Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC

Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC CAGLAR BERKEL, Ercan CACAN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1693902/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 High-grade serous ovarian carcinoma (HGSOC) is responsible for the majority of ovarian cancer-associated deaths. PARP (Poly-(ADP-ribose) polymerase) inhibitors (PARPi) (olaparib, rucaparib and niraparib) have been approved in recent years to be used in both the frontline setting as maintenance therapy and in the recurrent setting for patients with ovarian cancer (OC). Previous studies have reported increased expression of PARP1, a sensor for DNA damage, in OC; however, its compartment (epithelial tumor vs stroma)-, anatomical site (fallopian tube, omentum, ovary and serous tubal in situ carcinoma)- and histotype-specific expression have not been studied. Here, we showed that PARP1 protein levels are higher in epithelial tumors compared to adjacent stroma at four different anatomical locations in HGSOC, with the most significant difference in PARP1 levels in omental metastases. Furthermore, we found that PARP1 protein levels are higher in tumors compared to normal tissue in fallopian tube, omentum and ovary in patients with HGSOC, again with the most significant difference in omentum. We showed that S100A4 and CLDN7, transcriptional targets of PARP1, have the highest copy numbers in serous OC among all cancer types, showing the potential OC-specific roles of these two proteins. We also showed that PARP1 expression is increased in malignant and invasive stage in mouse ovarian surface epithelium cells, and that PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial OC. These findings indicate that a better understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in OC is needed, since PARP inhibitors which work by preventing DNA repair in cancer cells are currently being used in the treatment of patients with OC. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction High-grade serous ovarian carcinoma (HGSOC) is the most frequent and lethal type of ovarian carcinoma, and it is responsible for the majority of ovarian cancer-associated deaths in women (Siegel et al. 2018; Torre et al. 2018). HGSOC has a high proportion of stromal cells besides epithelial tumor cells in the tumor microenvironment (Eckert et al. 2019). It is known that the stromal cells support tumor cells adjacent to them, and co-evolve with the epithelial compartment during cancer progression and metastasis (Eckert et al. 2019; Polyak et al. 2009; Kalluri 2016). However, a better understanding of molecular differences between epithelial tumor cells and adjacent stromal cells in the tumor microenvironment, especially in terms of drug vulnerabilities, is needed to develop more effective targeted therapies. Standard frontline care for patients with ovarian cancer remains surgical debulking and platinum-based chemotherapy (such as with cisplatin); thus, overall survival has not changed significantly for several decades, despite a better understanding of the molecular mechanisms in HGSOC. More than 80% of women with HGSOC treated with these options ultimately experience disease recurrence, limiting the efficacy of the current treatment strategies (Salani et al. 2011; Zivanovic et al. 2009). PARP (Poly-(ADP-ribose) polymerase) inhibitors (PARPi) have been approved in recent years to be used in both the frontline setting as maintenance therapy and in the recurrent setting for patients with ovarian cancer (Kristeleit et al. 2017; Moore et al. 2018; Pujade-Lauraine et al. 2017). Cytotoxicity induced by PARPi is considered to be caused, at least in part, by the trapment of PARP1 on DNA lesions, limiting its activity in DNA damage response (Kristeleit et al. 2017; Moore et al. 2018; Pujade-Lauraine et al. 2017). HGSOC is almost always TP53-mutant, and approximately 50% of patients have deficiencies in homologous recombination (HR) DNA repair of DNA double strand breaks (DSB), most frequently due to the presence of mutations in BRCA1/2 (Konstantinopoulos et al. 2015). Tumours with deficiencies in HR display favourable responses to cisplatin chemotherapy and PARPi (Konstantinopoulos et al. 2015; Lord and Ashworth 2017; Scott et al. 2015). PARP enzymes are involved in DNA repair by promoting base excision repair and alternative end-joining pathways and by limiting non-homologous end-joining (NHEJ) pathway (Scott et al. 2015). The sensitivity of HR-deficient cells to PARPi relies on overactivation of the NHEJ pathway, impaired DNA replication fork protection and persistence of unrepaired collapsed forks (Scott et al. 2015; Ceccaldi et al. 2015; Swisher et al. 2017; Sanij et al. 2020). Since PARP inhibition with drugs such as olaparib is currently in use in the treatment of patients with HGSOC, PARP1 levels in epithelial tumor cells and adjacent stromal cells is of high clinical importance. Here, we showed that PARP1 protein levels are higher in epithelia tumor cells compared to adjacent stromal cells at 4 different anatomical locations in HGSOC, with the highest difference in PARP1 levels in omental metastases. Similarly, we found that PARP1 protein levels are higher in tumors compared to normal tissues in the fallopian tube, omentum and ovary in patients with HGSOC. We also report that S100A4 and CLDN7, transcriptional targets of PARP1, have the highest copy numbers (CNV gain) in serous ovarian cancer among all cancer types. Furthermore, we showed that PARP1 expression is higher in malignant and invasive stage compared to pre-neoplastic, non-malignant stage or to neoplastic, pre-invasive stage in mouse ovarian surface epithelium cells. Finally, we found that PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial ovarian cancer. Presented data in the current study highlight that a complete understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in OC is needed. Materials and Methods Proteome data Proteomics data for high grade serous ovarian carcinoma (HGSOC) patients, acquired using liquid chromatography (LC)-mass spectrometry (MS) from formalin-fixed, paraffin-embedded (FFPE) specimens were accessed from MaxQB - The MaxQuant DataBase (Max-Planck-Institut für Biochemie) (Eckert et al. 2019) (n = 107). [Dataset can be accessed