Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype

Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype Ako Yokoi, Toshihide Matsumoto, Yasuko Oguri, Yoshinori Hasegawa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.22626/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Jul, 2020 Read the published version in Cell Communication and Signaling → Version 2 posted 9 You are reading this latest preprint version Show more versions Abstract Background We previously demonstrated that ovarian high grade serous carcinomas (OHGSeCa) and ovarian clear cell carcinomas (OCCCa) with an HNF-1β+/p53+/ARID1A+ immunophenotype were associated with the worst unfavorable prognosis. To clarify the molecular mechanisms underlying this finding, we focused on alterations in the p53 signaling pathway in these tumors. Methods Changes in cell phenotype and function following knockdown of wild-type p53 (p53-KD) were assessed using OCCCa cells expressing endogenous HNF-1β and ARID1A. The prognostic significance of molecules that were deregulated following p53-KD was also examined using 129 OCCCa/OHGSeCa cases. Results p53-KD cells had increased expression of Snail, phospho-Akt (pAkt), and pGSK3β, and decreased E-cadherin expression, leading to epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) features. The cells also exhibited acceleration of cell motility and inhibition of cell proliferation and apoptosis. Next generation sequencing revealed that fibronectin (FN) expression was significantly increased in the p53 KD-cells, in line with our observation that wild-type p53 (but not mutant p53) repressed FN1 promoter activity. In addition, treatment of OCCCa cells with FN significantly increased cell migration capacity and decreased cell proliferation rate, independent of induction of EMT features. In clinical samples, FN/p53 scores were significantly higher in OCCCa/OHGSeCa with the HNF-1β+/p53+/ARID1A+ immunophenotype when compared to others. Moreover, high FN/high p53 expression was associated with the worst overall survival and progression-free survival in OCCCa/OHGSeCa patients. Conclusion These findings suggest that upregulation of FN following loss of p53 function may impact the biological behavior of OCCCa/OHGSeCa, particularly in tumors with an HNF-1β+/p53+/ARID1A+ immunophenotype, through alterations in cell mobility and cell proliferation. The accompanying induction of EMT/CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics. Cancer Biology Cell Communication and Signaling ovarian carcinoma p53 HNF-1 ARID1A fibronectin prognosis immunophenotype cell proliferation cell mobility apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Ovarian epithelial carcinomas (OECa) are among the most aggressive tumors and the leading cause of mortality among all types of malignancies in the female reproductive system [1]. Since the ovaries have a relatively inaccessible location and ovarian carcinoma patients very often lack symptoms in the early neoplastic stage, more than 75% of the patients are diagnosed with advanced stage disease that is characterized by metastasis to the peritoneal cavity [2]. In addition, approximately 80% of advanced stage patients have residual disease after surgery and receive front-line platinum-based combination chemotherapy; these individuals have a median progression-free survival (PFS) of 18 months [3]. p53 is widely acknowledged as the most frequently mutated gene in human malignancy and its mutational status is a prognostic marker in several tumor types [4]. Activated wild-type p53 (p53wt) acts as a checkpoint control for recognizing damaged DNA, allowing DNA repair and delayed entrance into the DNA replication phase of the cell cycle; together with observations that the incidence of tumorigenesis increases in p53 null or mutant tissues, these data confirm that p53 is a bona fide tumor suppressor [5]. Mutations in the TP53 gene are found in more than 50% of human malignancies and its inactivation can occur at various stages depending on the tissue that gives rise to the tumor. Therefore, loss of p53 function can promote neoplastic transformation as well as progression of established tumors to a more aggressive disease stage [6,7]. In OECa, and particularly in ovarian high-grade serous carcinomas (OHGSeCa), mutant p53 (p53mt) missense mutations are frequently found in the hotspot codon R175, R248, and R273 ( http://www-p53.iarc.fr/ ) that are critical contact residues in the p53 DNA-binding domain. The mutations occur early during tumorigenesis, most likely in precursor lesions of OECa, highlighting the importance of p53mt as a driver of the malignancy [8-11]. We previously developed an effective immunoprofiling classification system for OECa using only 4 immunohistochemical markers (HNF-1b, p53, ARID1A, and WT1) [12]. Using this system, we demonstrated that tumors with an HNF-1b+/p53+/ARID1A+ immunophenotype including OHGSeCa and ovarian clear cell carcinomas (OCCCa) were associated with the most unfavorable prognosis. In this study, we hypothesized that alterations in the p53 signaling pathway may play a key role in determining phenotypic characteristics in OECa with the HNF-1b+/p53+/ARID1A+ immunophenotype. To test this, we set out to first examine the effects of knocking down p53wt (p53-KD) in OCCCa cells expressing endogenous HNF-1b and ARID1A. Next, we applied a next generation sequencing (NGS) assay to identify the molecules associated with loss of p53 function. Finally, we examined associations between molecules that were differentially expressed following p53-KD, tumor phenotypic characteristics and prognostic significance in OHGSeCa and OCCCa. Methods Plasmids and cell lines The p53-specific short hairpin RNA (shRNA) oligonucleotides were designed as described previously [13]. Single-stranded p53 oligonucleotides were annealed and then cloned into BamH 1- EcoR V sites of RNAi-Ready pSIREN-RetroQ vector (Takara, Shiga, Japan), according to the manufacturer’s instructions. The p53mt (R248Q) was generated by PCR-based methods using a pCMV-p53wt construct. The human Fibronectin 1 promoter (UCSC genome browser, https://genome.ucsc.edu/) between -2028 and -23 (where +1 represents the transcription start site) was also generated by PCR and was cloned into the pGL3B vector (Promega, Madison, WT, USA). The primer sequences for the PCR reaction used in this study are listed in Table 1. pCMV-p53wt, pGL3B-(-1109/+36) Snail luc, pGL3B-(-899/+47) HNF-1b luc, and pGL3B-(-140/+216) HNF-1b luc were also used as described previously [14-16]. Four OCCCa cell lines, OVISE, ES2, OVTOKO, and TOV-21G were used as described previously [13,16,17], and two OHGSeCa cell lines, OVSAHO and OVCAR-3, were obtained from the National Institute of Biomedical Innovation (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA), respectively. p53 shRNA knockdown cells were established using OVISE cells, which have a wild-type p53 gene and abundant expression of endogenous HNF-1b and ARID1A (Supplementary Figure S1), as described previously [13,17]. In addition, spindle-shaped cells were defined as those that showed narrow and elongated phenotypes, along with weak or absent adhesions between cells, as described previously [17]. Antibodies and reagents Anti-p53, anti-p21 waf1 , anti-cyclin D1, and anti-bcl2 antibodies were purchased from Dako (Copenhagen, Denmark). Anti-HNF-1b, anti-GSK-3b, anti-Rb, anti-p27 kip1 , anti-XIAP, anti-bax, and anti-integrin b1 antibodies were obtained from BD Biosciences (San Jose, CA, USA). Anti-ARID1A, anti-cyclin B1, and anti-MDM2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail, anti-Akt, anti-phospho(p)Akt Serine473, anti-pGSK-3b Serine9, anti-pRb Serine807/811, anti-cleaved caspase-3, and anti-integrin b3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-fibronectin (FN), anti-E-cadherin, and anti-b-actin antibodies were obtained from Abcam (Cambridge, MA, USA), Takara (Shiga, Japan), and Sigma-Aldrich Chemicals (St. Louis, MO, USA), respectively. Anti-cyclin A2 and anti-integrin b2 antibodies were from Novocastra (Newcastle, UK) and Merck KGaA (Darmstadt, Germany), respectively. FN (catalog number #F2006) and cisplatin (CDDP: #479306) were purchased from Sigma-Aldrich Chemicals. Transfection Transfection was carried out using LipofectAMINE PLUS (Invitrogen, Carlsbad, CA, USA) as described previously [14-16]. Luciferase activity was assayed as described previously [14-16]. Reverse transcription (RT)-PCR cDNA was synthesized from 2 mg of total RNA. Amplification by RT-PCR was carried out in the exponential phase to allow comparison among cDNA synthesized from identical reactions using specific primers (Table 1). Primers for the HNF-1 b , Snail , and GAPDH genes were also applied, as described previously [14-16]. The signal intensity was analyzed by ImageJ software version 1.41 (NIH, Bethesda, MD, USA). For quantitative analysis, real-time RT-PCR was also conducted using a Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Fluorescent signals were detected using the ABI 7500 Real-time PCR System SDS Software (Applied Biosystems). Western blot assay Total cellular proteins were isolated using RIPA buffer [20 mM Tris-HCl (pH7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate]. Aliquots of the proteins were resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies, coupled with the ECL detection system (Amersham Pharmacia Biotechnology, Tokyo, Japan). Flow cytometry and Aldefluor assay Cells were fixed using 70% alcohol and stained with propidium iodide (Sigma) for cell cycle analysis. Aldehyde dehydrogenase 1 (ALDH1) enzyme activity in viable cells was determined using a fluorogenic dye-based Aldefluor assay (Stem Cell Technologies, Grenoble, France) according to the manufacturer’s instructions. The prepared cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences) and CellQuest Pro software version 3.3 (BD Biosciences). Cell Counting Kit-8 assay The quantitation of viable cell number in proliferation after CDDP treatment was carried out using a Cell Counting Kit-8 (CCK-8; Dojindo Lab, Kumamoto, Japan), according to the manufacturer’s instructions. Wound healing assay Cells were seeded into 24-well tissue culture plates, and grown to reach almost total confluence. After a cell monolayer formed, a wound was scratched with a sterile 200-ml tip. The area of the wound was analyzed by ImageJ software version 1.41 (NIH). Cell migration parameters were calculated in pixels as wound closure. Migration assay Cell migration was determined using 24-well Transwell chambers with an 8-mm pore size (Corning, NY, USA). The lower chamber was filled with medium containing 10% serum. Cell were suspended in serum-free medium with or without FN and transferred into the upper chamber. After 24 h, the number of cells stained by hematoxylin-eosin (HE) on the bottom surface of the polycarbonate membranes was counted visually using a light microscope. Apoptotic index Apoptotic cells were identified in HE-stained sections, according to the criteria of Kerr et al. [18]. A total 10 fields were randomly selected, and the number of apoptotic cells was calculated by counting the mean number of apoptotic figures per high power field (HPF). NGS assay Total RNAs were extracted from OV-shp53 and mock cells using the NucleoSpin RNA system (Takara). The concentration and quality of the RNA was verified with the Quantus Fluorometer (Promega) and Agilent 2100 Bioanalyzer, respectively. All the samples showed RIN values over 9. Total RNA (500 ng) was used for RNA library preparation, according to the instructions of the Quant Seq 3’ mRNA-seq library preparation kit FWD from Illumina (Lexogen, Vienna, Austria). The libraries were PCR-amplified for 12 cycles. Sequencing of the libraries (via single-end 75-bp reads) was conducted on the Illumina NextSeq500 system. All data analyses were conducted using Strand NGS (v3.2, Agilent Technologies). The adapter sequences were removed from the raw reads, and base trimming was performed from the 3’ end of each read to remove bases with quality below Q10 up to a minimum length of 25 bp. Each read was mapped to the reference human genome hg38 with default settings. Expression patterns of transcripts