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

Transcript levels of cellular senescence marker genes are increased based on high expression of gasdermin family of genes in breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcript levels of cellular senescence marker genes are increased based on high expression of gasdermin family of genes in breast cancer Caglar Berkel This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4884791/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version 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 highly regulated cell death mechanism with pro-inflammatory characteristics, mediated by gasdermin (GSDM) family of proteins, which has six members: GSDMA-E and PJVK. In the present study, I 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 three senescence marker genes. This high GSDM-dependent increases in the transcript levels of cellular senescence marker genes is more frequent in breast cancer cells than in non-malignant breast cells, suggesting that the association between gasdermin family of genes and senesence marker genes in terms of expression levels is more strong in the case of tumor. 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 an upstream regulatory pathway remains 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 certain contexts. Further mechanistic studies are required to better characterize molecular connections between senescence and pyroptosis in breast cancer. senescence pyroptosis breast cancer GSDME GSDMD inflammation p21 p16 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cellular senescence is a cell state characterized by a long-term and usually irreversible cell-cycle arrest with certain secretory features, macromolecular damage, and altered metabolism and morphology, contributing to pro-inflammatory milieu (Gorgoulis et al., 2019 ; Hernandez-Segura et al., 2018 ). Senescent cells carry out a pro-inflammatory response termed the senescence-associated secretory phenotype (SASP) which includes the production and secretion of many soluble and insoluble factors, including inflammatory cytokines (such as IL-6) and chemokines, into the extracellular space (Acosta et al., 2013 ; Acosta et al., 2008 ; Kuilman et al., 2008 ; Coppé et al., 2010 ). Similarly, in the opposite direction, inflammation has also been shown to stimulate cellular senescence; and the activation of the inflammasome controls the senescence secretome, and it is critical for cellular senescence and SASP (Acosta et al., 2013 ; Desdín-Micó et al., 2020 ; Li et al., 2023 ; Braumüller et al., 2013 ; Fernández-Duran et al., 2022 ; Zhou et al., 2024 ). Besides, secretion of certain inflammatory cytokines including IL-1β has been shown to induce cellular senescence (Tominaga and Suzuki, 2019 ; Shang et al., 2020 ; Martínez-Zamudio et al., 2017 ). Senescent cells are found primarily in tissues that experience prolonged inflammation (Coppé et al., 2010 ). Pyroptosis is a lytic and highly regulated cell death mechanism with pro-inflammatory characteristics (Broz et al., 2020 ). It is mediated by gasdermin (GSDM) family of proteins (6 members in humans: GSDMA, GSDMB, GSDMC, GSDMD, GSDME (DFNA5) and PJVK (Pejvakin, DFNB59)) following their proteolytic cleavage and activation (Broz et al., 2020 ; Chen et al., 2016 ; Yu et al., 2021 ; Shao, 2021; Lu et al., 2022 ). After the cleavage of the linker region between NT and CT domains by specific inflammatory caspases (incl. caspase-1), free NT domain of GSDMs, translocates, for instance, to the plasma membrane and binds to lipids and oligomerizes to form membrane pores, resulting in rapid membrane rupture and release of danger-associated molecular patterns (DAMPs) and pro-inflammatory cytokines including mature IL-18 and IL-1β (Broz et al., 2020 ; Chen et al., 2016 ; Yu et al., 2021 ; Shao, 2021; Lu et al., 2022 ). However, recently, the release of large molecules such as HMGB1 and LDH has also been shown to additionally require NINJ1-mediated plasma membrane rupture following gasdermin pore formation, since the sizes of pores formed by GSDM-NTs are not suitable for the passage of these large proteins (Kayagaki et al., 2021 ). Immune cells recognize certain molecules released from pyroptotic cells, and then initiate a cascade of immune responses, including the activation and infiltration of certain immune cells to the places where pyroptotic cells are located. At the upstream, upon certain stimuli, pyroptosis can be initiated by the activation of inflammasomes such as NLRP3 inflammasome, which activates caspase-1 that then processes GSDMD (or some other GSDMs) as well as cytokines, such as pro-IL-1β and pro-IL-18, promoting pyroptotic cell death (Berkel, 2024a ; Toldo and Abbate, 2024 ; Faria et al., 2021 ). Pyroptosis has been shown to be highly regulated in malignant transformation and tumor progression in cancer including breast and gynecological cancers (Berkel, 2024b ; Berkel and Cacan, 2022). Previous research also suggested an association between senescence and pyroptosis. Senescent fibroblast-derived SASP factors can induce NLRP3-, caspase-1-, and GSDMD–dependent pyroptotic cell death in neighboring normal (noncancerous) mammary epithelial cells (Hom et al., 2023 ). In certain wound healing responses, pyroptotic macrophages might induce senescence in certain types of stem cells and thus increase senescent cell burden; and this pro-senescent effects are mostly mediated by IL‐1β and extracellular vesicles (containing HMGB1) released from pyroptotic macrophages (Li et al., 2023 ). Cells surrounding pyroptotic macrophages have been shown to have a significant increase in markers representing senescence in other studies, pointing that pyroptotic macrophages may induce senescence in surrounding cells (Zhao et al., 2021 ). In support, silencing of GSDMD in macrophages decreases the expression of senescence markers such as p16 and p21 (rescues senescence) in fibroblasts exposed to media from these macrophages with cleared pyroptosis (Zhao et al., 2021 ). In human ovaries, pyroptotic macrophages (those accumulated in middle-aged ovaries) have been also found to promote senescence in stromal cells, principal components of the ovarian stroma (Zhou et al., 2024 ). Cytosolic double-stranded DNA (dsDNA), a sensitive biomarker for cellular senescence in mouse embryonic fibroblasts (MEFs), induces pyroptosis in these senescent cells in certain contexts (Zhou et al., 2021 ). Cytosolic dsDNA accumulation is proportional to senescence status in these cells independent of senescence-inducing stimuli (Zhou et al., 2021 ). Besides, in aged mice urothelium, a canonical SASP