through http://maxqb.biochem.mpg.de/mxdb/project/show/9373012627500]. This dataset contains protein expression levels for epithelial tumor cells and surrounding non-malignant stromal cells at four different anatomical locations in patients with HGSOC (namely, omental metastasis, serous tubal in situ carcinoma (STIC), invasive fallopian tube (FT) lesions and invasive ovarian lesions) (Eckert et al. 2019). Copy number variation analysis Copy number variation (CNV) data for ovarian cancer patients was accessed from National Cancer Institute Genomic Data Commons Data Portal [https://portal.gdc.cancer.gov/] (Grossman et al. 2016; Huang et al. 2018; Berger et al. 2018). Cancer types were ordered based on the percentage of copy number gains in PARP1 gene from the highest to the lowest (left to right). These copy number alteration data for cancer patients were originally obtained in The Cancer Genome Atlas (TCGA) project (Huang et al. 2018; Berger et al. 2018). Transcriptome data Following transcriptomics datasets from Gene Expression Omnibus (GEO) were used in this study: GSE24789 (Creekmore et al. 2011, n = 36), GSE14764 (Denkert et al. 2009), GSE20565 (Meyniel et al. 2010), GSE2109, GSE26193 (Mieulet et al. 2010), GSE30161 (Ferriss et al. 2012), GSE44104 (Wu et al. 2015), GSE6008 (Wu et al. 2016), GSE6822, GSE8842 (Marchini et al. 2008) and GSE9891 (Tothill et al. 2008). These datasets were loaded to R using curatedOvarianData Bioconductor package (Ganzfried et al. 2013). Each sample was indicated as a data point in the plots to show relative sample sizes. Data analysis and visualization Following R packages used in the data analysis and visualization (R Core Team, 2020): tidyverse (Wickham et al. 2019), readxl (Wickham and Bryan 2019), ggpubr (Kassambara 2020), tidytext (Silge and Robinson) and rmarkdown (Allaire et al. 2021). Following convention for star symbols indicating statistical significance (p values) was used in the analysis of data in the present study: non-significant (ns): p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001;****: p ≤ 0.0001 (ggpubr package)(Kassambara 2020). The normality test for the data used in each plot was performed using ggqqplot() (Quantile-Quantile plot) and shapiro.test() (Shapiro-Wilk normality test) functions in R (from ggpubr and stats R packages)(Kassambara 2020, R Core Team, 2020). When data is normally distributed, we used Student’s t test to compare group means; otherwise, we performed the analysis using two-sample Wilcoxon test (Mann-Whitney test). No observations were excluded from the analyses. Results PARP1 protein levels are higher in tumors compared to adjacent stroma at 4 different anatomical locations in HGSOC, with the most significant difference in PARP1 levels in omental metastases To investigate if PARP1 protein levels are different between epithelial tumor cells and stromal cells present in surrounding extracellular microenvironment, in patients with high-grade serous ovarian carcinoma ( HGSOC), we analyzed the proteomics data by Eckert et al. obtained using LC-MS (2019). We found that PARP1 protein expression is higher in tumor cells compared to adjacent stromal cells, at four different anatomical locations (fallopian tube, omentum, ovary and STIC (serous tubal in situ carcinoma)) in patients with HGSOC (Figure 1A). Between these anatomical sites, omentum represents a metastatic site called omental metastases, other three (fallopian tube, ovary and STIC) represent primary tumor tissues. In terms of anatomical locations, the highest difference in PARP1 protein levels between epithelial tumor compartment and stromal compartment was observed for omentum (p = 9.3e-08), followed by ovary (p = 3.2e-05), serous tubal in situ carcinoma (STIC) (p = 0.0025) and fallopian tube (p = 0.002) (Figure 1A). In other words, difference in PARP1 protein levels between epithelial tumor compartment and stromal compartment is the most significant in omental metastases. PARP1 protein levels are higher in tumors compared to normal tissue in fallopian tube, omentum and ovary in patients with HGSOC Next, we analyzed PARP1 expression at the protein level between HGSOC tumors and corresponding normal tissues in 3 different anatomic sites, namely, fallopian tube, omentum and ovary. We found that, for each of these sites, PARP1 protein levels are increased in tumor samples compared to samples from healthy tissues (Figure 1B). The most significant difference in PARP1 protein levels between tumor and normal samples was observed for omentum (p = 3.3e-05), followed by fallopian tube (p = 0.00021) and ovary (p = 0.028) (Figure 1B). Similar to the previous observation that PARP1 protein level differences between epithelial tumor compartment and stromal compartment is the most significant for omental metastases, PARP1 protein levels between normal and tumor tissues show the most significant difference in omentum. Collectively, these data may point that the contribution of PARP1, at least in part, to omental metastases. S100A4 and CLDN7, transcriptional targets of PARP1, have the highest copy numbers in serous ovarian cancer among all cancer types Choi et al. showed that PARP1 enhances lung adenocarcinoma metastasis through transcriptional upregulation of S100A4 and CLDN7, i.e. by mechanisms independently of its function in DNA repair (2016). Since we observed the most significant difference in PARP1 protein levels in omentum / omental metastases among other anatomical locations (others are primary tumor tissues, not metastases), between epithelial tumor and normal stromal compartments, and also between tumor and normal samples, we speculated that PARP1 may contribute, at least in part, to metastasis to omentum. We found that the transcriptional targets of PARP1, namely S100A4 and CLDN7, have the largest copy number gain percentages in serous ovarian cancer among other cancer types (Figure 2). This data indicates that these two proteins whose expression is upregulated by PARP1 may have more important functions in ovarian cancer compared to other cancer types. Supportingly, multiple studies reported the increased expression of these genes in ovarian cancer (Dahiya et al. 2011; Tassi et al. 2008; Kim et al. 2011; Soini and Talvensaari-Mattila 2006; Kleinberg et al. 2008; Link et al. 2019; Yan et al. 2016; Hiruta et