Transcript levels of cellular senescence marker genes are increased based on high expression of gasdermin family of genes in breast cancer

Abstract

Cellular senescence is a cell state characterized by a generally permanent cell-cycle arrest, generating a broad secretome of inflammatory factors, contributing to pro-inflammatory milieu. Pyroptosis is a lytic and highly regulated cell death mechanism with pro-inflammatory characteristics, mediated by gasdermin (GSDM) family of proteins, which has six members in humans: GSDMA-E and PJVK (GSDMF). The interplay between senescence and pyroptotic cell death has been shown in some contexts; however, crosstalk between senescence and pyroptosis has not been studied in breast cancer. In the present study, it was found that breast tumors with high gasdermin expression have higher expression of senescence marker genes, namely CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53). This is especially true for high GSDMD- or GSDME-expressing breast tumors, which show higher mRNA levels of all three senescence marker genes. This high GSDM-dependent increases in the transcript levels of cellular senescence marker genes is more frequent in breast cancer than in non-malignant breast tissue, suggesting that the association between gasdermin family of genes and senesence marker genes in terms of expression levels is stronger in the case of tumor rather than normal tissue. This might point that, in breast cancer, pyroptosis and senescence might be associated; however, whether pyroptosis regulates senescence or vice versa, whether these two processes both reciprocally regulate and control each other, or even whether they share a  common upstream regulatory pathway remain to be identified. These findings also support previous research demonstrating the promoting effect of pyroptosis on senesence, and that SASP (senescence-associated secretory phenotype) factors can induce GSDMD–dependent pyroptotic cell death in neighboring cells present in the same microenvironment, in certain contexts. Further mechanistic studies are required to better characterize the cellular and molecular connections between senescence and pyroptosis in breast cancer.

Keywords

Senescence / Pyroptosis / Breast cancer / GSDME / GSDMD / Inflammation / p21 / p16

References

[1] Acosta JC, Banito A, Wuestefeld T, Georgilis A, Janich P, Morton JP, Athineos D, Kang TW, Lasitschka F, Andrulis M, Pascual G, Morris KJ, Khan S, Jin H, Dharmalingam G, Snijders AP, Carroll T, Capper D, Pritchard C, Inman GJ, Longerich T, Sansom OJ, Benitah SA, Zender L, Gil J. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nature Cell Biology, 2013, 15(8): 978-990.

[2] Acosta JC, O’Loghlen A, Banito A, Guijarro MV, Augert A, Raguz S. et al.. Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell, 2008, 133: 1006-1018.

[3] Allaire, J., Xie, Y., Dervieux, C., McPherson, J., Luraschi, J., Ushey, K., Atkins, A., Wickham, H., Cheng, J., Chang, W., Iannone, R. (2023). _rmarkdown: Dynamic Documents for R_. R package version 2.21. Retrieved from https://github.com/rstudio/rmarkdown.

[4] Berkel C. An analysis of gasdermin family of genes in UCEC with respect to malignancy status, mutation percentages and histologic diagnosis. Genome Instability & Disease, 2024.

[5] Berkel C. Potential impact of climate change-induced alterations on pyroptotic cell death in animal cells: A review. Molecular Biotechnology, 2024.

[6] Berkel C, Cacan E. Analysis of longevity in Chordata identifies species with exceptional longevity among taxa and points to the evolution of longer lifespans. Biogerontology, 2021, 22(3): 329-343.

[7] Berkel C, Cacan E. Lower expression of NINJ1 (Ninjurin 1), a mediator of plasma membrane rupture, is associated with advanced disease and worse prognosis in serous ovarian cancer. Immunologic Research, 2023, 71: 15-28.

[8] Berkel, C. (2025). Potential reverse functions of GOLPH3 and GOLPH3L in pyroptotic cell death, with implications in resistance to radiotherapy and chemotherapy. Human Cell, 38(4), 1–4. https://doi.org/10.1007/s13577-025-01236-5. PMID: 40402191.

