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Single-cell transcriptome analysis revealing the intratumoral heterogeneity of ccRCC and validation of MT2A in pathogenesis

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Abstract

Clear-cell renal cell carcinoma (ccRCC) appears as the most common type of kidney cancer, the carcinogenesis of which has not been fully elucidated. Tumor heterogeneity plays a crucial role in cancer progression, which could be largely deciphered by the implement of scRNA-seq. The bulk and single-cell RNA expression profile is obtained from TCGA and study conducted by Young et al. We utilized UMAP, TSNE, and clustering algorithm Louvain for dimensionality reduction and FindAllMarkers function for determining the DEGs. Monocle2 was utilized to perform pseudo-time series analysis. SCENIC was implemented for transcription factor analysis of each cell subgroup. A series of WB, CFA, CCK-8, and EDU analysis was utilized for the validation of the role of MT2A in ccRCC carcinogenesis. We observed higher infiltration of T/NK and B cells in tumorous tissues, indicating the role of immune cells in ccRCC carcinogenesis. Transcription factor analysis revealed the activation of EOMES and ETS1 in CD8 + T cells, while CAFs were divided into myo-CAFs and i-CAFs, with i-CAFs showing distinct enrichment of ATF3, JUND, JUNB, EGR1, and XBP1. Through cell trajectory analysis, we discerned three distinct stages of cellular evolution, where State2 symbolizes normal renal tubular cells that underwent transitions into State1 and State3 as the CNV score ascended. Functional enrichment examination revealed an amplification of interferon gamma and inflammatory response pathways within tumor cells. The consensus clustering algorithm yielded two molecular subtypes, with cluster 2 being associated with advanced tumor stages and an abundance of infiltrated immune cells. We identified 17 prognostic genes through Cox and LASSO regression models and used them to construct a prognostic model, the efficacy of which was verified in multiple cohorts. Furthermore, we investigated the role of MT2A, one of our hub genes, in ccRCC carcinogenesis, and found it to regulate proliferation and migration of malignant cells. We depicted a detailed single-cell landscape of ccRCC, with special focus on CAFs, endothelial cells, and renal tubular cells. A prognostic model of high stability and accuracy was constructed based on the DEGs. MT2A was found to be actively implicated in ccRCC carcinogenesis, regulating proliferation and migration of the malignant cells.

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The original data can be obtained from Sun-Zhou.

References

  • Ayres JS, Vance RE (2012) Cellular teamwork in antibacterial innate immunity. Nat Immunol 13(2):115–117

    Article  CAS  PubMed  Google Scholar 

  • Cairns P (2011) Renal Cell Carcinoma 9(1–6):461–473

    Google Scholar 

  • Chang HH, Hemberg M, Barahona M, Ingber DE, Huang SJN (2008) Transcriptome-Wide Noise Controls Lineage Choice in Mammalian Progenitor Cells. Nature 453(7194):544–547

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen Y, Peng C, Chen J, Chen D, Yang B, He B, Hu W, Zhang Y, Liu H, Dai L, Xie H, Zhou L, Wu J, Zheng S (2019) WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1. Mol Cancer 18(1):127

    Article  PubMed  PubMed Central  Google Scholar 

  • Du ZL, Brett-Morris A, Aguila B, Kerner J, Hoppel CL, Puchowicz M, Serra D, Herrero L, Rini B (2017) HIF drives lipid deposition and cancer in ccRCC via repression of fatty acid metabolism. Nat Commun 8(1):1769

    Article  PubMed  PubMed Central  Google Scholar 

  • Elyada E, Bolisetty M, Laise P, Flynn WF, Courtois ET, Burkhart RA, Teinor JA, Belleau P, Biffi G, Lucito MS, Sivajothi S, Armstrong TD, Engle DD, Yu KH, Hao Y, Wolfgang CL, Park Y, Preall J, Jaffee EM, Califano A, Robson P, Tuveson DA (2019) Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts. Cancer Discov 9(8):1102–1123

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Feig C, Jones JO, Kraman M, Wells RJ, Deonarine A, Chan DS, Connell CM, Roberts EW, Zhao Q, Caballero OL, Teichmann SA, Janowitz T, Jodrell DI, Tuveson DA, Fearon DT (2013) Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci U S A 110(50):20212–20217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fiori ME, Di Franco S, Villanova L, Bianca P, Stassi G, De Maria R (2019) Cancer-associated fibroblasts as abettors of tumor progression at the crossroads of EMT and therapy resistance. Mol Cancer 18(1):70

    Article  PubMed  PubMed Central  Google Scholar 

  • Forma E, Krzeslak A, Wilkosz J, Jozwiak P, Szymczyk A, Rozanski W, Brys M (2012) Metallothionein 2A genetic polymorphisms and risk of prostate cancer in a Polish population. Cancer Gen 205(9):432–435