al. 2020; Deo et al. 2021; Xu et al. 2020; Kikuchi et al. 2006; Maekandsmo et al. 2009). PARP1 expression is increased in malignant and invasive stage in mouse ovarian surface epithelium cells To analyze PARP1 expression levels at different stages of malignancy, we used transcriptome data from mouse ovarian surface epithelial (MOSE) cells (Creekmore et al. 2011). Here, we used data from mice, since similar data from patients is not currently available. These mouse cells undergo spontaneous transformation in cell culture with increasing passage numbers, and represent phenotypes from pre−neoplastic, non−malignant to malignant and invasive (Creekmore et al. 2011). Since we observed themost significant difference in PARP1 levels in omental metastases when comparing its levels between tumors and adjacent stroma, we wanted to see if PARP1 expression increases in parallel to increasing malignancy. We found that PARP1 mRNA levels are indeed higher in malignant and invasive stage (late cells, passage 136, 142, and 143) compared to both pre−neoplastic, non−malignant stage (early cells, passage 13, 14, and 15) (p = 0.003) and neoplastic, pre−invasive stage (intermediate cells, passage 63, 71, and 73) (p = 0.0036) (Figure 3). PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial ovarian cancer Next, we analyzed if PARP1 mRNA levels differ between different histological types (histotypes) of epithelial ovarian cancer (EOC comprises of different histotypes: clear cell, endometrium, mucinous, serous and also very rare malignant Brenner tumors) (Berkel and Cacan 2021). We found that PARP1 expression is lower in mucinous histotype compared to serous histotype in 5 out of 6 datasets in which data is available for these histotypes (p values: 0.048, 0.0024, 0.027, 0.0017 and 9.1e-05) (Figure 4). Between other histological types, there seems to be no significant change in PARP1 mRNA levels which is consistent between datasets (Figure 5). Discussion In this study, we showed that PARP1 expression is increased in epithelial tumor compartment compared to adjacent stromal compartment at 4 different anatomical sites (fallopian tube, omentum, ovary and STIC (serous tubal in situ carcinoma)) in patients with HGSOC. We observed the most significant difference in PARP1 protein levels in omental metastases compared to primary tumor tissues (fallopian tube, ovary and STIC (serous tubal in situ carcinoma)). These data show that epithelial tumor cells have higher PARP1 protein expression compared to stromal cells in the tumor microenvironment, independently of their anatomical location. This is of clinical importance, since PARP inhibitors including olaparib are currently being used in the treatment of patients with ovarian cancer in both the frontline setting as maintenance therapy and in the recurrent setting (Kristeleit et al. 2017; Moore et al. 2018; Pujade-Lauraine et al. 2017). Because the difference in PARP1 protein levels between tumor and stroma is the most significant in omental metastases, PARP inhibition may promote synthetic lethality more favorably in metastases compared to primary sites. This is because epithelial tumor compartment will be more preferentially targeted than stromal compartment in omental metastases by the inhibition of PARP. However, further research is needed to make stronger inferences. Similarly, we observed that PARP1 protein levels are higher in tumors compared to normal tissue in fallopian tube, omentum and ovary in patients with HGSOC, again with the highest difference in omentum / omental metastases. Therefore, we can state that the most significant increase in PARP1 protein levels in the course of cancer progression occurs in omental metastasis compared to primary tumor sites, pointing to the potential contribution of PARP1, at least in part, to advanced disease and metastasis. Supportingly, we found that two transcriptional targets of PARP1, namely S100A4 and CLDN7, which enhances metastasis in lung adenocarcinoma (Choi et al. 2016), have the highest copy numbers in serous ovarian cancer among all cancer types, highlighting potential ovarian cancer-specific roles of these proteins. Indeed, multiple studies reported the increased expression of these proteins in ovarian cancer and their involvement in disease progression (Dahiya et al. 2011; Tassi et al. 2008; Kim et al. 2011; Soini and Talvensaari-Mattila 2006; Kleinberg et al. 2008; Link et al. 2019; Yan et al. 2016; Hiruta et al. 2020; Deo et al. 2021; Xu et al. 2020; Kikuchi et al. 2006; Maekandsmo et al. 2009). We also found that PARP1 expression is increased in malignant and invasive mouse ovarian surface epithelial cells compared to those with pre−neoplastic, non−malignant phenotype or with neoplastic, pre−invasive phenotype in vitro . This data is in line with a recent study showing that PARP1 overexpression promotes viability, migration, invasion, and tube formation in ovarian cancer cells (Chang et al. 2021). Wei et al. showed that hepatocyte growth factor (HGF) administration increases PARP1 levels and enhances cell invasion in a concentration- and time-dependent manner in ovarian cancer cells in vitro (Wei et al. 2018). They also reported that the silencing of PARP1 significantly reduces the impact of HGF on invasiveness in ovarian cancer cells, pointing to the role of PARP1 in the development of an invasive phenotype (Wei et al. 2018). Same group also found that PARP1 may enhance angiogenesis in epithelial ovarian cancer by upregulating VEGF-A (Wei et al. 2016). Our analysis also pointed out that mostly lower PARP1 mRNA levels are observed in mucinous histotype of epithelial ovarian cancer compared to serous histotype. This difference in PARP1 levels may contribute, at least to a certain extent, to the differences between these two histological types in terms of survival and response to chemotherapy and PARP inhibition (Overall, patients with mucinous histotype have a better prognosis compared to those with serous histotype in the early stage; however, patients with mucinous type have a worse prognosis at advanced stage compare to patients with serous histotype) (Peres et al. 2019; Monk et al. 2019; Simons et al. 2017). Combined, we showed that PARP1 protein levels are higher in tumor samples compared to both adjacent stroma or normal ovaries, in patients with