[9] Braumüller H, Wieder T, Brenner E, Aßmann S, Hahn M, Alkhaled M, Schilbach K, Essmann F, Kneilling M, Griessinger C, Ranta F, Ullrich S, Mocikat R, Braungart K, Mehra T, Fehrenbacher B, Berdel J, Niessner H, Meier F, van den Broek M, Häring HU, Handgretinger R, Quintanilla-Martinez L, Fend F, Pesic M, Bauer J, Zender L, Schaller M, Schulze-Osthoff K, Röcken M. T-helper-1-cell cytokines drive cancer into senescence. Nature, 2013, 494(7437): 361-365.

[10] Broz P, Pelegrín P, Shao F. The gasdermins, a protein family executing cell death and inflammation. Nature Reviews Immunology, 2020, 20(3): 143-157.

[11] Cancer Genome Atlas Research Network. et al.. The Cancer Genome Atlas Pan-Cancer analysis project. Nature Genetics, 2013, 45: 1113-1120.

[12] Cerami, E., Gao, J., Dogrusoz, U., Gross, B. E., Sumer, S. O., Aksoy, B. A., Jacobsen, A., Byrne, C. J., Heuer, M. L., Larsson, E., Antipin, Y., Reva, B., Goldberg, A. P., Sander, C., Schultz, N. (2012). The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discovery, 2(5), 401–404. https://doi.org/10.1158/2159-8290.CD-12-0095. Erratum in: Cancer Discovery 2012 Oct;2(10):960.

[13] Chen X, He WT, Hu L, Li J, Fang Y, Wang X, Xu X, Wang Z, Huang K, Han J. Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis. Cell Research, 2016, 26(9): 1007-1020.

[14] Coppé JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: The dark side of tumor suppression. Annual Review of Pathology: Mechanisms of Disease, 2010, 5: 99-118.

[15] David L, Borges JP, Hollingsworth LR, Volchuk A, Jansen I, Garlick E, Steinberg BE, Wu H. NINJ1 mediates plasma membrane rupture by cutting and releasing membrane disks. Cell, 2024, 187(9): 2224-2235.

[16] de Bruijn, I., Kundra, R., Mastrogiacomo, B., Tran, T. N., Sikina, L., Mazor, T., Li, X., Ochoa, A., Zhao, G., Lai, B., Abeshouse, A., Baiceanu, D., Ciftci, E., Dogrusoz, U., Dufilie, A., Erkoc, Z., Garcia Lara, E., Fu, Z., Gross, B.,…, Schultz, N. (2023). Analysis and visualization of longitudinal genomic and clinical data from the AACR project GENIE biopharma collaborative in cBioPortal. Cancer Research, 83(23), 3861–3867. https://doi.org/10.1158/0008-5472.CAN-23-0816

[17] Desdín-Micó G, Soto-Heredero G, Aranda JF, Oller J, Carrasco E, Gabandé-Rodríguez E, Blanco EM, Alfranca A, Cussó L, Desco M, Ibañez B, Gortazar AR, Fernández-Marcos P, Navarro MN, Hernaez B, Alcamí A, Baixauli F, Mittelbrunn M. T cells with dysfunctional mitochondria induce multimorbidity and premature senescence. Science, 2020, 368(6497): 1371-1376.

[18] Ding Y, Liu Y, Li D, Hu R, Tian Z, Yang L, Li Y, Lin Y, Qu Y. Melatonin ameliorates senescence of mouse auditory cell line HEI-OC1 cells by suppressing NLRP3 inflammasome-mediated pyroptosis. Molecular Neurobiology, 2025.

[19] Fali T, Fabre-Mersseman V, Yamamoto T, Bayard C, Papagno L, Fastenackels S, Zoorab R, Koup RA, Boddaert J, Sauce D, Appay V. Elderly human hematopoietic progenitor cells express cellular senescence markers and are more susceptible to pyroptosis. JCI Insight, 2018, 3(13): e95319.

