSU-IACUC-2023-001713.

Author contributions

The authors confirm contributions to the study as follows: study conception and design Tang M, Li J, Song L; data collection: Chen C, Zou H; analysis and interpretation of results: Feng R, Lin J, Chen X; draft manuscript preparation: Tang M, Yang M. All authors reviewed the results and approved the final version of the manuscript.

Data availability

All data generated or analyzed during this study are included in supplementary information files, and also available from the corresponding author upon reasonable request.

Acknowledgments

This work was supported by the Shenzhen Medical Research Fund (B2302046, B2502006) and Natural Science Foundation of China (No. 82330082, 82573270, 82203746, 82460551); The Special Program for Basic Research (Natural Science Foundation) of Shenzhen (No. JCYJ20230807114100001 to Hequn Zou).

Conflict of interest

The authors have no conflict of interest to declare.

Supplementary information accompanies this paper online at: https://doi.org/10.48130/targetome-0026-0007

References

[1] Tran L, Xiao JF, Agarwal N, Duex JE, Theodorescu D. 2021. Advances in bladder cancer biology and therapy. Nature Reviews Cancer 21:104-121

[2] Lenis AT, Lec PM, Chamie K, Mshs MD. 2020. Bladder Cancer: A Review. JAMA 324:1980-1991

[3] Dinney CPN, McConkey DJ, Millikan RE, Wu X, Bar-Eli M, et al. 2004. Focus on bladder cancer. Cancer Cell 6:111-116

[4] Kamat AM, Hahn NM, Efstathiou JA, Lerner SP, Malmström PU, et al. 2016. Bladder cancer. The Lancet 388:2796-2810

[5] Knowles MA, Hurst CD. 2015. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nature Reviews Cancer 15:25-41

[6] Galsky MD, Arija JÁA, Bamias A, Davis ID, De Santis M, et al. 2020. Atezolizumab with or without chemotherapy in metastatic urothelial cancer (IMvigor130): a multicentre, randomised, placebo-controlled phase 3 trial. The Lancet 395:1547-1557

[7] Patel VG, Oh WK, Galsky MD. 2020. Treatment of muscle-invasive and advanced bladder cancer in 2020. CA: A Cancer Journal for Clinicians 70:404-423

[8] Dyrskjøt L, Hansel DE, Efstathiou JA, Knowles MA, Galsky MD, et al. 2023. Bladder cancer. Nature Reviews Disease Primers 9:58

[9] Weinstein JN, Akbani R, Broom BM, Wang W, Verhaak RGW, et al. 2014. Comprehensive molecular characterization of urothelial bladder carci-noma. Nature 507:315-322

[10] Ross JS, Wang K, Gay LM, Al-Rohil RN, Nazeer T, et al. 2014. A high frequency of activating extracellular domain ERBB 2 (HER2) mutation in micropapillary urothelial carcinoma. Clinical Cancer Research 20:68-75

[11] Kim J, Akbani R, Creighton CJ, Lerner SP, Weinstein JN, et al. 2015. Inva-sive bladder cancer: genomic insights and therapeutic promise. Clini-cal Cancer Research 21:4514-4524

[12] Huan J, Grivas P, Birch J, Hansel DE. 2022. Emerging roles for mammalian target of rapamycin (mTOR) complexes in bladder cancer progression and therapy. Cancers 14:1555

[13] Rubenstein CS, Gard JMC, Wang M, McGrath JE, Ingabire N, et al. 2019. Gene editing of α6 integrin inhibits muscle invasive networks and increases cell-cell biophysical properties in prostate cancer. Cancer Research 79:4703-4714

[14] Harryman WL, Marr KD, Hernandez-Cortes D, Nagle RB, Garcia JGN, et al. 2021. Cohesive cancer invasion of the biophysical barrier of smooth muscle. Cancer and Metastasis Reviews 40:205-219

[15] Szarvas T, vom Dorp F, Ergün S, Rübben H. 2011. Matrix metallopro-teinases and their clinical relevance in urinary bladder cancer. Nature Reviews Urology 8:241-254

[16] Andersson KE, Arner A. 2004. Urinary bladder contraction and relax-ation: physiology and pathophysiology. Physiological Reviews 84:935-986

[17] Liu M, Gomez D. 2019. Smooth muscle cell phenotypic diversity. Arte-riosclerosis, Thrombosis, and Vascular Biology 39:1715-1723

[18] Allahverdian S, Chaabane C, Boukais K, Francis GA, Bochaton-Piallat ML. 2018. Smooth muscle cell fate and plasticity in atherosclerosis. Cardio-vascular Research 114:540-550

[19] Feil S, Fehrenbacher B, Lukowski R, Essmann F, Schulze-Osthoff K, et al. 2014. Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis. Circulation Research 115:662-667