HGSOC, with the most significant difference in omental metastases, compared to primary tumor tissues. Supportingly, we found that certain transcriptional targets of PARP1 which are also associated with PARP1-mediated metastasis in other cancers have the highest copy number gain percentage in ovarian cancer among other cancers. This points to a potential mechanism of PARP1 in promoting malignancy in ovarian cancer; however, mechanistic studies are needed to test these hypotheses. In parallel, we showed that malignant mouse ovarian surface epithelial cells have higher PARP1 expression cmpared to those with low malignancy. Furthermore, we observed differential levels of PARP1 in various histological types of epithelial ovarian cancer, possibly contributing to varying degrees of malignancy observed in these histotypes. This preliminary study highlights the need for a complete understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in ovarian cancer. 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Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine , 30 (1), 15–25. https://doi.org/10.1159/000199447 Berkel C, Cacan E (2021) GAB2 and GAB3 are expressed in a tumor stage-, grade- and histotype-dependent manner and are associated with shorter progression-free survival in ovarian cancer. Journal of cell communication and signaling , 15 (1), 57–70. https://doi.org/10.1007/s12079-020-00582-3 Berkel C, Kucuk B, Usta M et al. (2020) The Effect of Olaparib and Bortezomib Combination Treatment on Ovarian Cancer Cell Lines . European Journal of Biology , 79 (2) , 115-123. Chang H, Zhang X, Li B et al. (2021) PARP1 Is Targeted by miR-519a-3p and Promotes the Migration, Invasion, and Tube Formation of Ovarian Cancer Cells. Cancer biotherapy & radiopharmaceuticals , 10.1089/cbr.2020.4394. Advance online publication. https://doi.org/10.1089/cbr.2020.4394 Wei W, Lv S, Zhang C, et al. (2018). Potential role of HGF-PARP-1 signaling in invasion of ovarian cancer cells. International journal of clinical and experimental pathology , 11 (7), 3310–3317. Wei W, Li Y, Lv S et al. (2016) PARP-1 may be involved in angiogenesis in epithelial ovarian cancer. Oncology letters , 12 (6), 4561–4567. https://doi.org/10.3892/ol.2016.5226 Peres LC, Cushing-Haugen KL, Köbel M et al. (2019) Invasive Epithelial Ovarian Cancer Survival by Histotype and Disease Stage. Journal of the National Cancer Institute , 111 (1), 60–68. https://doi.org/10.1093/jnci/djy071 Monk BJ, Randall LM, Grisham RN (2019) The Evolving Landscape of Chemotherapy in Newly Diagnosed Advanced Epithelial Ovarian Cancer. American Society of Clinical Oncology educational book. American Society of Clinical Oncology. Annual Meeting , 39 , e141–e151. https://doi.org/10.1200/EDBK_239007 Simons M, Massuger L, Bruls J et al. (2017) Relatively Poor Survival of Mucinous Ovarian Carcinoma in Advanced Stage: A Systematic Review and Meta-analysis. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society , 27 (4), 651–658. https://doi.org/10.1097/IGC.0000000000000932 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 Advisory Board 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-1693902","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":109650447,"identity":"7ef7cd56-d456-420e-95a0-813bc53fb867","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":"https://orcid.org/0000-0003-4787-5157","institution":"Tokat Gaziosmanpasa University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"CAGLAR","middleName":"","lastName":"BERKEL","suffix":""},{"id":109650448,"identity":"a0a34a7a-168c-4030-b522-7ca9427260a8","order_by":1,"name":"Ercan CACAN","email":"","orcid":"","institution":"Tokat Gaziosmanpaşa University: Tokat Gaziosmanpasa Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ercan","middleName":"","lastName":"CACAN","suffix":""}],"badges":[],"createdAt":"2022-05-25 19:20:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1693902/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1693902/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":22181168,"identity":"d951db8b-2392-4017-ab62-c7e5a9991073","added_by":"auto","created_at":"2022-06-02 14:43:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePARP1 protein levels are higher in tumors compared to adjacent stroma inHGSOC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(A) PARP1 protein levels are higher in tumors compared to adjacent stroma at 4 different anatomical locations in high-grade serous ovarian carcinoma (HGSOC), with the most significant difference in PARP1 levels in omental metastases. (B) PARP1 protein levels are higher in tumors compared to normal tissue in fallopian tube, omentum and ovary in patients with HGSOC.\u003c/p\u003e\u003cp\u003enon-significant [ns]: p \u0026gt; 0.05; ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; ∗∗∗: p ≤ 0.001; ∗∗∗∗: p ≤ 0.0001.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSTIC: serous tubal in situ carcinoma. \u003c/em\u003e(n = 107)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1693902/v1/a62c80cff0c72a38db2b0bdd.png"},{"id":22181169,"identity":"bbbb6726-6bd4-4edd-b5c6-97b4f478adff","added_by":"auto","created_at":"2022-06-02 14:43:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS100A4 and CLDN7, transcriptional targets of PARP1, have the highest copy numbers in serous ovarian cancer among all cancer types\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u0026nbsp;LAML: Acute Myeloid Leukemia; ACC: Adrenocortical carcinoma; BLCA: Bladder Urothelial Carcinoma; LGG: Brain Lower Grade Glioma; BRCA: Breast invasive carcinoma; CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: Cholangiocarcinoma; LCML: Chronic Myelogenous Leukemia; COAD: Colon adenocarcinoma; ESCA: Esophageal carcinoma; GBM: Glioblastoma multiforme; HNSC: Head and Neck squamous cell carcinoma; KICH: Kidney Chromophobe; KIRC\u0026nbsp;\u0026nbsp;Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; DLBC: Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; MESO: Mesothelioma; OV: Ovarian serous cystadenocarcinoma; PAAD: Pancreatic adenocarcinoma; PCPG: Pheochromocytoma and Paraganglioma; PRAD: Prostate adenocarcinoma; READ: Rectum adenocarcinoma; SARC: Sarcoma; SKCM: Skin Cutaneous Melanoma; STAD: Stomach adenocarcinoma; TGCT: Testicular Germ Cell Tumors; THYM: Thymoma; THCA: Thyroid