[20] Fan CY, Ye FH, Peng M, Dong JJ, Chai WW, Deng WJ, Zhang H, Yang LC. Endogenous HMGB1 regulates GSDME-mediated pyroptosis via ROS/ERK1/2/caspase-3/GSDME signaling in neuroblastoma. American Journal of Cancer Research, 2023, 13(2): 436-451

[21] Faria SS, Costantini S, de Lima VCC, de Andrade VP, Rialland M, Cedric R, Budillon A, Magalhães KG. NLRP3 inflammasome-mediated cytokine production and pyroptosis cell death in breast cancer. Journal of Biomedical Science, 2021, 28(1): 26.

[22] Fernández-Duran I, Quintanilla A, Tarrats N, Birch J, Hari P, Millar FR, Lagnado AB, Smer-Barreto V, Muir M, Brunton VG, Passos JF, Acosta JC. Cytoplasmic innate immune sensing by the caspase-4 non-canonical inflammasome promotes cellular senescence. Cell Death and Differentiation, 2022, 29(6): 1267-1282.

[23] Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, Sun Y, Jacobsen A, Sinha R, Larsson E, Cerami E, Sander C, Schultz N. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Science Signaling, 2013.

[24] Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C, Campisi J, Collado M, Evangelou K, Ferbeyre G, Gil J, Hara E, Krizhanovsky V, Jurk D, Maier AB, Narita M, Niedernhofer L, Passos JF, Robbins PD, Schmitt CA, Sedivy J, Vougas K, von Zglinicki T, Zhou D, Serrano M, Demaria M. Cellular senescence: Defining a path forward. Cell, 2019, 179(4): 813-827.

[25] Győrffy B. Integrated analysis of public datasets for the discovery and validation of survival-associated genes in solid tumors. Innovation, 2024, 5(3): 100625.

[26] Győrffy B. Transcriptome-level discovery of survival-associated biomarkers and therapy targets in non-small-cell lung cancer. British Journal of Pharmacology, 2024, 181(3): 362-374.

[27] Hernandez-Segura A, Nehme J, Demaria M. Hallmarks of cellular senescence. Trends in Cell Biology, 2018, 28: 436-453.

[28] Hom LM, Sun S, Campbell J, Liu P, Culbert S, Murphy IM, Schafer ZT. A role for fibroblast-derived SASP factors in the activation of pyroptotic cell death in mammary epithelial cells. Journal of Biological Chemistry, 2023, 299(7): 104922.

[29] Hou W, Wei X, Liang J, Fang P, Ma C, Zhang Q, Gao Y. HMGB1-induced hepatocyte pyroptosis expanding inflammatory responses contributes to the pathogenesis of acute-on-chronic liver failure (ACLF). Journal of Inflammation Research, 2021, 23(14): 7295-7313.

[30] Joshi CS, Salazar AM, Wang C, Ligon MM, Chappidi RR, Fashemi BE, Felder PA, Mora A, Grimm SL, Coarfa C, Mysorekar IU. D-Mannose reduces cellular senescence and NLRP3/GasderminD/IL-1β-driven pyroptotic uroepithelial cell shedding in the murine bladder. Developmental Cell, 2024, 59(1): 33-47.

[31] Kassambara, A. (2023). _ggpubr: 'ggplot2' Based Publication Ready Plots_. R package version 0.6.0. Retrieved from https://CRAN.R-project.org/package=ggpubr

[32] Kayagaki, N., Kornfeld, O. S., Lee, B. L., Stowe, I. B., O’Rourke, K., Li, Q., Sandoval, W., Yan, D., Kang, J., Xu, M., Zhang, J., Lee, W. P., McKenzie, B. S., Ulas, G., Payandeh, J., Roose-Girma, M., Modrusan, Z., Reja, R., Sagolla, M., … Dixit, V. M. (2021). NINJ1 mediates plasma membrane rupture during lytic cell death. Nature, 591(7848), 131–136. https://doi.org/10.1038/s41586-021-03218-7