along with continuous NLRP3-inflammasome- and GSDMD-dependent pyroptotic cell death has been recently identified (Joshi et al., 2024 ). A natural monosaccharide, D-mannose, both reverses the SASP and reduces NLRP3/GSDMD/IL-1β-driven pyroptotic epithelial cell shedding in the bladder in aged mice (Joshi et al., 2024 ). Nlrp3 inflammasome activation has been also shown to lead to SASP and immunesenescence (T cell senescence) (Latz and Duewell, 2018 ; Youm et al., 2012; Spadaro et al., 2016 ; Marín-Aguilar et al., 2020 ). Others showed that GSDMD might contribute to LPS-induced senescence, however, this might involve a mechanism other than the cleavage and liberation (thus, activation) of GSDMD NT domain (Fernández-Duran et al., 2022 ). Studies mentioned above points that cellular senescence and pyroptosis might be interconnected in certain contexts. In the present study, I found that breast cancer cells with high gasdermin (GSDM) 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 cancer cells, which show higher expression of all three senescence marker genes. This high GSDM-dependent increases in the expression of senescence marker genes is more frequent in breast cancer cells than in non-malignant breast cells; in other words, the association between gasdermin family of genes and senesence marker genes in terms of expression levels is more strong in the case of tumor rather than non-malignant tissue. This might suggest that, in breast cancer, pyroptosis and senescence might be associated; however, whether pyroptosis regulates senescence or vice versa , whether these two processes reciprocally regulate and control each other, or whether they share an upstream regulatory pathway remains to be determined. This also supports previous research demonstrating the promoting effect of pyroptosis on senesence, and that SASP factors can induce GSDMD–dependent pyroptotic cell death in neighboring cells present in the same microenvironment. Further mechanistic studies are required to determine the cellular and molecular connections between senescence and pyroptosis in breast cancer. Gasdermins and pyroptosis Methods Datasets In this study, I used transcriptomics and clinical datasets from TCGA (The Cancer Genome Atlas)-BRCA (Breast cancer) project (Cancer Genome Atlas Network, 2012 ; The Cancer Genome Atlas Research Network, 2013 ), accessed programmatically via ExperimentHub Bioconductor package (Morgan and Shepherd, 2022 ), as reported previously (Berkel, 2024b ). It contains expression data for 1119 breast tumors and 113 normal (non-malignant) breast tissues for 23368 genes. Samples (tumor or normal) with higher expression than the median expression value for each gene are defined as samples with “high expression”; therefore, half of the samples for each gene are categorized as “high expression” and the other half as “low expression” for each gene separately. Alternatively processed and compiled RNA-Seq and associated clinical data for patient samples from TCGA can also be found in GEO with accession ID of GSE62944 (Rahman et al., 2015 ). Gene expression values were shown in log10 scale in y-axes of plots. Mean gene expression values for each group (low vs high expression) compared were also shown on boxplots. Data analysis Data analysis and visualization were completely performed in R language in the present study using Rstudio IDE (version 2023.3.0.386; https://cran.r-project.org/ ) (R Core Team, 2022 ; Posit team, 2023 ). Following R / Bioconductor packages were used throughout the analysis: SummarizedExperiment (package version of 1.26.1) (Morgan et al., 2022 ), tidyverse (2.0.0) (Wickham et al., 2019 ), ggpubr (0.6.0) (Kassambara, 2023 ), magick (2.7.4) (Ooms, 2023 ), knitr (1.42) (Xie, 2023 ) and rmarkdown (2.21) (Allaire et al., 2023 ). Shapiro-Wilk normality test was performed to analyze the distribution of the expression data (using ggqqplot() and shapiro.test() functions), and if the resultant p value is less than 0.05, normality could not be assumed and therefore wilcox test was performed to statistically compare the group means (Kassambara, 2023 ). Results Breast tumors with high GSDMD or GSDME expression have higher transcript levels of cellular senescence markers I found that breast cancer cells from patients with high expression of GSDMD or GSDME have significantly increased mRNA levels of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) compared to those with low expression of GSDMD or GSDME, respectively (Figs. 1 and 2 , first panels). In normal (non-malignant) breast tissue, some of these GSDMD- and GSDME-dependent differences in the expression of cellular senescence marker genes observed in breast tumors are lost, especially for CDKN1A (p21) (Figs. 1 and 2 , second panels). CDKN2A (p16) expression is higher in breast tumors with high GSDMB or GSDMC expression Next, I showed that CDKN2A (p16) transcript levels are increased in breast cancer cells from patients with high expression of GSDMB or GSDMC, compared to those with low expression of these genes, respectively (Figs. 3 and 4 , first panels). Interestingly, this GSDMB- or GSDMC-dependent difference in CDKN2A expression is lost in healthy breast tissue (Figs. 3 and 4 , second panels). The other two cellular senescence marker genes (CDKN1A and TP53) did not show GSDMB- or GSDMC-dependent expression in breast cancer (Figs. 3 and 4 , first panels). In normal breast tissue, only significant difference was observed for TP53: its expression was found to be higher in breast tissue with high GSDMC expression compared to that with low GSDMC expression (Fig. 4 , second panel). CDKN1A (p21) and TP53 (p53) expression are higher in breast tumors with high expression of GSDMA or PJVK I found that, although the expression of CDKN1A (p21) and TP53 (p53) is not higher in breast tumors with high GSDMB or GSDMC expression (as reported above), the expression of these two cellular senescence marker genes are increased based on high expression of other two gasdermin genes, GSDMA and PJVK, in breast tumors (Figs. 5 and 6 , first panels). These significant differences in the expression of CDKN1A and TP53 based on GSDMB or GSDMC expression were mostly lost in non-malignant breast tissues (Figs. 5 and 6 , second panels). In contrast, the expression of the other marker gene (CDKN2A) did not show GSDMA or PJVK expression-dependent changes either in breast tumors or in normal breast tissue (Figs. 5 and 6 , first panels). High NINJ1-expressing breast cancer cells or non-malignant breast cells have higher expression of senescence markers Lastly, I compared the expression of same three senescence marker genes at the mRNA