carcinoma; UCS: Uterine Carcinosarcoma; UCEC: Uterine Corpus Endometrial Carcinoma; UVM: Uveal Melanoma\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1693902/v1/9d61806d5d4ad8eecff54e58.png"},{"id":22181171,"identity":"f057e0e6-6d2f-4248-872b-16cb9e01e588","added_by":"auto","created_at":"2022-06-02 14:43:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePARP1 expression is increased in malignant and invasive stage in mouse ovarian surface epithelium (MOSE) cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eDifferent colors given in figure legend indicate different probes used in order to measure PARP1 expression. (n = 36)\u003c/p\u003e\u003cp\u003enon-significant [ns]: p \u0026gt; 0.05; ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; ∗∗∗: p ≤ 0.001; ∗∗∗∗: p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1693902/v1/c9fad54d7bea7bf5e709bd3d.png"},{"id":22181170,"identity":"9aeb240f-1cbb-4597-adf5-de4f114dcd9d","added_by":"auto","created_at":"2022-06-02 14:43:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":454506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial ovarian cancer\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eDifferent histological types (histotypes) of epithelial ovarian cancer (EOC): clearcell: clear cell; endo: endometrium, mucinous; ser: serous. Each sample was indicated as a data point in the plots to\u0026nbsp;show relative sample sizes.\u003c/p\u003e\u003cp\u003enon-significant [ns]: p \u0026gt; 0.05; ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; ∗∗∗: p ≤ 0.001; ∗∗∗∗: p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1693902/v1/e58e9405ee8423d393ec4f1d.png"},{"id":22181173,"identity":"e3b02fe2-8a27-45ab-8e8b-1b17585ed41f","added_by":"auto","created_at":"2022-06-02 14:43:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":459038,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1693902/v1/cf4434a0-f58c-4dda-a495-71180f96414c.pdf"}],"financialInterests":"","formattedTitle":"Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHigh-grade serous ovarian carcinoma (HGSOC) is the most frequent and lethal type of ovarian carcinoma, and it is responsible for the majority of ovarian cancer-associated deaths in women (Siegel et al. 2018; Torre et al. 2018). HGSOC has a high proportion of stromal cells besides epithelial tumor cells in the tumor microenvironment (Eckert et al. 2019). It is known that the stromal cells support tumor cells adjacent to them, and co-evolve with the epithelial compartment during cancer progression and metastasis (Eckert et al. 2019; Polyak et al. 2009; Kalluri 2016). However, a better understanding of molecular differences between epithelial tumor cells and adjacent stromal cells in the tumor microenvironment, especially in terms of drug vulnerabilities, is needed to develop more effective targeted therapies.\u003c/p\u003e\n\u003cp\u003eStandard frontline care for patients with ovarian cancer remains surgical debulking and platinum-based chemotherapy (such as with cisplatin); thus, overall survival has not changed significantly for several decades, despite a better understanding of the molecular mechanisms in HGSOC. More than 80% of women with HGSOC treated with these options ultimately experience disease recurrence, limiting the efficacy of the current treatment strategies (Salani et al. 2011; Zivanovic et al. 2009). PARP (Poly-(ADP-ribose) polymerase) inhibitors (PARPi) have been approved in recent years to be used in both the frontline setting as maintenance therapy and in the recurrent setting for patients with ovarian cancer (Kristeleit et al. 2017; Moore et al. 2018; Pujade-Lauraine et al. 2017). Cytotoxicity induced by PARPi is considered to be caused, at least in part, by the trapment of PARP1 on DNA lesions, limiting its activity in DNA damage response (Kristeleit et al. 2017; Moore et al. 2018; Pujade-Lauraine et al. 2017).\u003c/p\u003e\n\u003cp\u003eHGSOC is almost always TP53-mutant, and approximately 50% of patients have deficiencies in homologous recombination (HR) DNA repair of DNA double strand breaks (DSB), most frequently due to the presence of mutations in BRCA1/2 (Konstantinopoulos et al. 2015). Tumours with deficiencies in HR display favourable responses to cisplatin chemotherapy and PARPi (Konstantinopoulos et al. 2015;\u0026nbsp;Lord and Ashworth 2017; Scott et al. 2015). PARP enzymes are involved in DNA repair by promoting base excision repair and alternative end-joining pathways and by limiting non-homologous end-joining (NHEJ) pathway (Scott et al. 2015). The sensitivity of HR-deficient cells to PARPi relies on overactivation of the NHEJ pathway, impaired DNA replication fork protection and persistence of unrepaired collapsed forks (Scott et al. 2015; Ceccaldi et al. 2015; Swisher et al. 2017; Sanij et al. 2020).\u003c/p\u003e\n\u003cp\u003eSince PARP inhibition with drugs such as olaparib is currently in use in the treatment of patients with HGSOC, PARP1 levels in epithelial tumor cells and adjacent stromal cells is of high clinical importance. Here, we showed that PARP1 protein levels are higher in epithelia tumor cells compared to adjacent stromal cells at 4 different anatomical locations in HGSOC, with the highest difference in PARP1 levels in omental metastases. Similarly, we found that PARP1 protein levels are higher in tumors compared to normal tissues in the fallopian tube, omentum and ovary in patients with HGSOC. We also report that S100A4 and CLDN7, transcriptional targets of PARP1, have the highest copy numbers (CNV gain) in serous ovarian cancer among all cancer types. Furthermore, we showed that PARP1 expression is higher in malignant and invasive stage compared to pre-neoplastic, non-malignant stage or to neoplastic, pre-invasive stage in mouse ovarian surface epithelium cells. Finally, we found that PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial ovarian cancer. Presented data in the current study highlight that a complete understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in OC is needed.