[33] Kayagaki N, Stowe IB, Alegre K, Deshpande I, Wu S, Lin Z, Kornfeld OS, Lee BL, Zhang J, Liu J, Suto E, Lee WP, Schneider K, Lin W, Seshasayee D, Bhangale T, Chalouni C, Johnson MC, Joshi P, Mossemann J, Zhao S, Ali D, Goldenberg NM, Sayed BA, Steinberg BE, Newton K, Webster JD, Kelly RL, Dixit VM. Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury. Nature, 2023, 618(7967): 1072-1077.

[34] Kuilman T, Michaloglou C, Vredeveld LC, Douma S, van Doorn R, Desmet CJ. et al.. Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell, 2008, 133: 1019-1031.

[35] Latz E, Duewell P. NLRP3 inflammasome activation in inflammaging. Seminars in Immunology, 2018, 40: 61-73.

[36] Li B, Dewey CN. RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics, 2011, 12: 323.

[37] Li H, Ning S, Ghandi M, Kryukov GV, Gopal S, Deik A, Souza A, Pierce K, Keskula P, Hernandez D, Ann J, Shkoza D, Apfel V, Zou Y, Vazquez F, Barretina J, Pagliarini RA, Galli GG, Root DE, Hahn WC, Tsherniak A, Giannakis M, Schreiber SL, Clish CB, Garraway LA, Sellers WR. The landscape of cancer cell line metabolism. Nature Medicine, 2019, 25(5): 850-860.

[38] Li J, Wang X, Yao Z, Yuan F, Liu H, Sun Z, Yuan Z, Luo G, Yao X, Cui H, Tu B, Sun Z, Fan C. NLRP3-dependent crosstalk between pyroptotic macrophage and senescent cell orchestrates trauma-induced heterotopic ossification during aberrant wound healing. Advanced Science, 2023, 10(19): e2207383.

[39] Liao LZ, Chen ZC, Wang SS, Liu WB, Zhao CL, Zhuang XD. NLRP3 inflammasome activation contributes to the pathogenesis of cardiocytes aging. Aging, 2021, 13(16): 20534-20551.

[40] Liao Y, Smyth GK, Shi W. The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. Nucleic Acids Research, 2019, 47. e47

[41] Liu L, Wang N, Kalionis B, Xia S, He Q. HMGB1 plays an important role in pyroptosis induced blood brain barrier breakdown in diabetes-associated cognitive decline. Journal of Neuroimmunology, 2022, 15(362): 577763.

[42] Lu L. et al.. Emerging mechanisms of pyroptosis and its therapeutic strategy in cancer. Cell Death Discovery, 2022, 8: 1-13.

[43] Marín-Aguilar F, Lechuga-Vieco AV, Alcocer-Gómez E, Castejón-Vega B, Lucas J, Garrido C, Peralta-Garcia A, Pérez-Pulido AJ, Varela-López A, Quiles JL, Ryffel B, Flores I, Bullón P, Ruiz-Cabello J, Cordero MD. NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice. Aging Cell, 2020, 19(1): e13050.

[44] Martínez-Zamudio RI, Robinson L, Roux PF, Bischof O. SnapShot: Cellular senescence pathways. Cell, 2017, 170: 816-816.e811.

[45] Morgan, M., Obenchain, V., Hester, J., & Pagès, H. (2022). _SummarizedExperiment: SummarizedExperiment container_. R package version 1.26.1. Retrieved from https://bioconductor.org/packages/SummarizedExperiment

[46] Morgan, M., & Shepherd, L. (2022). _ExperimentHub: Client to access ExperimentHub resources_. R package version 2.4.0.