[20] Hong X, Margariti A, Le Bras A, Jacquet L, Kong W, et al. 2017. Transdif-ferentiated human vascular smooth muscle cells are a new potential cell source for endothelial regeneration. Scientific Reports 7:5590

[21] Murgai M, Ju W, Eason M, Kline J, Beury DW, et al. 2017. KLF4-depen-dent perivascular cell plasticity mediates pre-metastatic niche forma-tion and metastasis. Nature Medicine 23:1176-1190

[22] Cao L, Wu G, Zhu J, Tan Z, Shi D, et al. 2019. Genotoxic stress-triggered β-catenin/JDP2/PRMT 5 complex facilitates reestablishing glutathione homeostasis. Nature Communications 10:3761

[23] Jiang S, Wang J, Yang C, Tan R, Hou J, et al. 2019. Continuous culture of urine-derived bladder cancer cells for precision medicine. Protein & Cell 10:902-907

[24] Naito T, Higuchi T, Shimada Y, Kakinuma C. 2020. An improved mouse orthotopic bladder cancer model exhibiting progression and treat-ment response characteristics of human recurrent bladder cancer. Oncology Letters 19:833-839

[25] Li C, Yan R, Bao J, Yuan G, Feng Y, et al. 2006. Characterization of a novel transplantable orthotopic murine xenograft model of a human bladder transitional cell tumor (BIU-87). Cancer Biology & Therapy 5:394-398

[26] Lorenzatti Hiles G, Cates AL, El-Sawy L, Day KC, Broses LJ, et al. 2019. A surgical orthotopic approach for studying the invasive progression of human bladder cancer. Nature Protocols 14:738-755

[27] Hayashi T, Gust KM, Wyatt AW, Goriki A, Jäger W, et al. 2016. Not all NOTCH is created equal: the oncogenic role of NOTCH2 in bladder cancer and its implications for targeted therapy. Clinical Cancer Research 22:2981-2992

[28] Hu C, Liu Y, Lin Y, Liang JK, Zhong WW, et al. 2018. Intravenous injec-tions of the oncolytic virus M1 as a novel therapy for muscle-invasive bladder cancer. Cell Death & Disease 9:274

[29] Liu X, Zhang Y, Chen Y, Li M, Zhou F, et al. 2017. In situ capture of chro-matin interactions by biotinylated dCas9. Cell 170:1028-1043.e19

[30] Li G, Wang D, Zhai Y, Pan C, Zhang J, et al. 2024. Glycometabolic repro-gramming-induced XRCC1 lactylation confers therapeutic resistance in ALDH1A3-overexpressing glioblastoma. Cell Metabolism 36:1696-1710.e10

[31] Weidner N. 1995. Current pathologic methods for measuring intratu-moral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Research and Treatment 36:169-180

[32] Monteiro-Reis S, Ferreira JPS, Pires RA, Lobo J, Carvalho JA, et al. 2023. Bladder wall stiffness after cystectomy in bladder cancer patients: a preliminary study. Cancers 15:359

[33] Xu Y, Zhang H, Chen Y, Pober JS, Zhou M, et al. 2024. SRF SUMOylation modulates smooth muscle phenotypic switch and vascular remodel-ing. Nature Communications 15:6919

[34] Salmon M, Gomez D, Greene E, Shankman L, Owens GK. 2012. Coopera-tive binding of KLF4, pELK-1, and HDAC2 to a G/C repressor element in the SM22α promoter mediates transcriptional silencing during SMC phenotypic switching in vivo. Circulation Research 111:685-696

[35] Reddy A, Winther S, Tran N, Xiao H, Jakob J, et al. 2024. Monocarboxy-late transporters facilitate succinate uptake into brown adipocytes. Nature Metabolism 6:567-577

[36] Gudgeon N, Munford H, Bishop EL, Hill J, Fulton-Ward T, et al. 2022. Succinate uptake by T cells suppresses their effector function via inhi-bition of mitochondrial glucose oxidation. Cell Reports 40:111193

[37] Peruzzotti-Jametti L, Bernstock JD, Vicario N, Costa ASH, Kwok CK, et al. 2018. Macrophage-derived extracellular succinate licenses neural stem cells to suppress chronic neuroinflammation. Cell Stem Cell 22:355-368.e13

[38] Reddy A, Bozi LHM, Yaghi OK, Mills EL, Xiao H, et al. 2020. pH-gated succinate secretion regulates muscle remodeling in response to exercise. Cell 183:62-75.e17

[39] Meng S, Zhou G, Gu Q, Chanda PK, Ospino F, et al. 2016. Transdifferen-tiation requires iNOS activation: role of RING1A S-nitrosylation. Circula-tion Research 119:e129-e138