level between low and high NINJ1-expressing cells (both breast cancer cells (left) or normal / non-malignant breast cells (right)) (Fig. 7 ). I showed that breast cancer cells with high NINJ1 expression have higher transcript levels of CDKN1A and TP53, compared to breast cancer cells with low NINJ1 expression (Fig. 7 , left). Besides, normal breast cells with high NINJ1 expression were fount to have increased expression of CDKN2A and TP53, compared to low NINJ1-expressing normal breast cells (Fig. 7 , right). Discussion Data in this analysis show that breast tumors with high gasdermin (GSDM) expression have higher expression of senescence marker genes, namely CDKN1A, CDKN2A and TP53. This is especially valid for breast tumors with high GSDMD- or GSDME expression, which show increased expression of all three senescence marker genes. This high gasdermin expression-dependent increases in the expression of senescence marker genes is more common in breast tumors than in non-malignant breast tissue; in other words, the association between gasdermin family of genes and cellular senesence marker genes in terms of transcript levels is more strong in breast tumors compared to normal breast tissue. Previous research suggested that SASP factors released from senescent cells can induce pyroptotic cell death in other cells (Hom et al., 2023 ; Li et al., 2023 ; Zhao et al., 2021 ). The observation in the present study might be due to the fact that certain senescent cells in breast tumor microenvironment can activate pyroptotic cell death (via released factors) by upregulating gasdermin gene expression in other cells present nearby. Since bulk RNA-Seq data was used in this study, not allowing the identification of specific cell types with particular expression profiles, data from single-cell RNA-Seq technologies might help identify cell types with SASP and cell types with pyroptotic characteristics, enabling the determination of possible senescent cell-pyroptotic cell interactions in tumor. Another explanation for the observations in the present study might be that pyroptotic cells themselves (with increased mRNA levels of gasdermin family of genes) or increased inflammation due to pro-inflammatory pyroptotic cell death might induce senescence in surrounding cells in breast tumor microenvironment, as suggested previously in some other contexts (Acosta et al., 2013 ; Desdín-Micó et al., 2020 ; Li et al., 2023 ; Braumüller et al., 2013 ; Fernández-Duran et al., 2022 ; Zhou et al., 2024 ; Tominaga and Suzuki, 2019 ; Shang et al., 2020 ; Martínez-Zamudio et al., 2017 ; Li et al., 2023 ; Zhao et al., 2021 ; Zhou et al., 2021 ; Fernández-Duran et al., 2022 ). For instance, inflammatory cytokines such as IL-18 and IL-1β released from gasdermin pores in the plasma membranes of pyroptotic cells might promote cellular senescence in neighboring cells or even in themselves (Tominaga and Suzuki, 2019 ; Shang et al., 2020 ; Martínez-Zamudio et al., 2017 ). Fernández-Duran et al. suggested that GSDMD might contribute to cellular senescence independent of its role in pyroptosis, because cleavage and pyroptotic activation of GSDMD is not required for it to induce senescence LPS-induced senescence (2021). Therefore, it can be suggested that pyroptotic or non-pyroptotic functions of gasdermins might be important depending on senescence-inducing stimuli. Since I observed that breast cancer cells with high GSDMD- or GSDME expression have increased expression of all three senescence marker genes, these two gasdermin genes might be relatively more important compared to other members of the family in terms of their influence on senescence. GSDMD has been shown to mediate the continous export of SASP factors such as IL-33 and IL-1β from senescent hepatic stellate cells, by forming cell membrane pores, in models of obesity-induced hepatocellular carcinoma (Yamagishi et al., 2022 ). Others showed that NLRP1 inflammasome promotes senescence (induced by irradiation) by regulating the expression of p16, p21 and p53 (CDKN2A, CDKN1A and TP53; the same senescence marker genes studied in the current study), and SASP in GSDMD-dependent manner since these responses are reduced in conditions of NLRP1 insufficiency or GSDMD inhibition (Muela-Zarzuela et al., 2023). Therefore, it can be proposed that particular gasdermin family members might be involved in the release of certain SASP factors from senescence cells (possibly via the formation of pores) and also in the regulation of senescence responses such as the regulation of p16, p21 and p53 expression. In the case of breast cancer, GSDMD and GSDME might be more capable of promoting senescence or of contributing to SASP. Since NLRP3 inflammasome activation has been also shown to lead to SASP and senescence in certain cells (Latz and Duewell, 2018 ; Youm et al., 2012; Spadaro et al., 2016 ; Marín-Aguilar et al., 2020 ), NLPR3 inflammasome might be a shared upstream regulator / inducer of both senescence and pyroptosis in breast cancer. Other inflammasomes such as NLRP1 might also be involved in the joint regulation of cellular senescence and pyroptotic cell death in certain contexts and cells including breast cancer cells (Muela-Zarzuela et al., 2023). A better understanding of the association between senescence and pyroptosis, and of the possible common regulators of both cell states is needed. HMGB1 is a pro-inflammatory protein (an alarmin) released from cells following NINJ1-mediated plasma mebrane rupture after gasdermin pore formation (Kayagaki et al., 2021 ; Berkel and Cacan, 2023 ; Kayagaki et al., 2023 ; David et al., 2024 ). Sofiadis et al. showed that HMGB1 controls the expression of genes that are central to the senescent program and influences the availability of senescence-relevant mRNAs (2021). Authors also found that senescence entry is marked by HMGB1 nuclear loss and secretion; in other words, its depletion in cells underlies induction of the senescence program (Sofiadis et al., 2021 ). Considering the role of HMGB1 in senescence and its involvement in induction of pyroptosis in certain contexts ( Hou et al., 2021 ; Shang et al., 2023 ; Fan et al., 2023 ; Liu et al., 2022 ), HMGB1 might be a link between senescence and pyroptosis. Senescent cells have essentially no nuclear HMGBs ( Sofiadis et al., 2021 ), and high levels of HMGB1 in the cytoplasm might lead to the increased activation of NLRP3 inflammasome, resulting in enhanced levels of pyroptosis in these cells. Since high gasdermin expression-dependent increases in the expression of senescence marker genes were observed to be more common in breast tumors than in non-malignant breast tissue, it can be suggested that senescence – pyroptosis association might be more strong in cancer; however, it should also be noted that sample size for breast cancer patients is higher than patients without breast cancer in the current analysis. Further comparative mechanistic studies are neeeded to better identify differences between breast tumor and normal breast tissue in this respect. Declarations Conflict of Interests The author declares no conflicts of interest. Funding No funding has been received for the present study. Author Contribution C.B. conceptualized the study, performed the data analysis and visualization, and wrote the paper. 