    Article  CAS  Google Scholar 

  • Gieniec KA, Butler LM, Worthley DL, Woods SL (2019) Cancer-associated fibroblasts-heroes or villains? Br J Cancer 121(4):293–302

    Article  PubMed  PubMed Central  Google Scholar 

  • Kasashima H, Duran A, Martinez-Ordoñez A, Nakanishi Y, Kinoshita H, Linares JF, Reina-Campos M, Kudo Y, L’Hermitte A, Yashiro M, Ohira M, Bao F, Tauriello DVF, Batlle E, Diaz-Meco MT, Moscat J (2021) Stromal SOX2 upregulation promotes tumorigenesis through the generation of a sfrp1/2-expressing cancer-associated fibroblast population. Dev Cell 56(1):95-110.e10

    Article  CAS  PubMed  Google Scholar 

  • Kobayashi H, Enomoto A, Woods SL, Burt AD, Takahashi M, Worthley DL (2019) Cancer-associated fibroblasts in gastrointestinal cancer. Nat Rev Gastroenterol Hepatol 16(5):282–295

    Article  PubMed  Google Scholar 

  • Krześlak A, Forma E, Jóźwiak P, Szymczyk A, Smolarz B, Romanowicz-Makowska H, Różański W, Bryś M (2014) Metallothionein 2A genetic polymorphisms and risk of ductal breast cancer. Clin Exp Med 14:107–113

    Article  PubMed  Google Scholar 

  • Lähnemann D, Köster J, Szczurek E, McCarthy DJ, Hicks SC, Robinson MD, Vallejos CA, Campbell KR, Beerenwinkel N, Mahfouz A, Pinello L, Skums P, Stamatakis A, Attolini CS, Aparicio S, Baaijens J, Balvert M, Barbanson B, Cappuccio A, Corleone G, Dutilh BE, Florescu M, Guryev V, Holmer R, Jahn K, Lobo TJ, Keizer EM, Khatr I, Kielbasa SM, Korbel JO, Kozlov AM, Kuo TH, Lelieveldt BPF, Mandoiu II, Marioni JC, Marschall T, Mölder F, Niknejad A, Raczkowski L, Reinders M, Ridder J, Saliba AE, Somarakis A, Stegle O, Theis FJ, Yang H, Zelikovsky A, McHardy AC, Raphael BJ, Shah SP, Schönhuth A (2020) Eleven grand challenges in single-cell data science. Genome Biol 21(1):31

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu Q, Bai X, Li H, Zhang Y, Zhao Y, Zhang X, Ye L (2013) The oncoprotein HBXIP upregulates Lin28B via activating TF II D to promote proliferation of breast cancer cells. Int J Cancer 133(6):1310–1322

    Article  CAS  PubMed  Google Scholar 

  • Muglia VF, Prando A (2015) Renal cell carcinoma: histological classification and correlation with imaging findings. Radial Bras 48:166–174

    Article  Google Scholar 

  • Navin NE (2015) Delineating cancer evolution with single-cell sequencing. Sci Transl Med 7(296):296fs29

    Article  PubMed  PubMed Central  Google Scholar 

  • Nielsen AE, Bohr A, Penkowa M (2006) The balance between life and death of cells: roles of metallothioneins. Biomarker Insights 1:117727190600100020

    Article  Google Scholar 

  • Öhlund D, Handly-Santana A, Biffi G, Elyada E, Almeida AS, Ponz-Sarvise M, Corbo V, Oni TE, Hearn SA, Lee EJ, Chio II, Hwang CI, Tiriac H, Baker LA, Engle DD, Feig C, Kultti A, Egeblad M, Fearon DT, Crawford JM, Clevers H, Park Y, Tuveson DA (2017) Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J Exp Med 214(3):579–596

    Article  PubMed  PubMed Central  Google Scholar 

  • Pötzl J, Roser D, Bankel L, Hömberg N, Geishauser A, Brenner CD, Weigand M, Röcken M, Mocikat R (2017) Reversal of tumor acidosis by systemic buffering reactivates NK cells to express IFN-γ and induces NK cell-dependent lymphoma control without other immunotherapies. Int J Cancer 140(9):2125–2133

    Article  PubMed  Google Scholar 

  • Saunders NA, Simpson F, Thompson EW, Hill MM, Endo-Munoz L, Leggatt G, Minchin RF, Guminski A (2012) Role of intratumoural heterogeneity in cancer drug resistance: molecular and clinical perspectives. Embo Mol Med 4(8):675–684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sebastian A, Hum NR, Martin KA, Gilmore SF, Peran I, Byers SW, Wheeler EK, Coleman MA, Loots GG (2020) Single-cell transcriptomic analysis of tumor-derived fibroblasts and normal tissue-resident fibroblasts reveals fibroblast heterogeneity in breast cancer. Cancers 12(5):1307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Siegel RL, Miller KD, Jemal A (2019) Cancer Statistics 69(1):7–34