[40] Mossa AH, Velasquez Flores M, Cammisotto PG, Campeau L. 2017. Succinate, increased in metabolic syndrome, activates GPR91 receptor signaling in urothelial cells. Cellular Signalling 37:31-39

[41] Ren R, Fan Y, Peng Z, Wang S, Jiang Y, et al. 2022. Characterization and perturbation of CTCF-mediated chromatin interactions for enhancing myogenic transdifferentiation. Cell Reports 40:111206

[42] Xu Q, Sun J, Holden CM, Neto HCF, Wang T, et al. 2024. Cellular commu-nication network factor 2 regulates smooth muscle cell transdifferenti-ation and lipid accumulation in atherosclerosis. Cardiovascular Research 120:2191-2207

[43] Stomberski CT, Hess DT, Stamler JS. 2019. Protein S-nitrosylation: deter-minants of specificity and enzymatic regulation of S-nitrosothiol-based signaling. Antioxidants & Redox Signaling 30:1331-1351

[44] Lin Z, Zhao S, Li X, Miao Z, Cao J, et al. 2023. Cathepsin B S-nitrosylation promotes ADAR1-mediated editing of its own mRNA transcript via an ADD1/MATR3 regulatory axis. Cell Research 33:546-561

[45] Guo Y, Wen J, He A, Qu C, Peng Y, et al. 2023. iNOS contributes to heart failure with preserved ejection fraction through mitochondrial dysfunction and Akt S-nitrosylation. Journal of Advanced Research 43:175-186

[46] Sha Y, Marshall HE. 2012. S-nitrosylation in the regulation of gene transcription. Biochimica et Biophysica Acta (BBA) - General Subjects 1820:701-711

[47] Liu X, Zhao G, Sun S, Fan C, Feng X, et al. 2022. Biosynthetic pathway and metabolic engineering of succinic acid. Frontiers in Bioengineering and Biotechnology 10:843887

[48] Fernández-Veledo S, Ceperuelo-Mallafré V, Vendrell J. 2021. Rethinking succinate: an unexpected hormone-like metabolite in energy home-ostasis. Trends in Endocrinology & Metabolism 32:680-692

[49] Zhang J, Wang YT, Miller JH, Day MM, Munger JC, et al. 2018. Accumu-lation of succinate in cardiac ischemia primarily occurs via canonical krebs cycle activity. Cell Reports 23:2617-2628

[50] Bisbach CM, Hass DT, Robbings BM, Rountree AM, Sadilek M, et al. 2020. Succinate can shuttle reducing power from the hypoxic retina to the O2-rich pigment epithelium. Cell Reports 31:107606

[51] Chouchani ET, Pell VR, Gaude E, Aksentijević D, Sundier SY, et al. 2014. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 515:431-435

[52] Taegtmeyer H. 1978. Metabolic responses to cardiac hypoxia. Increased production of succinate by rabbit papillary muscles. Circulation Research 43:808-815

[53] Spinelli JB, Rosen PC, Sprenger HG, Puszynska AM, Mann JL, et al. 2021. Fumarate is a terminal electron acceptor in the mammalian electron transport chain. Science 374:1227-1237

[54] Borst P. 2020. The malate-aspartate shuttle (Borst cycle): How it started and developed into a major metabolic pathway. IUBMB Life 72:2241-2259

[55] Yang H, Zhou L, Shi Q, Zhao Y, Lin H, et al. 2015. SIRT3-dependent GOT2 acetylation status affects the malate-aspartate NADH shuttle activity and pancreatic tumor growth. The EMBO Journal 34:1110-1125

[56] Bulusu V, Jayaraman V, Balaram H. 2011. Metabolic fate of fumarate, a side product of the purine salvage pathway in the intraerythrocytic stages of Plasmodium falciparum. Journal of Biological Chemistry 286:9236-9245

[57] Watts RWE. 1983. Some regulatory and integrative aspects of purine nucleotide biosynthesis and its control: an overview. Advances in Enzyme Regulation 21:33-51

[58] Taeusch HW, Smith BT. 1987. Surfactant sufficiency for immature infants-prenatal induction vs. postnatal treatment. European Journal of Respiratory Diseases Supplement 153:249-254

[59] Banerjee R, Kumar R. 2022. Gas regulation of complex II reversal via electron shunting to fumarate in the mammalian ETC. Trends in Biochemical Sciences 47:689-698

[60] Massague J, Seoane J, Wotton D. 2005. Smad transcription factors. Genes & Development 19:2783-2810

[61] Schmierer B, Hill CS. 2007. TGFβ-SMAD signal transduction: molecular specificity and functional flexibility. Nature Reviews Molecular Cell Bio-logy 8:970-982