[47] Muela-Zarzuela I, Suarez-Rivero JM, Gallardo-Orihuela A, Wang C, Izawa K, de Gregorio-Procopio M, Couillin I, Ryffel B, Kitaura J, Sanz A, von Zglinicki T, Mbalaviele G, Cordero MD. NLRP1 inflammasome modulates senescence and senescence-associated secretory phenotype. bioRxiv, 2023.

[48] Network CGA. Comprehensive molecular portraits of human breast tumours. Nature, 2012, 490(7418): 61-70.

[49] Nikolayeva O, Robinson MD. edgeR for differential RNA-seq and ChIP-seq analysis: An application to stem cell biology. Methods in Molecular Biology, 2014, 1150: 45-79.

[50] Nusinow DP, Szpyt J, Ghandi M, Rose CM, McDonald ER3rd, Kalocsay M, Jané-Valbuena J, Gelfand E, Schweppe DK, Jedrychowski M, Golji J, Porter DA, Rejtar T, Wang YK, Kryukov GV, Stegmeier F, Erickson BK, Garraway LA, Sellers WR, Gygi SP. Quantitative proteomics of the cancer cell line encyclopedia. Cell, 2020, 180(2): 387-402.e16.

[51] Ooms, J. (2023). _magick: Advanced Graphics and Image-Processing in R_. R package version 2.7.4. Retrieved from https://CRAN.R-project.org/package=magick.

[52] Posit Team. (2023). RStudio: Integrated development environment for R. Posit Software, PBC, Boston, MA. Retrieved from http://www.posit.co/.

[53] R Core Team. (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Retrieved from https://www.R-project.org/.

[54] Rahman M, Jackson LK, Johnson WE, Li DY, Bild AH, Piccolo SR. Alternative preprocessing of RNA-Sequencing data in The Cancer Genome Atlas leads to improved analysis results. Bioinformatics, 2015, 31(22): 3666-3672.

[55] Shang D, Hong Y, Xie W, Tu Z, Xu J. Interleukin-1β drives cellular senescence of rat astrocytes induced by oligomerized amyloid β peptide and oxidative stress. Frontiers in Neurology, 2020, 27(11): 929.

[56] Shang J, Zhao F, Cao Y, Ping F, Wang W, Li Y. HMGB1 mediates lipopolysaccharide-induced macrophage autophagy and pyroptosis. BMC Molecular and Cell Biology, 2023, 24(1): 2.

[57] Shao, F. (2021). Gasdermins: making pores for pyroptosis. Nature Reviews Immunology, 21(10), 620–621. https://doi.org/10.1038/s41577-021-00602-2. PMID: 34580452.

[58] Shimizu K, Inuzuka H, Tokunaga F. The interplay between cell death and senescence in cancer. Seminars in Cancer Biology, 2025, 108: 1-16.

[59] Smyth G.K. (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol., 3, Article3.

[60] Sofiadis K, Josipovic N, Nikolic M, Kargapolova Y, Übelmesser N, Varamogianni-Mamatsi V, Zirkel A, Papadionysiou I, Loughran G, Keane J, Michel A, Gusmao EG, Becker C, Altmüller J, Georgomanolis T, Mizi A, Papantonis A. HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence. Molecular Systems Biology, 2021, 17(6): e9760.

[61] Spadaro O, Goldberg EL, Camell CD, Youm YH, Kopchick JJ, Nguyen KY, Bartke A, Sun LY, Dixit VD. Growth Hormone Receptor Deficiency Protects against Age-Related NLRP3 Inflammasome Activation and Immune Senescence. Cell Reports, 2016, 14(7): 1571-1580.

[62] Toldo S, Abbate A. The role of the NLRP3 inflammasome and pyroptosis in cardiovascular diseases. Nature Reviews. Cardiology, 2024, 21(4): 219-237.

[63] Tominaga K, Suzuki HI. TGF-β signaling in cellular senescence and aging-related pathology. International Journal of Molecular Sciences, 2019, 20(20): 5002.