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Yu, P., Zhang, X., Liu, N., Tang, L., Peng, C., & Chen, X. (2021). Pyroptosis: Mechanisms and diseases. Signal Transduction and Targeted Therapy, 6(1), 128. https://doi.org/10.1038/s41392-021-00507-5 . PMID: 33776057; PMCID: PMC8005494. Zhao P, Yue Z, Nie L, Zhao Z, Wang Q, Chen J, Wang Q. Hyperglycaemia-associated macrophage pyroptosis accelerates periodontal inflamm-aging. J Clin Periodontol. 2021;48(10):1379–1392. doi: 10.1111/jcpe.13517 . Epub 2021 Aug 18. PMID: 34219262. 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. Adv Sci (Weinh). 2024;11(4):e2305175. doi: 10.1002/advs.202305175 . Epub 2023 Nov 30. PMID: 38036420; PMCID: PMC10811476. 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 Cell. 2021;72:101554. doi: 10.1016/j.tice.2021.101554 . Epub 2021 Apr 30. PMID: 33991763. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4884791","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":341407286,"identity":"bd270aa5-545d-41c7-966a-260f43020c4f","order_by":0,"name":"Caglar Berkel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYHACxgNAIoGBgY2B4UEFREiCkB6EloQzcC0GRGpJbCNCC3/78QsHfu5hyOOXPpb4IHFerZw5A/PB2zwMf/JxaZE4k1NwsOcZQ7FkX9phg8Rtx40tG9iSrXkYDCwbcGgxYMhJOMBzgCFxwxn2NonEbccSNxzgMZMGasHpMgP+NwkH/wC17D/D3v4jcQ5IC/83/Fok0g8cBtvCw3aMIbGhBmQLG14tEjfeMByWOSBRLHGGLVki4dgBY8tmNmPLOQbGOLXw96c/fPjmgE0efw+b4YcPNXVy5uzND2+8qZDDEzE8IDl4dB9mMGCGBAsewP4AmVdHINpHwSgYBaNgJAIAJTxXRAfiVSMAAAAASUVORK5CYII=","orcid":"","institution":"Tokat Gaziosmanpaşa University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Caglar","middleName":"","lastName":"Berkel","suffix":""}],"badges":[],"createdAt":"2024-08-09 06:29:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4884791/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4884791/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65787863,"identity":"8c9bfc8a-133b-47ad-ad56-01c8f72873b7","added_by":"auto","created_at":"2024-10-02 16:45:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":243919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) based on low and high expression GSDMD in breast tumors (left) and normal breast tissue (right).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ens: p \u0026gt; 0.05; *: p \u0026lt;= 0.05; **: p \u0026lt;= 0.01; ***: p \u0026lt;= 0.001; ****: p \u0026lt;= 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4884791/v1/27ae8a25119ac20f7225ca1f.png"},{"id":65787862,"identity":"a2880729-26e1-4ccc-a533-f2bafc5d8901","added_by":"auto","created_at":"2024-10-02 16:45:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) based on low and high expression GSDME in breast tumors (left) and normal breast tissue (right).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ens: p \u0026gt; 0.05; *: p \u0026lt;= 0.05; **: p \u0026lt;= 0.01; ***: p \u0026lt;= 0.001; ****: p \u0026lt;= 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4884791/v1/d560f4087a50af5eabaf8c87.png"},{"id":65789232,"identity":"aa47f01b-b800-4e0c-85ab-760ebc08c57f","added_by":"auto","created_at":"2024-10-02 16:53:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) based on low and high expression GSDMB in breast tumors (left) and normal breast tissue (right).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ens: p \u0026gt; 0.05; *: p \u0026lt;= 0.05; **: p \u0026lt;= 0.01; ***: p \u0026lt;= 0.001; ****: p \u0026lt;= 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4884791/v1/14fb2df4298399d1f3396cfc.png"},{"id":65789229,"identity":"66fbb3e5-fa12-4b5e-9264-6b79a2eec484","added_by":"auto","created_at":"2024-10-02 16:53:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":244701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) based on low and high expression GSDMC in breast tumors (left) and normal breast tissue (right).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ens: p \u0026gt; 0.05; *: p \u0026lt;= 0.05; **: p \u0026lt;= 0.01; ***: p \u0026lt;= 0.001; ****: p \u0026lt;= 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4884791/v1/0b4a55ce5089c452db60754b.png"},{"id":65787866,"identity":"fc54f412-40ac-43b0-8c0c-3bc231ad530e","added_by":"auto","created_at":"2024-10-02 16:45:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":244230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) based on low and high expression GSDMA in breast tumors (left) and normal breast tissue (right).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ens: p \u0026gt; 0.05; *: p \u0026lt;= 0.05; **: p \u0026lt;= 0.01; ***: p \u0026lt;= 0.001; ****: p \u0026lt;= 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4884791/v1/fd6b348d1c0c9f4e7c1f0ba5.png"},{"id":65787868,"identity":"722b82d3-1d3f-495e-9147-f75f5fcb2810","added_by":"auto","created_at":"2024-10-02 16:45:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":212687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) based on low and high expression PJVK (Pejvakin) in breast tumors (left) and normal breast tissue (right).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ens: p \u0026gt; 0.05; *: p \u0026lt;= 0.05; **: p \u0026lt;= 0.01; ***: p \u0026lt;= 0.001; ****: p \u0026lt;= 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4884791/v1/57a78047b416182fe5f2ae0f.png"},{"id":65787867,"identity":"6883264a-c49f-4141-b82f-2c6a3cdc2173","added_by":"auto","created_at":"2024-10-02 16:45:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":211978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) based on low and high expression NINJ1 (Ninjurin) in breast tumors (left) and normal breast tissue (right).