were compared after normalization of DESeq [19] using default settings. TCGA data analysis The Cancer Genome Atlas (TCGA) OHGSeCa annotated TP53 gene alteration and mRNA expression data (RNA Seq V2 PSEM) for HNF-1b and ARID1A were extracted from cBioportal for Cancer Genomics ( http://www.cbioportal.org/ ) for 398 OHGSeCa cases. Clinical cases A total of 199 cases of OECa, surgically resected at Kitasato University Hospital between 2006 and 2017, were selected from our patient records according to the criteria of the 2014 World Health Organization classification [20]. All patients underwent oophorectomy with or without hysterectomy. None of the patients had received chemotherapy or any other preoperative treatment, while most patients had received Paclitaxel/Carboplatin-based chemotherapy after surgical treatment. Of these, 99 cases including 41 OHGSeCa and 58 OCCCa showed complete resection of the tumors, while 28 cases including 17 OHGSeCa and 11 OCCCa had residual tumors after debulking surgery. Evaluation of relapse and disease progression was conducted on the basis of radiologic image findings. The tumor cases investigated were comprised of 58 OHGSeCa, 9 ovarian low grade serous carcinoma, 29 ovarian endometroid carcinomas, 71 OCCCa, and 30 ovarian mucinous carcinomas. All tissues were routinely fixed in 10% formalin and processed for embedding in paraffin wax. Approval for this study was given by the Ethics Committee of the Kitasato University School of Medicine (B16-10). Immunohistochemistry (IHC) IHC was performed using a combination of the microwave-oven heating and polymer immunocomplex (Envision, Dako) methods, as described previously [14-16]. For evaluation of IHC findings, scoring of nuclear/cytoplasmic immunoreactivity was performed, on the basis of the percentage of immunopositive cells and the immunointensity, with multiplication of values of the two parameters, as described previously [14-16]. In addition, FN/p53 score was generated by multiplication of the values of the two scores. To evaluate the prognostic significance of FN and p53 expression, the scores were divided into two categories (high and low) with the mean values as the cut-off in each category (Table 2). With regard to p53, cases that were completely negative for p53 immunoreactivity were categorized into the high p53 score group (score = 12), since combining 2 IHC labeling patterns associated with p53 mutations (0% and 60-100% positive cells) correctly identified a mutation in 94% of cases [ 21]. Statistics Comparative data were analyzed using the Mann-Whitney U -test. Overall survival (OS) was calculated as the time between onset and death or the date of the last follow-up evaluation. PFS was also examined from the onset of treatment until relapse, disease progression, or last follow-up evaluation. OS and PFS were estimated using the Kaplan-Meier methods, and the statistical comparisons were made using the log rank test. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. The cut-off for statistical significance was set as p < 0.05. Results Loss of p53 function leads to induction of EMT features To examine the phenotypic characteristics of OECa cells with the HNF-1b+/p53+/ARID1A+ immunophenotype, we first established two independent cell lines in which p53 expression was blocked by a p53-specific shRNA (OV-shp53#2 and #8) using OVISE (OV) cells. The OV-p53-KD cells showed increased expression of both HNF-1b and ARID1A, in contrast to decreased expression of MDM2 and p21 waf1 , which are p53 target genes (Figure 1A). HNF-1 b mRNA expression was also increased in the OV-p53-KD cells as compared to the mock cells (Figure 1B), in line with the observation of dose-dependent repression of HNF-1 b promoter activity following transfection of p53wt. In contrast, the repressive effects were not evident when p53mt was transfected (Figure 1C). The OV-p53-KD cells also demonstrated a significant switch towards a fibroblastic morphology (Figure 2A), along with increased expression of Snail, pAkt, and pGSK-3b, and decreased E-cadherin expression (Figure 2B). Although Snail promoter activity was inhibited by p53wt, but not p53mt (Figure 2C), changes in mRNA expression were relatively minor in OV-p53-KD cells as compared to the mock cells (Figure 2D). These findings suggest that loss of p53 function contributes to increased expression of HNF-1b and ARID1A, leading to induction of epithelial-mesenchymal transition (EMT) features, probably through post-translational regulation of Snail expression. Loss of p53 function is associated with CSC features and acceleration of cell mobility To examine whether p53-KD affects cell proliferation, the two independent OV-p53-KD cell lines were seeded at low density. OV-p53-KD cells tended to proliferate more slowly, particularly in the exponential growth phase, along with an increased proportion of cells in G2/M phase of the cell cycle (Figure 3A). To further examine alterations in expression of several cell cycle-related molecules during cell growth, the OV-p53-KD cells were rendered quiescent by serum starvation and were subsequently stimulated with serum. At 6, 12, and 24 h after release into the cell cycle, p27 kip1 expression was substantially increased in OV-p53-KD cells relative to the mock cells, in contrast to the progressive reduction of cyclin B1 expression in the former (Figure 3B). Next, we examined the association between loss of p53 function and apoptotic features in response to cytotoxic effects. Treatment of OV-p53-KD cells with CDDP showed decreased apoptotic cells as compared to the mock (Figure 3C), in line with the results of increased cell viability during CDDP treatment (Figure 3D). The expression of cleaved caspase-3, as well as bax and bcl-2, were also apparently decreased in the OV-p53-KD cells as compared to mock cells, in contrast to increased pAkt, but not XIAP, expression (Figure 3E). Since EMT promotes stem cell properties and further generates cells with cancer stem cell (CSC)-like features [22], we examined the association between loss of p53 function and CSC properties. As shown in