    Google Scholar 

  • Starska K, Krześlak A, Forma E, Olszewski J, Lewy-Trenda I, Osuch-Wójcikiewicz E, Bryś M (2014) Genetic polymorphism of metallothionein 2A and risk of laryngeal cancer in a Polish population. Med Oncol 31:1–10

    Article  CAS  Google Scholar 

  • Starska K, Bryś M, Forma E, Olszewski J, Pietkiewicz P, Lewy-Trenda I, Stasikowska-Kanicka O, Danilewicz M, Krześlak A (2015) Metallothionein 2A core promoter region genetic polymorphism and its impact on the risk, tumor behavior, and recurrences of sinonasal inverted papilloma (Schneiderian papilloma). Tumor Biol 36:8559–8571

    Article  CAS  Google Scholar 

  • Takahashi SJMMR (2015) Positive and Negative Regulators of the Metallothionein Gene. Mol Med Rep 12(1):795–799

    Article  CAS  PubMed  Google Scholar 

  • Tang Q, Chen J, Di Z, Yuan W, Zhou Z, Liu Z, Han S, Liu Y, Ying G, Shu X, Di M (2020) TM4SF1 promotes EMT and cancer stemness via the Wnt/β-catenin/SOX2 pathway in colorectal cancer. J Exp Clin Cancer Res : CR 39(1):232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang F, Xu J, Zhu Q, Qin X, Cao Y, Lou J, Xu Y, Ke X, Li Q, Xie E, Zhang L, Sun R, Chen L, Fang B, Pan S (2013) Downregulation of IFNG in CD4(+) T cells in lung cancer through hypermethylation: a possible mechanism of tumor-induced immunosuppression. PLoS ONE 8(11):e79064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Y, Zhou S, Sun W, McClung K, Pan Y, Liang G, Tan Y, Zhao Y, Liu Q, Sun J, Cai L (2014) Metabolism, inhibition of JNK by novel curcumin analog C66 prevents diabetic cardiomyopathy with a preservation of cardiac metallothionein expression. Am J Physiol-Endocrinol Metab 306(11):1239–1247

    Article  Google Scholar 

  • Wolf MM, Kimryn Rathmell W, Beckermann KE (2020) Modeling clear cell renal cell carcinoma and therapeutic implications. Oncogene 39(17):3413–3426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang DC, Chen CH (2020) In Potential new therapeutic approaches for renal cell carcinoma. Semin Nephrol 40(1):86–97

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank Dr. Chen for his technical support.

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Author notes

  1. Jie Wang, Zili Zuo, Zongze Yu, and Zhigui Chen are equal contributors in the making of this manuscript.

Authors and Affiliations

  1. Department of Urology, The Second People’s Hospital of Meishan City, Meishan, 620500, Sichuan, China

    Jie Wang, Zili Zuo, Zongze Yu & Zhigui Chen

  2. Department of Urology, China-Japan Union Hospital of Jilin University, Changchun, 130000, Jilin, China

    Jie Wang, Xiangdi Meng, Zhaosen Ma, Jiqiang Niu, Rui Guo & Zhou Sun

  3. Department of General, Visceral, and Transplant Surgery, Ludwig Maximilians University Munich, 81377, Munich, Germany

    Lisa Jia Tran

  4. Division of Basic Biomedical Sciences, The University of South Dakota Sanford School of Medicine, Vermillion, SD, 57069, USA

    Jing Zhang & Tianxiao Jiang

  5. Department of Urology, Huashan Hospital, Fudan University, Shanghai, China

    Fangdie Ye

  6. Department of Urology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, China

    Baoluo Ma

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Contributions

Ma-Baoluo, Ye-Fangdie, and Sun-Zhou were involved in the final development of the project and manuscript preparation. Jie Wang, Zili Zuo, Zongze Yu, and Zhigui Chen wrote the manuscript draft and analyzed the data. Xiangdi Meng, Zhaosen Ma, Jiqiang Niu, Rui Guo, Lisa Jia Tran, Jing Zhang, and Tianxiao Jiang participate in the completion of the work.

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Correspondence to Fangdie Ye, Baoluo Ma or Zhou Sun.

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Wang, J., Zuo, Z., Yu, Z. et al. Single-cell transcriptome analysis revealing the intratumoral heterogeneity of ccRCC and validation of MT2A in pathogenesis. Funct Integr Genomics 23, 300 (2023). https://doi.org/10.1007/s10142-023-01225-7

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  • DOI: https://doi.org/10.1007/s10142-023-01225-7

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