[64] Tsherniak A, Vazquez F, Montgomery PG, Weir BA, Kryukov G, Cowley GS, Gill S, Harrington WF, Pantel S, Krill-Burger JM, Meyers RM, Ali L, Goodale A, Lee Y, Jiang G, Hsiao J, Gerath WFJ, Howell S, Merkel E, Ghandi M, Garraway LA, Root DE, Golub TR, Boehm JS, Hahn WC. Defining a cancer dependency map. Cell, 2017, 170(3): 564-576.

[65] Wagner GP. et al.. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theor. Biosci., 2012, 131: 281-285.

[66] Wickham H, Averick M, Bryan J, Chang W, McGowan LD, François R, Grolemund G, Hayes A, Henry L, Hester J, Kuhn M, Pedersen TL, Miller E, Bache SM, Müller K, Ooms J, Robinson D, Seidel DP, Spinu V, Takahashi K, Vaughan D, Wilke C, Woo K, Yutani H (2019). “Welcome to the tidyverse.” _Journal of Open Source Software_, *4*(43), 1686. https://doi.org/10.21105/joss.01686 <https://doi.org/10.21105/joss.01686>.

[67] Xie Y. knitr: A General-Purpose Package for Dynamic Report Generation in R. R Package Version, 2023, 1: 42

[68] Yamagishi R, Kamachi F, Nakamura M, Yamazaki S, Kamiya T, Takasugi M, Cheng Y, Nonaka Y, Yukawa-Muto Y, Thuy LTT, Harada Y, Arai T, Loo TM, Yoshimoto S, Ando T, Nakajima M, Taguchi H, Ishikawa T, Akiba H, Miyake S, Kubo M, Iwakura Y, Fukuda S, Chen WY, Kawada N, Rudensky A, Nakae S, Hara E, Ohtani N. Gasdermin D-mediated release of IL-33 from senescent hepatic stellate cells promotes obesity-associated hepatocellular carcinoma. Sci Immunol. 2022 Jun 24;7(72):eabl7209. https://doi.org/10.1126/sciimmunol.abl7209

[69] Youm YH, Kanneganti TD, Vandanmagsar B, Zhu X, Ravussin A, Adijiang A, Owen JS, Thomas MJ, Francis J, Parks JS, Dixit VD. (2012). The Nlrp3 inflammasome promotes age-related thymic demise and immunosenescence. Cell Reports, 1(1), 56–68. https://doi.org/10.1016/j.celrep.2011.11.005. Epub 2012 Jan 26. PMID: 22832107; PMCID: PMC3883512.

[70] Yu P, Zhang X, Liu N, Tang L, Peng C, Chen X. Pyroptosis: Mechanisms and diseases. Signal Transduction and Targeted Therapy, 2021, 6(1): 128.

[71] Zhang H. NDRG2 promotes lens epithelial cells senescence via NLRP3/Caspase1-mediated pyroptosis. Applied Biochemistry and Biotechnology, 2024, 196(8): 5435-5446.

[72] Zhao P, Yue Z, Nie L, Zhao Z, Wang Q, Chen J, Wang Q. Hyperglycaemia-associated macrophage pyroptosis accelerates periodontal inflamm-aging. Journal of Clinical Periodontology, 2021, 48(10): 1379-1392.

[73] Zhou C, Guo Q, Lin J, Wang M, Zeng Z, Li Y, Li X, Xiang Y, Liang Q, Liu J, Wu T, Zeng Y, He S, Wang S, Zeng H, Liang X. Single-cell atlas of human ovaries reveals the role of the pyroptotic macrophage in ovarian aging. Advanced Science, 2024, 11(4): e2305175.

[74] Zhou R, Xie X, Qin Z, Li X, Liu J, Li H, Zheng Q, Luo Y. Cytosolic dsDNA is a novel senescence marker associated with pyroptosis activation. Tissue and Cell, 2021, 72: 101554.

RIGHTS & PERMISSIONS

Shenzhen University School of Medicine; Fondazione Istituto FIRC di Oncologia Molecolare