Figure 4A, there was a significant increase in the ALDH high population, which includes a high percentage of CSC-like cells, in the p53-KD cells compared to the mock cells. To further examine whether loss of p53 function contributes to cell motility, we carried out scratch and migration assays. The OV-p53-KD cells refilled wounded empty spaces more rapidly (Figure 4B), in line with the significantly increased migration rates as compared to the mock cells (Figure 4C). These findings suggest that loss of p53 function also engenders CSC features and accelerates cell motility in OVISE cells; these changes are accompanied by inhibition of cell proliferation and susceptibility to apoptosis. Upregulation of FN expression by loss of p53 function To identify genes that are differentially expressed following p53-KD, NGS assays were carried out using total RNAs extracted from OV-p53-KD cells. A total of 12051 and 13094 genes in OV-shp53#2 and OV-shp53#8 cells were dysregulated, respectively. Of these, 57 and 83 genes were upregulated or downregulated over 5-fold, respectively, in the p53-KD cells as compared to the mock cells. As shown Figure 5A, hierarchical clustering revealed that the genes could be readily categorized into eleven groups, and we focused on the FN1 gene in group IV that was overexpressed by 17-fold. FN1 mRNA and protein expression were apparently increased in OV-p53-KD cells, along with increased expression of integrin b1, b3, and b3 (Figure 5B, C). Moreover, FN1 promoter activity was repressed by transfection of p53wt, but not p53mt (Figure 5D). Since FN is an EMT-related marker [23,24], we asked whether there was an association of FN with either EMT or cell motility. OVISE cells treated with FN did not show any changes in cell morphology or expression of E-cadherin, Snail, Akt and GSK-3b (Figure 6A); the expression of apoptosis-related molecules was also unchanged (Figure 6B). In contrast, both scratch and migration assays revealed that FN treatment resulted in a significant increase in migration capacity (Figure 6C,D), along with a decrease in proliferation at later stages (Figure 6E). These findings suggest that FN is an important determinant of cellular function in p53-KD cells due to its effects on cell mobility and proliferation, rather than via modulation of EMT or apoptosis. Prognostic significances of FN and p53 expression in OCCCa/OHGSeCa Representative IHC findings for FN and p53 in OCCCa and OHGSeCa are illustrated in Figure 7A, demonstrating cytoplasmic immunostaining for FN and nuclear staining for p53. FN score was significantly higher in OCCCa as compared to that of OHGSeCa, in contrast to a significantly higher p53 score in the latter (Supplementary Figure S2A). Previously, we used hierarchical clustering analysis to identify seven immunopurified groups (IPGs) in OECa including OCCCa, OHGSeCa, OLGSeCa, OEmCa, and OMuCa [12]. Here, we observed that average FN/p53 scores were significantly higher in IPG VII, which includes OCCCa/OHGSeCa with the HNF-1b+/p53+/ARID1A immunophenotype, and lower in the IPGs IV, V, and VI (Figure 7B). Similar findings were also observed in p53, but not FN, scores among IPGs including the five OECa histotypes (Supplementary Figure S2B). The FN scores were also significantly associated with tumor histotype, tumor size, and lymph node metastasis in OCCCa; p53 score was also significantly correlated with clinical stage, histotype, tumor size, and distant metastasis in OHGSeCa (Table 2). The Kaplan-Meier curves showed that patients with high FN and p53 scores had poorer OS and PFS when compared to patients with low scores in the OCCCa/OHGSeCa category (Figure 8A, B), although such associations were not observed in p53 scores in OHGSeCa (Supplementary Figure S3). Patients with a combination of high FN and high p53 scores also had the worst OS and PFS in OCCCa/OHGSeCa, whereas patients with low values of both scores had the best prognosis (Figure 8C). Univariate Cox progression hazards regression revealed that FN, p53, age, tumor histotype, FIGO stage, lymph node metastasis, distant metastasis, and residual tumors after surgical treatment were significant prognostic factors for OS or PFS in OCCCa/OHGSeCa. In addition, multivariate Cox regression analysis showed that FN, FIGO stage, and distant metastasis were significant and independent prognostic factors for OS or PFS (Table 3). These findings suggest that a combined IHC analysis of FN and p53 expression is useful for prognostic prediction of OCCCa/OHGSeCa. Discussion Although extensive studies on the gain-of-function (GOF) effects of p53mt have been conducted using in vitro cell culture systems, several in vivo models have indicated that the primary effect of p53 mutation is the loss of p53wt function, with little or no GOF effect on tumorigenesis. Thus, the GOF of a particularly p53mt is likely to be determined by tissue- and tumor type-specific factors. For example, MMTV- HrasTP53 R172H/R172H and MMTV- Hras/TP53 -/- mice were very similar with regard to age of salivary tumor onset, tumor growth rate, tumor histopathological features, and response to a DNA-damaging agent [25]. This was also the case in a K-ras -driven lung cancer model, as well as in the context of WAP-Cre -induced expression of the p53R270H mutant in p53-null-mouse mammary glands, and in p53R172H homozygous knock-in mouse models [26-28]. In addition, the GOF activity of any particular p53mt is largely dependent on multiple signals required for its post-translational stabilization: the presence of such signals is likely to vary among both normal and tumor cells [29]. Based on the above evidence, we examined alterations in the p53 signaling pathway using cells following shRNA-mediated knockdown of p53wt. Here, we provide clear evidence that p53 loss leads to upregulation of HNF-1b, as well as ARID1A, at both mRNA and protein levels in OCCCa cells. Moreover, transfection of p53wt, but not p53mt, represses HNF-1 b promoter activity, suggesting that alterations in the p53 gene may play an important role in development of OCCCa that have the HNF-1b+/p53+ /ARID1A+ immunophenotype. However, analysis of the TCGA database revealed that expression of HNF1 b and ARID1A mRNA was not correlated with p53 status in OHGSeCa. Given the evidence that HNF-1b is a sensitive and specific marker for OCCCa and is not expressed in OHGSeCa with clear cell changes [30], it appears that some cell type-specific factors may also be required for establishment of OHGSeCa with the immunophenotypic features we specify above. We also found that p53-KD cells have dramatically altered cell morphology and are more fibroblast-like in appearance; these changes are accompanied by increased expression of E-cadherin-repressor Snail, as well as pAkt and pGSK-3b, and decreased E-cadherin expression. Although Snail promoter activity was specifically repressed by p53wt, we did not observe differences in Snail mRNA levels between p53-KD and the mock-transfected cells. In general, Snail expression is decreased through GSK-3b-mediated phosphorylation/degradation [31], while GSK-3b activity is inhibited following activation of Akt [32]. Given the mutual antagonism between the p53 and Akt networks [33-35], it is suggested that loss of p53 function leads to post-translational upregulation of Snail through activation of the Akt/GSK-3b axis, which in turn leads to induction of EMT. Interestingly, p53mt can directly bind and trans-repress the promoter of miR-130b , a microRNA that specifically downregulates ZEB1, leading to the upregulation of BMI-1 and Snail [36]. In addition, p53wt can induce MDM2 mediated degradation of Snail [37]. Here, we found that p53-KD cells had a reduced proliferative rate and enhanced migration capability, along with enhanced G2/M arrest and increased p27 kip1 expression. Moreover, cyclin B1 expression also decreased progressively after serum stimulation in p53-KD cells. Our findings are consistent with those of previous reports. First, p27 kip1 is important for the initial activation of G2/M checkpoint in response to low-dose ionizing radiation [38], in line with evidence that Cdc2, which is essential for entry into mitosis, physically interacts with, and is inhibited by p27 kip1 [39,40]. In addition, binding of Cdc2 to cyclin B1 is required for its activity and repression of cyclin B1 contributes to blocking entry into mitosis [C]. Second, hematopoietic cells expressing p53wt arrest in both G1 and G2 phase of the cell cycle, while p53-null cells or cells overexpressing p53mt exhibited only G2 arrest [42]. Third, reduced levels of p53 correlate with increased G2/M phase arrest in response to paclitaxel treatment in normal human fibroblast depleted of functional p53 [43]. Finally, migratory cells have a lower proliferation rate in comparison with cells in the tumor core, indicating an inverse correlation between cell proliferation and mobility [44-46]. Our findings also revealed that susceptibility to apoptosis in response to CDDP treatment was significantly inhibited in p53-KD cells. This may be explained by the prolonged high levels of pAkt in response to loss of p53 function, because there is an ‘all-or-none’ switching behavior between a pro-survival cellular state (low p53 and high Akt levels) and a pro-apoptotic state (high p53 and low Akt levels) [33], as well as decreased expression of the pro-apoptotic protein, bax [47]. An important finding in this study was that FN1 mRNA and protein expression were significantly increased in p53-KD cells, while FN1 promoter activity was repressed by p53wt, but not p53mt, in line with other studies [48,49]. Moreover, treatment of OCCCa cells with FN resulted in an enhanced migration capability and a reduced proliferation rate. In contrast, the effects of FN on cell morphology, expression of EMT-related molecules, and susceptibility to apoptosis were minimal. Taken together with our results that p53-KD increases the expression of the FN receptors integrins b1, b2, and b3 [50], we conclude that FN upregulation due to loss of p53 function is closely associated with enhancement of cell mobility, but not induction of EMT and apoptotic features. However, we could not demonstrate a direct correlation between FN and p53 scores in the OCCCa/OHGSeCa category, indicating that a p53-independent pathway must regulate FN expression in the tumors, particularly in OCCCa. In fact, FN expression is upregulated through the PI3K/Akt pathway in tamoxifen-resistant breast cancer cells [51]. Finally, patients with a combination of high FN and high p53 IHC scores had significantly worse OS and PFS than did patients with low values for both scores in OCCCa/OHGSeCa. Both FN and p53 scores were also significantly associated with several unfavorable clinicopathological factors in the tumors. Moreover, multivariate Cox regression analysis also showed that FN, but not p53, was a significant and independent unfavorable prognostic factor for OS and PFS. Furthermore, there was a positive association between high FN score, enlarged tumor size and nodal metastasis, suggesting that combined IHC analysis for FN and p53 expression may have great utility in OCCCa/OHGSeCa prediction and prognosis. Conclusion Upregulation of FN following loss of p53 function may influence the malignant properties of OCCCa/OHGSeCa, particularly in those tumors with an HNF-1b+/p53+/ARID1A+ immunophenotype. The accompanying induction of EMT/CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics (Figure 9). List Of Abbreviations OECa: ovarian epithelial carcinomas; OHGSeCa: ovarian high grade serous carcinoma; OCCCa: ovarian clear cell carcinoma; KD: knockdown; p53wt: wild-type p53; p53mt: mutant type p53; IHC; immunohistochemistry; EMT: epithelial-mesenchymal transition; FN: fibronectin; OS: overall survival; PFS: progression-free survival; IPG; immunoprofile group Declarations Ethics approval and consent to participate Approval for this study was given by the Ethics Committee of the Kitasato University School of Medicine (B16-10). Consent for publication Not applicable Availability of data and materials Data and materials will be shared. Competing interests The authors declare that they have no competing interest. Funding This study was supported by a grant from JSPS KAKENHI Grant Number 17K08703. Authors’ contributions AY, TM, and MS carried out the majority of the experiments, analyzed the data, and wrote the manuscript. They were helped by YO, YH, MT, and MN. All authors reviewed and approved the final manuscript. References Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin. 2007;57:43-63. Wang Y, Helland A, Holm R, Skomedal H, Abeler VM, Danielsen HE, Trope CG, Borresen-Dale A-L, Kristensen GB. TP53 mutations in early-stage ovarian carcinoma, relation to long-term survival. Br J Cancer. 