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ens: p \u0026gt; 0.05; *: p \u0026lt;= 0.05; **: p \u0026lt;= 0.01; ***: p \u0026lt;= 0.001; ****: p \u0026lt;= 0.0001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4884791/v1/1ab6642669cc5fcb226441b2.png"},{"id":65790116,"identity":"ed4534d3-0bd6-443c-aa85-7ddfa8bab951","added_by":"auto","created_at":"2024-10-02 17:01:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2450990,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4884791/v1/5cdbb746-7a45-48ba-ae7e-fb457a50d7a3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTranscript levels of cellular senescence marker genes are increased based on high expression of gasdermin family of genes in breast cancer\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCellular senescence is a cell state characterized by a long-term and usually irreversible cell-cycle arrest with certain secretory features, macromolecular damage, and altered metabolism and morphology, contributing to pro-inflammatory milieu (Gorgoulis et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hernandez-Segura et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Senescent cells carry out a pro-inflammatory response termed the senescence-associated secretory phenotype (SASP) which includes the production and secretion of many soluble and insoluble factors, including inflammatory cytokines (such as IL-6) and chemokines, into the extracellular space (Acosta et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Acosta et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kuilman et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Copp\u0026eacute; et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Similarly, in the opposite direction, inflammation has also been shown to stimulate cellular senescence; and the activation of the inflammasome controls the senescence secretome, and it is critical for cellular senescence and SASP (Acosta et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Desd\u0026iacute;n-Mic\u0026oacute; et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Braum\u0026uuml;ller et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fern\u0026aacute;ndez-Duran et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Besides, secretion of certain inflammatory cytokines including IL-1β has been shown to induce cellular senescence (Tominaga and Suzuki, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mart\u0026iacute;nez-Zamudio et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Senescent cells are found primarily in tissues that experience prolonged inflammation (Copp\u0026eacute; et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePyroptosis is a lytic and highly regulated cell death mechanism with pro-inflammatory characteristics (Broz et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is mediated by gasdermin (GSDM) family of proteins (6 members in humans: GSDMA, GSDMB, GSDMC, GSDMD, GSDME (DFNA5) and PJVK (Pejvakin, DFNB59)) following their proteolytic cleavage and activation (Broz et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shao, 2021; Lu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). After the cleavage of the linker region between NT and CT domains by specific inflammatory caspases (incl. caspase-1), free NT domain of GSDMs, translocates, for instance, to the plasma membrane and binds to lipids and oligomerizes to form membrane pores, resulting in rapid membrane rupture and release of danger-associated molecular patterns (DAMPs) and pro-inflammatory cytokines including mature IL-18 and IL-1β (Broz et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shao, 2021; Lu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, recently, the release of large molecules such as HMGB1 and LDH has also been shown to additionally require NINJ1-mediated plasma membrane rupture following gasdermin pore formation, since the sizes of pores formed by GSDM-NTs are not suitable for the passage of these large proteins (Kayagaki et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Immune cells recognize certain molecules released from pyroptotic cells, and then initiate a cascade of immune responses, including the activation and infiltration of certain immune cells to the places where pyroptotic cells are located. At the upstream, upon certain stimuli, pyroptosis can be initiated by the activation of inflammasomes such as NLRP3 inflammasome, which activates caspase-1 that then processes GSDMD (or some other GSDMs) as well as cytokines, such as pro-IL-1β and pro-IL-18, promoting pyroptotic cell death (Berkel, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Toldo and Abbate, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Faria et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Pyroptosis has been shown to be highly regulated in malignant transformation and tumor progression in cancer including breast and gynecological cancers (Berkel, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e; Berkel and Cacan, 2022).\u003c/p\u003e \u003cp\u003ePrevious research also suggested an association between senescence and pyroptosis. Senescent fibroblast-derived SASP factors can induce NLRP3-, caspase-1-, and GSDMD\u0026ndash;dependent pyroptotic cell death in neighboring normal (noncancerous) mammary epithelial cells (Hom et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In certain wound healing responses, pyroptotic macrophages might induce senescence in certain types of stem cells and thus increase senescent cell burden; and this pro-senescent effects are mostly mediated by IL‐1β and extracellular vesicles (containing HMGB1) released from pyroptotic macrophages (Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Cells surrounding pyroptotic macrophages have been shown to have a significant increase in markers representing senescence in other studies, pointing that pyroptotic macrophages may induce senescence in surrounding cells (Zhao et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In support, silencing of GSDMD in macrophages decreases the expression of senescence markers such as p16 and p21 (rescues senescence) in fibroblasts exposed to media from these macrophages with cleared pyroptosis (Zhao et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In human ovaries, pyroptotic macrophages (those accumulated in middle-aged ovaries) have been also found to promote senescence in stromal cells, principal components of the ovarian stroma (Zhou et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cytosolic double-stranded DNA (dsDNA), a sensitive biomarker for cellular senescence in mouse embryonic fibroblasts (MEFs), induces pyroptosis in these senescent cells in certain contexts (Zhou et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cytosolic dsDNA accumulation is proportional to senescence status in these cells independent of senescence-inducing stimuli (Zhou et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Besides, in aged mice urothelium, a canonical SASP along with continuous NLRP3-inflammasome- and GSDMD-dependent