[62] Moustakas A, Souchelnytskyi S, Heldin CH. 2001. Smad regulation in TGF-β signal transduction. Journal of Cell Science 114:4359-4369

[63] Colak S, Ten Dijke P. 2017. Targeting TGF-β signaling in cancer. Trends in Cancer 3:56-71

[64] Bedford MT, Richard S. 2005. Arginine methylation an emerging regula-tor of protein function. Molecular Cell 18:263-272

[65] Casadio F, Lu X, Pollock SB, LeRoy G, Garcia BA, et al. 2013. H3R42me2a is a histone modification with positive transcriptional effects. Proceed-ings of the National Academy of Sciences of the United States of America 110:14894-14899

[66] Si Y, Bon C, Barbachowska M, Cadet-Daniel V, Jallet C, et al. 2022. A novel screening strategy to identify histone methyltransferase inhibitors reveals a crosstalk between DOT1L and CARM1. RSC Chemi-cal Biology 3:456-467

[67] Wu J, Xu W. 2012. Histone H3R17me2a mark recruits human RNA poly-merase-associated factor 1 complex to activate transcription. Proceed-ings of the National Academy of Sciences of the United States of America 109:5675-5680

[68] Chen FX, Xie P, Collings CK, Cao K, Aoi Y, et al. 2017. PAF 1 regulation of promoter-proximal pause release via enhancer activation. Science 357:1294-1298

[69] Chen FX, Woodfin AR, Gardini A, Rickels RA, Marshall SA, et al. 2015. PAF1, a molecular regulator of promoter-proximal pausing by RNA polymerase II. Cell 162:1003-1015

[70] Alfred Witjes J, Lebret T, Compérat EM, Cowan NC, De Santis M, et al. 2017. Updated 2016 EAU guidelines on muscle-invasive and metastatic bladder cancer. European Urology 71:462-475

[71] Haas G, Fan S, Ghadimi M, De Oliveira T, Conradi LC. 2021. Different forms of tumor vascularization and their clinical implications focusing on vessel co-option in colorectal cancer liver metastases. Frontiers in Cell and Developmental Biology 9:612774

[72] Das RN, Tevet Y, Safriel S, Han Y, Moshe N, et al. 2022. Generation of specialized blood vessels via lymphatic transdifferentiation. Nature 606:570-575

[73] Sayed N, Wong WT, Ospino F, Meng S, Lee J, et al. 2015. Transdifferenti-ation of human fibroblasts to endothelial cells: role of innate immunity. Circulation 131:300-309

[74] Wu JY, Huang TW, Hsieh YT, Wang YF, Yen CC, et al. 2020. Cancer-derived succinate promotes macrophage polarization and cancer metastasis via succinate receptor. Molecular Cell 77:213-227.e5

[75] Tannahill GM, Curtis AM, Adamik J, Palsson-McDermott EM, McGettrick AF, et al. 2013. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature 496:238-242

[76] Guzy RD, Sharma B, Bell E, Chandel NS, Schumacker PT. 2008. Loss of the SdhB, but Not the SdhA, subunit of complex II triggers reactive oxygen species-dependent hypoxia-inducible factor activation and tumorigenesis. Molecular and Cellular Biology 28:718-731

[77] Rubic T, Lametschwandtner G, Jost S, Hinteregger S, Kund J, et al. 2008. Triggering the succinate receptor GPR91 on dendritic cells enhances immunity. Nature Immunology 9:1261-1269

[78] Markovich D, Murer H. 2004. The SLC 13 gene family of sodium sulphate/carboxylate cotransporters. Pflügers Archiv 447:594-602

[79] Bertrand-Chapel A, Caligaris C, Fenouil T, Savary C, Aires S, et al. 2022. SMAD2/ 3 mediate oncogenic effects of TGF-β in the absence of SMAD4. Communications Biology 5:1068

[80] Deng Z, Fan T, Xiao C, Tian H, Zheng Y, et al. 2024. TGF-β signaling in health, disease, and therapeutics. Signal Transduction and Targeted Therapy 9:61

[81] Massagué J, Sheppard D. 2023. TGF-β signaling in health and disease. Cell 186:4007-4037

[82] Hua W, Ten Dijke P, Kostidis S, Giera M, Hornsveld M. 2020. TGFβ-induced metabolic reprogramming during epithelial-to-mesenchymal transition in cancer. Cellular and Molecular Life Sciences 77:2103-2123

[83] Derynck R, Zhang YE. 2003. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 425:577-584

[84] Sun X, Zhang X, Zhai H, Zhang D, Ma S. 2019. Chicoric acid (CA) induces autophagy in gastric cancer through promoting endoplasmic reticu-lum (ER) stress regulated by AMPK. Biomedicine & Pharmacotherapy 118:109144