2004;90:678-85. Salani R, Backes FJ, Fung MF, Holschneider CH, Parker LP, Bristow RE, Goff BA. 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TGF-b-mediated LEFTY/Akt/GSK-3b/Snail axis modulates epithelial-mesenchymal transition and cancer stem cell properties in ovarian clear cell carcinomas. Mol Carcinog. 2018;57:957-67. Kerr JF, Winterford CM, Harmon BV. Apoptosis: its significance in cancer and cancer therapy. Cancer. 1994;73:2013-26. Aders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11:R106. Longacre TA, Wells M, Bell DA, Malpica A, Prat J, Ronnet BM. Tumours of the tumours of the ovary. In: Kurman RJ, Carcangiu ML, Herrington CS, Young RH, (ed). WHO Classification of Tumours of Female Reproductive Organs. Lyon: France; 2014. P11-86. Yemelyanova A, Vang R, Kshirsagar M, Lu D, Marks MA, Shih leM, Kurman RJ. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for TP53 mutations in ovarian carcinomas: an immunohistochemical and nucleotide sequencing analysis. Mod Pathol. 2011;24:1248-53. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133;704-715. Park J, Schwarzbauer JE. Mammary epithelial cell interactions with fibronectin stimulate epithelial-mesenchymal transition. Oncogene. 2014;33:1649-57. Li C-L, Yang D, Cao X, wang F, Hong D-Y, Wang J, Shen X-C, Chen Y. Fibronectin induces epithelial-mesenchymal transition in human breast cancer MCF-7 cells via activation of calpain. Oncol Lett. 2017;13:3889-95. Jiang D, Dumur CI, Massey HD, Ramakrishan V, Subler MA, Windle JJ. Comparison of effect of p53 null and gain-of-function mutations on salivary tumors in MMTV-Hras transgenic mice. PLoS One. 2015;10:e0118029. Jackson EL, Olive KP, Tuveson DA, Bronson R, Crowley D, Brown M, Jacks T. The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res. 2005;65:10280-8. Wijnhoven SW, Zwart E, Speksnijder EN, Beems RB, Olive KP, Tuveson DA, Jonkers J, Schaap MM, van den Berg J, Jacks T, van Steeg H, de Vries A. Mice expressing a mammary gland-specific R270H mutation in the p53 tumor suppressor gene mimic human breast cancer development. Cancer Res. 2005;65:8166-73. Liu G, McDonnell TJ, Montes de Oca Luna R, Kapoor M, Mims B, El-Naggar AK, Lozano G. High metastatic potential in mice inheriting a targeted p53 missense mutation. Proc Natl Acad Sci USA. 2000;97:4174-9. Kim MP, Zhang Y, Lozano G. mutant p53: multiple mechanisms define biologic activity in cancer. Front Oncol 2015;5:249. DeLair D, Han G, Irving JA, Leung S, Ewanowich CA, Longacre TA, Gilks CB, Soslow RA. HNF-1b in ovarian carcinomas with serous and clear cell change. Int J Gynecol Pathol. 2013;32:541-6. Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M, Hung MC. Dual regulation of Snail by GSK-3b-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol. 2004;6:931-40. Lin JX, Xie XS, Weng XF, Qiu SL, Xie JW, Wang JB, Lu J, Chen QY, Cao LL, Lin M, Tu RH, Li P, Huang CM, Zheng CH. Overexpression of IC53d promotes the proliferation of gastric cancer cells by activating the AKT/GSK-3b/cyclin D1 signaling pathway. Oncol Rep. 2019;41:2739-52. Wee KB, Aguda BD. Akt versus p53 in a network of oncogenes and tumor suppressor genes regulating cell survival and death. Biophys J. 2006;91:857-65. Wee KB, Surana U, Aguda BD. Oscillations of the p53-Akt network: implications on cell survival and death. PLoS ONE. 2009;4:e4407. Gottlieb TM, Leal JFM, Seger R, Taya Y, Oren M. Cross-talk between Akt, p53 and Mdm2: possible implications for the regulation of apoptosis. Oncogene. 2002;21:1299-303. Dong P, Karaayvaz M, Jia N, Kaneuchi M, Hamada J, Watari H, Sudo S, Ju J, Sakuragi N. Mutant p53 gain-of-function induces epithelial-mesenchymal transition through modulation of the miR-130b-ZEB1 axis. Oncogene. 2013;32:3286-95. Lim S-O, Kim H, Jung G. p53 inhibits tumor cell invasion via the degradation of snail protein in hepatocellular carcinoma. FEBS Lett. 2010;584:2231-6. Payne SR, Zhang S, Tsuchiya K, Moser R, Gurley KE, Longton G, DeBoer J, Kemp CJ. p27 kip1 deficiency impairs G 2 /M arrest in response to DNA damage leading to an increase in genetic instability. Mol Cell Biol. 2008;28:258-68. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990;344:503-8. Nakayama K, Nagahama H, Minamishima A, Miyake S, Ishida N, Hatakeyama S, Kitagawa M, Iemura S, Natsume T, Nakayama KI. Skp2-mediated degradation of p27 regulates progression into mitosis. Dev Cell. 2004;6:661-72. Taylor WR, Stark GR. Regulation of the G2/M transition by p53. Oncogene. 2001;20:1803-15. Kastan MB, Onvekwere O, Sidransky D, Vogelstein B, Craig RW. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991;51:6304-11. Wahl AF, Donaldson KL, Faircnild C, Lee FYF, Foster SA, Demers GW, Galloway DA. Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis. Nat Med. 1996;2:72-9. Giese A, Loo MA, Tran N, Haskett D, Coons SW, Berens ME. Dichotomy of astrocytoma migration and proliferation. Int J Cancer. 1996;67:275-82. Giese A, Bjerkvig R, Berens ME, Westphal M. Cost of migration: invasion of malignant glioma and implications for treatment. J Clin Oncol. 2003;21:1624-36. Merzak A, McCrea S, Koocheckpour S, Pilkington GJ. Control of human glioma cell growth, migration and invasion in vitro by transforming growth factor beta 1. Br J Cancer. 1994;70:199-203. Pawloaski J, Kraft AS. Bax-induced apoptotic cell death. Proc Natl Acad Sci USA. 2000;97:529-31. Iotsova V, Stehelin D. Down-regulation of fibronectin gene expression by the p53 tumor suppressor protein. Cell Growth Differ. 