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eProteome data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteomics data for high grade serous ovarian carcinoma (HGSOC) patients, acquired using liquid chromatography (LC)-mass spectrometry (MS) from formalin-fixed, paraffin-embedded (FFPE) specimens were accessed from MaxQB - The MaxQuant DataBase (Max-Planck-Institut für Biochemie) (Eckert et al. 2019) (n = 107). [Dataset can be accessed through http://maxqb.biochem.mpg.de/mxdb/project/show/9373012627500]. This dataset contains protein expression levels for epithelial tumor cells and surrounding non-malignant stromal cells at four different anatomical locations in patients with HGSOC \u0026nbsp;(namely, omental metastasis, serous tubal in situ carcinoma (STIC), invasive fallopian tube (FT) lesions and invasive ovarian lesions) (Eckert et al. 2019).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eCopy number variation analysis\u003c/h3\u003e\n\u003cp\u003eCopy number variation (CNV) data for ovarian cancer patients was accessed from National Cancer Institute Genomic Data Commons Data Portal [https://portal.gdc.cancer.gov/] (Grossman et al. 2016; Huang et al. 2018; Berger et al. 2018). Cancer types were ordered based on the percentage of copy number gains in PARP1 gene from the highest to the lowest (left to right). These copy number alteration data for cancer patients were originally obtained in The Cancer Genome Atlas (TCGA) project (Huang et al. 2018; Berger et al. 2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing transcriptomics datasets from Gene Expression Omnibus (GEO) were used in this study: GSE24789 (Creekmore et al. 2011, n = \u0026nbsp;36), GSE14764 (Denkert et al. 2009), GSE20565 (Meyniel et al. 2010), GSE2109, GSE26193 (Mieulet et al. 2010), GSE30161 (Ferriss et al. 2012), GSE44104 (Wu et al. 2015), GSE6008 (Wu et al. 2016), GSE6822, GSE8842 (Marchini et al. 2008) and GSE9891 (Tothill et al. 2008). These datasets were loaded to R using\u0026nbsp;curatedOvarianData Bioconductor package (Ganzfried et al. 2013). Each sample was indicated as a data point in the plots to show relative sample sizes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis and visualization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing R packages used in the data analysis and visualization (R Core Team, 2020): tidyverse (Wickham et al. 2019), readxl (Wickham and Bryan 2019), ggpubr (Kassambara 2020), tidytext (Silge and Robinson) and rmarkdown (Allaire et al. 2021).\u003c/p\u003e\n\u003cp\u003eFollowing convention for star symbols indicating statistical significance (p values) was used in the analysis of data in the present study: non-significant (ns): p \u0026gt; 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001;****: p ≤ 0.0001 (ggpubr package)(Kassambara 2020). The normality test for the data used in each plot was performed using ggqqplot() (Quantile-Quantile plot) and shapiro.test() (Shapiro-Wilk normality test) functions in R (from ggpubr and stats R packages)(Kassambara 2020, R Core Team, 2020). When data is normally distributed, we used Student’s t test to compare group means; otherwise, we performed the analysis using two-sample Wilcoxon test (Mann-Whitney test). No observations were excluded from the analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePARP1 protein levels are higher in tumors compared to adjacent stroma at 4 different anatomical locations in HGSOC, with the most significant difference in PARP1 levels in omental metastases\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate if PARP1 protein levels are different between epithelial tumor cells and stromal cells present in surrounding extracellular microenvironment, in patients with high-grade serous ovarian carcinoma\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eHGSOC), we analyzed the proteomics data by Eckert et al. obtained using LC-MS (2019). We found that PARP1 protein expression is higher in tumor cells compared to adjacent stromal cells, at four different anatomical locations (fallopian tube, omentum, ovary and STIC (serous tubal in situ carcinoma)) in patients with HGSOC (Figure 1A). Between these anatomical sites, omentum represents a metastatic site called omental metastases, other three (fallopian tube, ovary and STIC) represent primary tumor tissues. In terms of anatomical locations, the highest difference in PARP1 protein levels between epithelial tumor compartment and stromal compartment was observed for omentum (p = 9.3e-08), followed by ovary (p = 3.2e-05), serous tubal in situ carcinoma (STIC) (p = 0.0025) and fallopian tube (p = 0.002) (Figure 1A). In other words, difference in PARP1 protein levels between epithelial tumor compartment and stromal compartment is the most significant in omental metastases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePARP1 protein levels are higher in tumors compared to normal tissue in fallopian tube, omentum and ovary in patients with HGSOC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we analyzed PARP1 expression at the protein level between HGSOC tumors and corresponding normal tissues in 3 different anatomic sites, namely, fallopian tube, omentum and ovary. We found that, for each of these sites, PARP1 protein levels are increased in tumor samples compared to samples from healthy tissues (Figure 1B). The most significant difference in PARP1 protein levels between tumor and normal samples was observed for omentum (p = 3.3e-05), followed by fallopian tube (p = 0.00021) and ovary (p = 0.028) (Figure 1B). Similar to the previous observation that PARP1 protein level differences between epithelial tumor compartment and stromal compartment is the most significant for omental metastases, PARP1 protein levels between normal and tumor tissues show the most significant difference in omentum. Collectively, these data may point that the contribution of PARP1, at least in part, to omental metastases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS100A4 and CLDN7, transcriptional targets of PARP1, have the highest copy numbers in serous ovarian cancer among all cancer types\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChoi et al. showed that PARP1 enhances lung adenocarcinoma metastasis through transcriptional upregulation of S100A4 and CLDN7, i.e. by mechanisms independently of its function in DNA repair (2016). Since we observed the most significant difference in PARP1 protein levels in omentum / omental metastases among other anatomical locations (others are primary tumor tissues, not metastases), between epithelial tumor and normal stromal compartments, and also between tumor and normal samples, we speculated that PARP1 may contribute, at least in part, to metastasis to omentum. We found that the transcriptional targets of PARP1, namely S100A4 and CLDN7, have the largest copy number gain percentages in serous ovarian cancer among other cancer types (Figure 2). This data indicates that these two proteins whose expression is upregulated by PARP1 may have more important functions in ovarian cancer compared to other cancer types. Supportingly, multiple studies reported the increased expression of these genes in ovarian cancer (Dahiya et al. 2011; Tassi et al. 2008; Kim et al. 2011; Soini and Talvensaari-Mattila 2006; Kleinberg et al. 2008; Link et al. 2019; Yan et al. 2016; Hiruta et al. 2020; Deo et al. 2021; Xu et al. 2020; Kikuchi et al. 2006; Maekandsmo et al. 2009). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePARP1 expression is increased in malignant and invasive stage in mouse ovarian surface epithelium cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze PARP1 expression levels at different stages of malignancy, we used transcriptome data from mouse ovarian surface epithelial (MOSE) cells (Creekmore et al. 2011). Here, we used data from mice, since similar data from patients is not currently available. These mouse cells undergo spontaneous transformation in cell culture with increasing passage numbers, and represent phenotypes from pre−neoplastic, non−malignant to malignant and invasive (Creekmore et al. 2011). Since we observed themost significant difference in PARP1 levels in omental metastases when comparing its levels between tumors and adjacent stroma, we wanted to see if PARP1 expression increases in parallel to increasing malignancy. We found that PARP1 mRNA levels are indeed higher in malignant and invasive stage (late cells, passage 136, 142, and 143) compared to both pre−neoplastic, non−malignant stage (early cells, passage 13, 14, and 15) (p = 0.003) and neoplastic, pre−invasive stage (intermediate cells, passage 63, 71, and 73) (p = 0.0036) (Figure 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial ovarian cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we analyzed if PARP1 mRNA levels differ between different histological types (histotypes) of epithelial ovarian cancer (EOC comprises of \u0026nbsp; different histotypes: clear cell, endometrium, mucinous, serous and also very rare malignant Brenner tumors) (Berkel and Cacan 2021). We found that PARP1 expression is lower in mucinous histotype compared to serous histotype in 5 out of 6 datasets in which data is available for these histotypes (p values: 0.048, 0.0024, 0.027, 0.0017 and 9.1e-05) (Figure 4). Between other histological types, there seems to be no significant change in PARP1 mRNA levels which is consistent between datasets (Figure 5). \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we showed that PARP1 expression is increased in epithelial tumor compartment compared to adjacent stromal compartment at 4 different anatomical sites (fallopian tube, omentum, ovary and STIC (serous tubal in situ carcinoma)) in patients with HGSOC. We observed the most significant difference in PARP1 protein levels in omental metastases compared to primary tumor tissues (fallopian tube, ovary and STIC (serous tubal in situ carcinoma)). These data show that epithelial tumor cells have higher PARP1 protein expression compared to stromal cells in the tumor microenvironment, independently of their anatomical location. This is of clinical importance, since PARP inhibitors including olaparib are currently being used in the treatment of patients with ovarian cancer in both the frontline setting as maintenance therapy and in the recurrent setting (Kristeleit et al. 2017; Moore et al. 2018; Pujade-Lauraine et al. 2017). Because the difference in PARP1 protein levels between tumor and stroma is the most significant in omental metastases, PARP inhibition may promote synthetic lethality more favorably in metastases compared to primary sites. This is because epithelial tumor compartment will be more preferentially targeted than stromal compartment in omental metastases by the inhibition of PARP. However, further research is needed to make stronger inferences.\u003c/p\u003e\n\u003cp\u003eSimilarly, we observed that PARP1 protein levels are higher in tumors compared to normal tissue in fallopian tube, omentum and ovary in patients with HGSOC, again with the highest difference in omentum / omental metastases. Therefore, we can state that the most significant increase in PARP1 protein levels in the course of cancer progression occurs in omental metastasis compared to primary tumor sites, pointing to the potential contribution of PARP1, at least in part, to advanced disease and metastasis. Supportingly, we found that two transcriptional targets of PARP1, namely S100A4 and CLDN7, which enhances metastasis in lung adenocarcinoma (Choi et al. 2016), have the highest copy numbers in serous ovarian cancer among all cancer types, highlighting potential ovarian cancer-specific roles of these proteins. Indeed, multiple studies reported the increased expression of these proteins in ovarian cancer and their involvement in disease progression (Dahiya et al. 2011; Tassi et al. 2008; Kim et al. 2011; Soini and Talvensaari-Mattila 2006; Kleinberg et al. 2008; Link et al. 2019; Yan et al. 2016; Hiruta et al. 2020; Deo et al. 2021; Xu et al. 2020; Kikuchi et al. 2006; Maekandsmo et al. 2009).\u003c/p\u003e\n\u003cp\u003eWe also found that PARP1 expression is increased in malignant and invasive mouse ovarian surface epithelial cells compared to those with pre−neoplastic, non−malignant phenotype or with neoplastic, pre−invasive phenotype\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e. This data is in line with a recent study showing that PARP1 overexpression promotes viability, migration, invasion, and tube formation in ovarian cancer cells (Chang et al. 2021). Wei et al. showed that hepatocyte growth factor (HGF) administration increases PARP1 levels and enhances cell invasion in a concentration- and time-dependent manner in ovarian cancer cells \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e(Wei et al. 2018). They also reported that the silencing of PARP1 significantly reduces the impact of HGF on invasiveness in ovarian cancer cells, pointing to the role of PARP1 in the development of an invasive phenotype (Wei et al. 2018). Same group also found that PARP1 may enhance angiogenesis in epithelial ovarian cancer by upregulating VEGF-A (Wei et al. 2016).