pyroptotic cell death has been recently identified (Joshi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A natural monosaccharide, D-mannose, both reverses the SASP and reduces NLRP3/GSDMD/IL-1β-driven pyroptotic epithelial cell shedding in the bladder in aged mice (Joshi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nlrp3 inflammasome activation has been also shown to lead to SASP and immunesenescence (T cell senescence) (Latz and Duewell, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Youm et al., 2012; Spadaro et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mar\u0026iacute;n-Aguilar et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Others showed that GSDMD might contribute to LPS-induced senescence, however, this might involve a mechanism other than the cleavage and liberation (thus, activation) of GSDMD NT domain (Fern\u0026aacute;ndez-Duran et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies mentioned above points that cellular senescence and pyroptosis might be interconnected in certain contexts.\u003c/p\u003e \u003cp\u003eIn the present study, I found that breast cancer cells with high gasdermin (GSDM) 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 cancer cells, which show higher expression of all three senescence marker genes. This high GSDM-dependent increases in the expression of senescence marker genes is more frequent in breast cancer cells than in non-malignant breast cells; in other words, the association between gasdermin family of genes and senesence marker genes in terms of expression levels is more strong in the case of tumor rather than non-malignant tissue. This might suggest that, in breast cancer, pyroptosis and senescence might be associated; however, whether pyroptosis regulates senescence or \u003cem\u003evice versa\u003c/em\u003e, whether these two processes reciprocally regulate and control each other, or whether they share an upstream regulatory pathway remains to be determined. This also supports previous research demonstrating the promoting effect of pyroptosis on senesence, and that SASP factors can induce GSDMD\u0026ndash;dependent pyroptotic cell death in neighboring cells present in the same microenvironment. Further mechanistic studies are required to determine the cellular and molecular connections between senescence and pyroptosis in breast cancer.\u003c/p\u003e"},{"header":"Gasdermins and pyroptosis","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eDatasets\u003c/h2\u003e \u003cp\u003eIn this study, I used transcriptomics and clinical datasets from TCGA (The Cancer Genome Atlas)-BRCA (Breast cancer) project (Cancer Genome Atlas Network, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; The Cancer Genome Atlas Research Network, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), accessed programmatically via ExperimentHub Bioconductor package (Morgan and Shepherd, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), as reported previously (Berkel, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). It contains expression data for 1119 breast tumors and 113 normal (non-malignant) breast tissues for 23368 genes. Samples (tumor or normal) with higher expression than the median expression value for each gene are defined as samples with \u0026ldquo;high expression\u0026rdquo;; therefore, half of the samples for each gene are categorized as \u0026ldquo;high expression\u0026rdquo; and the other half as \u0026ldquo;low expression\u0026rdquo; for each gene separately. Alternatively processed and compiled RNA-Seq and associated clinical data for patient samples from TCGA can also be found in GEO with accession ID of GSE62944 (Rahman et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Gene expression values were shown in log10 scale in y-axes of plots. Mean gene expression values for each group (low vs high expression) compared were also shown on boxplots.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData analysis and visualization were completely performed in R language in the present study using Rstudio IDE (version 2023.3.0.386; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cran.r-project.org/\u003c/span\u003e\u003cspan address=\"https://cran.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (R Core Team, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Posit team, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Following R / Bioconductor packages were used throughout the analysis: SummarizedExperiment (package version of 1.26.1) (Morgan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), tidyverse (2.0.0) (Wickham et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), ggpubr (0.6.0) (Kassambara, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), magick (2.7.4) (Ooms, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), knitr (1.42) (Xie, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and rmarkdown (2.21) (Allaire et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Shapiro-Wilk normality test was performed to analyze the distribution of the expression data (using ggqqplot() and shapiro.test() functions), and if the resultant p value is less than 0.05, normality could not be assumed and therefore wilcox test was performed to statistically compare the group means (Kassambara, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eBreast tumors with high GSDMD or GSDME expression have higher transcript levels of cellular senescence markers\u003c/b\u003e \u003c/p\u003e \u003cp\u003eI found that breast cancer cells from patients with high expression of GSDMD or GSDME have significantly increased mRNA levels of cellular senescence marker genes (CDKN1A (encoding p21), CDKN2A (encoding p16) and TP53 (encoding p53)) compared to those with low expression of GSDMD or GSDME, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, first panels). In normal (non-malignant) breast tissue, some of these GSDMD- and GSDME-dependent differences in the expression of cellular senescence marker genes observed in breast tumors are lost, especially for CDKN1A (p21) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, second panels).