\u003c/p\u003e\n\u003cp\u003eOur analysis also pointed out that mostly lower PARP1 mRNA levels are observed in mucinous histotype of epithelial ovarian cancer compared to serous histotype. This difference in PARP1 levels may contribute, at least to a certain extent, to the differences between these two histological types in terms of survival and response to chemotherapy and PARP inhibition (Overall, patients with mucinous histotype have a better prognosis compared to those with serous histotype in the early stage; however, patients with mucinous type have a worse prognosis at advanced stage compare to patients with serous histotype) (Peres et al. 2019; Monk et al. 2019; Simons et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCombined, we showed that PARP1 protein levels are higher in tumor samples compared to both adjacent stroma or normal ovaries, in patients with HGSOC, with the most significant difference in omental metastases, compared to primary tumor tissues. Supportingly, we found that certain transcriptional targets of PARP1 which are also associated with PARP1-mediated metastasis in other cancers have the highest copy number gain percentage in ovarian cancer among other cancers. This points to a potential mechanism of PARP1 in promoting malignancy in ovarian cancer; however, mechanistic studies are \u0026nbsp;needed to test these hypotheses. In parallel, we showed that malignant mouse ovarian surface epithelial cells have higher PARP1 expression cmpared to those with low malignancy. Furthermore, we observed differential levels of PARP1 in various histological types of epithelial ovarian cancer, possibly contributing to varying degrees of malignancy observed in these histotypes. This preliminary study highlights the need for a complete understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in ovarian cancer. For instance, how PARP1 expression is upregulated in ovarian tumors compared to normal ovaries or surrounding stromal cells, or the mechanisms by which PARP1 promotes malignancy in ovarian cancer should be studied in more detail.This is of high clinical importance since PARP inhibitors are currently being used in the treatment of ovarian cancer, and mechanisms leading to PARPi resistance still remain to be identified.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCB is funded by the 2211-E programme of The Scientific and Technological Research Council of Turkey.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo new data was generated during this study. R code written to analyze datasets was given as a supplementary file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Jemal A (2018) Cancer statistics, 2018. \u003cem\u003eCA: a cancer journal for clinicians\u003c/em\u003e, \u003cem\u003e68\u003c/em\u003e(1), 7\u0026ndash;30. https://doi.org/10.3322/caac.21442\u003c/li\u003e\n\u003cli\u003eTorre LA, Trabert B, DeSantis CE et al. (2018) Ovarian cancer statistics, 2018. \u003cem\u003eCA: a cancer journal for clinicians\u003c/em\u003e, \u003cem\u003e68\u003c/em\u003e(4), 284\u0026ndash;296. https://doi.org/10.3322/caac.21456\u003c/li\u003e\n\u003cli\u003eEckert MA, Coscia F, Chryplewicz A, et al. 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(2017) Relatively Poor Survival of Mucinous Ovarian Carcinoma in Advanced Stage: A Systematic Review and Meta-analysis. \u003cem\u003eInternational journal of gynecological cancer : official journal of the International Gynecological Cancer Society\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e(4), 651\u0026ndash;658. https://doi.org/10.1097/IGC.0000000000000932\u003c/li\u003e\n\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":"","lastPublishedDoi":"10.21203/rs.3.rs-1693902/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1693902/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"High-grade serous ovarian carcinoma (HGSOC) is responsible for the majority of ovarian cancer-associated deaths. PARP (Poly-(ADP-ribose) polymerase) inhibitors (PARPi) (olaparib, rucaparib and niraparib) have been approved in recent years to be used in both the frontline setting as maintenance therapy and in the recurrent setting for patients with ovarian cancer (OC). Previous studies have reported increased expression of PARP1, a sensor for DNA damage, in OC; however, its compartment (epithelial tumor vs stroma)-, anatomical site (fallopian tube, omentum, ovary and serous tubal in situ carcinoma)- and histotype-specific expression have not been studied. Here, we showed that PARP1 protein levels are higher in epithelial tumors compared to adjacent stroma at four different anatomical locations in HGSOC, with the most significant difference in PARP1 levels in omental metastases. Furthermore, we found that PARP1 protein levels are higher in tumors compared to normal tissue in fallopian tube, omentum and ovary in patients with HGSOC, again with the most significant difference in omentum. We showed that S100A4 and CLDN7, transcriptional targets of PARP1, have the highest copy numbers in serous OC among all cancer types, showing the potential OC-specific roles of these two proteins. We also showed that PARP1 expression is increased in malignant and invasive stage in mouse ovarian surface epithelium cells, and that PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial OC. These findings indicate that a better understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in OC is needed, since PARP inhibitors which work by preventing DNA repair in cancer cells are currently being used in the treatment of patients with OC.","manuscriptTitle":"Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-02 14:43:37","doi":"10.21203/rs.3.rs-1693902/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":"June 2nd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-06-02T14:43:37+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-02 14:43:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1693902","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1693902","identity":"rs-1693902","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}