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCDKN2A (p16) expression is higher in breast tumors with high GSDMB or GSDMC expression\u003c/h2\u003e \u003cp\u003eNext, I showed that CDKN2A (p16) transcript levels are increased in breast cancer cells from patients with high expression of GSDMB or GSDMC, compared to those with low expression of these genes, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, first panels). Interestingly, this GSDMB- or GSDMC-dependent difference in CDKN2A expression is lost in healthy breast tissue (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, second panels). The other two cellular senescence marker genes (CDKN1A and TP53) did not show GSDMB- or GSDMC-dependent expression in breast cancer (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, first panels). In normal breast tissue, only significant difference was observed for TP53: its expression was found to be higher in breast tissue with high GSDMC expression compared to that with low GSDMC expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, second panel).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCDKN1A (p21) and TP53 (p53) expression are higher in breast tumors with high expression of GSDMA or PJVK\u003c/b\u003e \u003c/p\u003e \u003cp\u003eI found that, although the expression of CDKN1A (p21) and TP53 (p53) is not higher in breast tumors with high GSDMB or GSDMC expression (as reported above), the expression of these two cellular senescence marker genes are increased based on high expression of other two gasdermin genes, GSDMA and PJVK, in breast tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, first panels). These significant differences in the expression of CDKN1A and TP53 based on GSDMB or GSDMC expression were mostly lost in non-malignant breast tissues (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, second panels). In contrast, the expression of the other marker gene (CDKN2A) did not show GSDMA or PJVK expression-dependent changes either in breast tumors or in normal breast tissue (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, first panels).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHigh NINJ1-expressing breast cancer cells or non-malignant breast cells have higher expression of senescence markers\u003c/h2\u003e \u003cp\u003eLastly, I compared the expression of same three senescence marker genes at the mRNA level between low and high NINJ1-expressing cells (both breast cancer cells (left) or normal / non-malignant breast cells (right)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). I showed that breast cancer cells with high NINJ1 expression have higher transcript levels of CDKN1A and TP53, compared to breast cancer cells with low NINJ1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, left). Besides, normal breast cells with high NINJ1 expression were fount to have increased expression of CDKN2A and TP53, compared to low NINJ1-expressing normal breast cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, right).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eData in this analysis show that breast tumors with high gasdermin (GSDM) expression have higher expression of senescence marker genes, namely CDKN1A, CDKN2A and TP53. This is especially valid for breast tumors with high GSDMD- or GSDME expression, which show increased expression of all three senescence marker genes. This high gasdermin expression-dependent increases in the expression of senescence marker genes is more common in breast tumors than in non-malignant breast tissue; in other words, the association between gasdermin family of genes and cellular senesence marker genes in terms of transcript levels is more strong in breast tumors compared to normal breast tissue. Previous research suggested that SASP factors released from senescent cells can induce pyroptotic cell death in other cells (Hom et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The observation in the present study might be due to the fact that certain senescent cells in breast tumor microenvironment can activate pyroptotic cell death (via released factors) by upregulating gasdermin gene expression in other cells present nearby. Since bulk RNA-Seq data was used in this study, not allowing the identification of specific cell types with particular expression profiles, data from single-cell RNA-Seq technologies might help identify cell types with SASP and cell types with pyroptotic characteristics, enabling the determination of possible senescent cell-pyroptotic cell interactions in tumor.\u003c/p\u003e \u003cp\u003eAnother explanation for the observations in the present study might be that pyroptotic cells themselves (with increased mRNA levels of gasdermin family of genes) or increased inflammation due to pro-inflammatory pyroptotic cell death might induce senescence in surrounding cells in breast tumor microenvironment, as suggested previously in some other contexts (Acosta et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Desd\u0026iacute;n-Mic\u0026oacute; et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Braum\u0026uuml;ller et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fern\u0026aacute;ndez-Duran et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tominaga and Suzuki, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mart\u0026iacute;nez-Zamudio et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fern\u0026aacute;ndez-Duran et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For instance, inflammatory cytokines such as IL-18 and IL-1β released from gasdermin pores in the plasma membranes of pyroptotic cells might promote cellular senescence in neighboring cells or even in themselves (Tominaga and Suzuki, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mart\u0026iacute;nez-Zamudio et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Fern\u0026aacute;ndez-Duran et al. suggested that GSDMD might contribute to cellular senescence independent of its role in pyroptosis, because cleavage and pyroptotic activation of GSDMD is not required for it to induce senescence LPS-induced senescence (2021). Therefore, it can be suggested that pyroptotic or non-pyroptotic functions of gasdermins might be important depending on senescence-inducing stimuli. Since I observed that breast cancer cells with high GSDMD- or GSDME expression have increased expression of all three senescence marker genes, these two gasdermin genes might be relatively more important compared to other members of the family in terms of their influence on senescence. GSDMD has been shown to mediate the continous export of SASP factors such as IL-33 and IL-1β from senescent hepatic stellate cells, by forming cell membrane pores, in models of obesity-induced hepatocellular carcinoma (Yamagishi et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Others showed that NLRP1 inflammasome promotes senescence (induced by irradiation) by regulating the expression of p16, p21 and p53 (CDKN2A, CDKN1A and TP53; the same senescence marker genes studied in the current study), and SASP in GSDMD-dependent manner since these responses are reduced in conditions of NLRP1 insufficiency or GSDMD inhibition (Muela-Zarzuela et al., 2023). Therefore, it can be proposed that particular gasdermin family members might be involved in the release of certain SASP factors from senescence cells (possibly via the formation of pores) and also in the regulation of senescence responses such as the regulation of p16, p21 and p53 expression. In the case of breast cancer, GSDMD and GSDME might be more capable of promoting senescence or of contributing to SASP.\u003c/p\u003e \u003cp\u003eSince NLRP3 inflammasome activation has been also shown to lead to SASP and senescence in certain cells (Latz and Duewell, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Youm et al., 2012; Spadaro et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mar\u0026iacute;n-Aguilar et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), NLPR3 inflammasome might be a shared upstream regulator / inducer of both senescence and pyroptosis in breast cancer. Other inflammasomes such as NLRP1 might also be involved in the joint regulation of cellular senescence and pyroptotic cell death in certain contexts and cells including breast cancer cells (Muela-Zarzuela et al., 2023). A better understanding of the association between senescence and pyroptosis, and of the possible common regulators of both cell states is needed.\u003c/p\u003e \u003cp\u003eHMGB1 is a pro-inflammatory protein (an alarmin) released from cells following NINJ1-mediated plasma mebrane rupture after gasdermin pore formation (Kayagaki et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Berkel and Cacan, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kayagaki et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; David et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Sofiadis et al. showed that HMGB1 controls the expression of genes that are central to the senescent program and influences the availability of senescence-relevant mRNAs (2021). Authors also found that senescence entry is marked by HMGB1 nuclear loss and secretion; in other words, its depletion in cells underlies induction of the senescence program (Sofiadis et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eConsidering the role of HMGB1 in senescence and its involvement in induction of pyroptosis in certain contexts (\u003c/span\u003eHou et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eHMGB1 might be a link between senescence and pyroptosis. Senescent cells have essentially no nuclear HMGBs (\u003c/span\u003eSofiadis et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand high levels of HMGB1 in the cytoplasm might lead to the increased activation of NLRP3 inflammasome, resulting in enhanced levels of pyroptosis in these cells.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eSince high gasdermin expression-dependent increases in the expression of senescence marker genes were observed to be more common in breast tumors than in non-malignant breast tissue, it can be suggested that senescence \u0026ndash; pyroptosis association might be more strong in cancer; however, it should also be noted that sample size for breast cancer patients is higher than patients without breast cancer in the current analysis. Further comparative mechanistic studies are neeeded to better identify differences between breast tumor and normal breast tissue in this respect.\u003c/span\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interests\u003c/h2\u003e \u003cp\u003eThe author declares no conflicts of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNo funding has been received for the present study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.B. conceptualized the study, performed the data analysis and visualization, and wrote the paper.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eIn this study, I used transcriptomics and clinical datasets from TCGA (The Cancer Genome Atlas)-BRCA (Breast cancer) project (\u003c/span\u003eCancer Genome Atlas Network, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe\u003c/span\u003e Cancer Genome Atlas Research Network, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), accessed programmatically via ExperimentHub Bioconductor package (\u003c/span\u003eMorgan and Shepherd, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcosta 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. 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PMID: 33991763.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"senescence, pyroptosis, breast cancer, GSDME, GSDMD, inflammation, p21, p16","lastPublishedDoi":"10.21203/rs.3.rs-4884791/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4884791/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCellular 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 highly regulated cell death mechanism with pro-inflammatory characteristics, mediated by gasdermin (GSDM) family of proteins, which has six members: GSDMA-E and PJVK. In the present study, I 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 three senescence marker genes. This high GSDM-dependent increases in the transcript levels of cellular senescence marker genes is more frequent in breast cancer cells than in non-malignant breast cells, suggesting that the association between gasdermin family of genes and senesence marker genes in terms of expression levels is more strong in the case of tumor. This might point that, in breast cancer, pyroptosis and senescence might be associated; however, whether pyroptosis regulates senescence or \u003cem\u003evice versa\u003c/em\u003e, whether these two processes both reciprocally regulate and control each other, or even whether they share an upstream regulatory pathway remains 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\u0026ndash;dependent pyroptotic cell death in neighboring cells present, in certain contexts. Further mechanistic studies are required to better characterize molecular connections between senescence and pyroptosis in breast cancer.\u003c/p\u003e","manuscriptTitle":"Transcript levels of cellular senescence marker genes are increased based on high expression of gasdermin family of genes in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-02 16:45:45","doi":"10.21203/rs.3.rs-4884791/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a0e95b1-9956-43f6-9190-7c530bd8a89b","owner":[],"postedDate":"October 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-19T03:53:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-02 16:45:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4884791","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4884791","identity":"rs-4884791","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}