1996;7:629-34. You D, Jung SP, Jeong Y, Bae SY, Kim S. Wild-type p53 controls the level of fibronectin expression in breast cancer cells. Oncol Rep. 2017;38:2551-7. Wang JP, Hielscher A. Fibronectin: how its aberrant expression in tumors may improve therapeutic targeting. J Cancer. 2017;8:674-82. You D, Jung SP, Jeong Y, Bae SY, Lee JE, Kim S. Fibronectin expression is upregulated by PI-3K/Akt activation in tamoxifen-resistant breast cancer cells. BMB Rep. 2017;50:615-20. Tables Due to technical limitations, tables are only available as a download in the supplemental files section Additional File Information Supplementary Figure S1. p53, HNF-1 b , and ARID1A expression in OCCCa cells. (A) Western blot analysis for the indicated proteins in total lysates from four OCCCa cell lines. Note p53 mutation was only presented in ES-2 cells. wt, wild-type. (B) Analysis of TCGA data for associations between p53 gene abnormalities with expression of HNF-1 b and ARID1A mRNAs (left and right, respectively). Supplementary Figure S2. FN and p53 expression in OECa. (A) FN and p53 scores in OECa. (B) FN/p53 IHC scores in the immunoprofile groups (IPGs) of OECa including OCCCa, OHGSeCa, OLGSeCa, OEmCa, and OMuCa. OMuCa are excluded from IPG VII (w/o OMuCa). The data shown are as means±SDs. Supplementary Figure S3. Relationship between FN and p53 expression and prognosis in OCCCa or OHGSeCa. (A) OS (left) and PFS (right) relative to FN and p53 expression (upper and lower, respectively) in OCCCa. B) OS (left) and PFS (right) relative to FN and p53 expression (upper and lower, respectively) in OHGSeCa. N, number of cases. Supplementary Files SupFigureS3.tif SupFigureS2.tif SupFigureS1.tif Table1.xlsx RevisedTable3.xlsx RevisedTable2.xlsx Cite Share Download PDF Status: Published Journal Publication published 07 Jul, 2020 Read the published version in Cell Communication and Signaling → Version 2 posted Editorial decision: Accept 10 Apr, 2020 Review # 2 received at journal 09 Apr, 2020 Reviewer # 2 agreed at journal 02 Apr, 2020 Review # 1 received at journal 31 Mar, 2020 Reviewer # 1 agreed at journal 27 Mar, 2020 Editor assigned by journal 25 Mar, 2020 Reviewers invited by journal 25 Mar, 2020 Submission checks completed at journal 24 Mar, 2020 Editor invited by journal 24 Mar, 2020 You are reading this latest preprint version Show more versions 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. 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Also discoverable on Platform About 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-13200","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":445664,"identity":"b5dd610c-4455-4a79-a2b1-550754f0e3e2","order_by":1,"name":"Ako Yokoi","email":"","orcid":"","institution":"Kitasato Daigaku Igakubu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ako","middleName":"","lastName":"Yokoi","suffix":""},{"id":445665,"identity":"fd361fe4-30fe-4e11-bf69-0b554d502480","order_by":2,"name":"Toshihide Matsumoto","email":"","orcid":"","institution":"Kitasato Daigaku Igakubu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Toshihide","middleName":"","lastName":"Matsumoto","suffix":""},{"id":445666,"identity":"647841a8-b7f8-48e8-a137-730d5c2ebb71","order_by":3,"name":"Yasuko Oguri","email":"","orcid":"","institution":"Kitasato Daigaku Igakubu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasuko","middleName":"","lastName":"Oguri","suffix":""},{"id":445667,"identity":"552a3603-9e6b-4835-b01b-9d1bef6cf1b8","order_by":4,"name":"Yoshinori Hasegawa","email":"","orcid":"","institution":"Kazusa DNA Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yoshinori","middleName":"","lastName":"Hasegawa","suffix":""},{"id":445668,"identity":"63d15220-73a9-4e40-8899-b8e17c18c2a2","order_by":5,"name":"Masataka Tochimoto","email":"","orcid":"","institution":"Kitasato Daigaku Igakubu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masataka","middleName":"","lastName":"Tochimoto","suffix":""},{"id":445669,"identity":"1f7ffc9b-5705-49fb-8f84-68bb284799df","order_by":6,"name":"Mayu Nakagawa","email":"","orcid":"","institution":"Kitasato Daigaku Igakubu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mayu","middleName":"","lastName":"Nakagawa","suffix":""},{"id":445670,"identity":"fec5172f-3b2e-4c67-96b8-9d5a5ad1481b","order_by":7,"name":"Makoto Saegusa","email":"data:image/png;base64,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","orcid":"","institution":"Kitasato Daigaku Igakubu","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Saegusa","suffix":""}],"badges":[],"createdAt":"2020-02-03 13:24:38","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.2.22626/v2","doiUrl":"https://doi.org/10.21203/rs.2.22626/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-020-00580-3","type":"published","date":"2020-07-07T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":791921,"identity":"73c47e4f-f793-4422-a490-c6d78a8d62ee","added_by":"auto","created_at":"2020-03-31 03:21:09","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1214861,"visible":true,"origin":"","legend":"Expression of HNF-1 and ARID1A in p53-KD cells. (A) Western blot analysis for the indicated proteins in total lysates from OV-shp53 and control cells (Con). (B) Analysis of endogenous HNF-1 mRNA expression by conventional (upper) and real time RT-PCR assay (lower) for OV-shp53 and control cells (Con). The fold changes in mRNA expression detected by real time RT-PCR are presented as means±SDs. The experiment was performed in triplicate. (C) OVISE cells were transfected with two HNF-1 reporter constructs, respectively, together with either p53wt or p53mt. Relative activity was determined based on arbitrary luciferase light units normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means±SDs. The experiment was performed in duplicate.","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Figure 1.tif"},{"id":791923,"identity":"10c4850a-51cc-4af9-8a0d-3c3fbd640997","added_by":"auto","created_at":"2020-03-31 03:21:09","extension":"tif","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1914980,"visible":true,"origin":"","legend":"Relationship between cell phenotypic characteristics and p53-KD. (A) Left: phase contrast images of OV-shp53 cells. Note the changes in cell morphology toward fibroblastic appearances in OV-shp53 cells. Right: the numbers of spindle-shaped cells are presented as means±SDs. (B) Western blot analysis for the indicated proteins in total lysates from OV-shp53 and control cells (Con). (C) OVISE cells were transfected with Snail reporter constructs, together with either p53wt or p53mt. Relative activity was determined based on arbitrary luciferase light units normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means±SDs. The experiment was performed in duplicate. (D) Analysis of endogenous Snail mRNA expression by conventional (left) and real time RT-PCR assay (right) for OV-shp53 and control cells (Con). The signals of endogenous Snail mRNA expression in the conventional RT-PCR assay were normalized to GAPDH. The fold changes in mRNA expression detected by both assays are presented as means±SDs. The experiment was performed in triplicate.","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Figure 2.tif"},{"id":791925,"identity":"5db12e3e-99bf-42df-929c-f8e8759802a8","added_by":"auto","created_at":"2020-03-31 03:21:10","extension":"tif","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4443908,"visible":true,"origin":"","legend":"Relationship between p53-KD and cell proliferation or apoptosis. (A) Left: two independent OV-shp53 and control cell lines were seeded at low density. The cell numbers are presented as means±SDs. P0, P3, P6, and P8 are 0, 3, 6, and 8 days after seeding, respectively. Right: FACS analysis of OV-shp53 and control cells at 3 days after seeding (P3). (B) Western blot analysis for the indicated proteins in total lysates from OV-shp53 and the mock cells. (C) Left: after treatment with 10 M CDDP, OV-shp53 and control cells undergoing apoptosis are indicated by arrows. Original magnification, x400. Right: the numbers of apoptotic cells are demonstrated as means±SDs. Con, control. (D) Upper: treatment of OV-shp53 and control cells with 10 M CDDP for the times shown. Cell viability was measured using the CCK-8 kit. The viability in the absence of CDDP treatment (0 h) is set as 100%. Lower: treatment of OV-shp53 and control cells with 10 M CDDP for the times shown. The numbers of trypan blue-positive cells (non-viable cells) are presented as mean±SD. This experiment was performed in triplicate using independent samples. (E) Western blot analysis for the indicated proteins in total lysates from OV-shp53 and control cells treated with 10 M CDDP.","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Figure 3.tif"},{"id":791927,"identity":"95edeca7-9c09-433e-98a5-738c461f823f","added_by":"auto","created_at":"2020-03-31 03:21:10","extension":"tif","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5028353,"visible":true,"origin":"","legend":"Relationship between p53-KD and cancer stem cell features or cell migration. (A) Aldefluor analysis in OV-shp53 and control cells. Note the R1 populations including the ALDHhigh population with cancer stem cell-like features. (B) Left: wound-healing assay with OV-shp53 and control cells. A scratch ‘wound’ was introduced to the middle of wells containing cell growth to confluency, and phase contrast images were taken after 6, 9, and 12 h. Right: the values of wound areas in 0 h were set as 1. The fold wound areas are presented as means±SDs. C, control. (C) Migration rate measured using transwell assay. Left: the OV-shp53 and control cells were seeded in a 24-well transwell plates and incubated for 24 h in medium without serum. Cells were stained with HE and counted using a light microscope. Right: the numbers of migrated cells are presented as means±SDs (right).","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Figure 4.tif"},{"id":791929,"identity":"6954cc64-6256-4fbf-bbd9-8acd8a892421","added_by":"auto","created_at":"2020-03-31 03:21:11","extension":"tif","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1202975,"visible":true,"origin":"","legend":"Relationship between p53 and FN expression. (A) Unsupervised hierarchical clustering of mRNA expression detected by next generation sequencing in OV-shp53 and control cells (Con). The expression level of each mRNA is colored; red, black, and green indicated high (\u003e4), neutral (1-4), and low (\u003c1), respectively. Major clusters are shown as group I to XI. (B) Analysis of endogenous FN1 mRNA expression by conventional (left) and real time RT-PCR assay (right) in OV-shp53 and control cells (Con). The values of endogenous FN1 mRNA expression detected by conventional RT-PCR assay were normalization to GAPDH. The fold changes in mRNA expression for both assays are presented as means±SDs. The experiment was performed in triplicate. (C) Western blot analysis for the indicated proteins in total lysates from OV-shp53 and control cells (Con). (D) OVISE cells were transfected with FN1 reporter constructs, together with either p53wt or p53mt. Relative activity was determined based on arbitrary luciferase light units normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means±SDs. The experiment was performed in duplicate.","description":"","filename":"Figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Figure 5.tif"},{"id":791931,"identity":"8b26e6b0-70de-4b66-8e51-565573704aab","added_by":"auto","created_at":"2020-03-31 03:21:11","extension":"tif","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5094786,"visible":true,"origin":"","legend":"Relationship between FN and cell kinetics. (A) Upper: phase-contrast images of OVISE cells treated with 20 g/ml FN for 96 h. Note there was no alteration in cell morphology during the treatment. Lower: western blot analysis for the indicated proteins in total lysates from OVISE cells with or without 20 g/ml FN treatment. (B) Western blot analysis of the indicated proteins in total lysates from OVISE cells with or without 10 M CDDP treatment in the presence or the absence of 20 g/ml FN. C) Left: wound-healing assay with OVISE cells with or without 20 g/ml FN treatment. A scratch ‘wound’ was made in the middle of cells grown to confluency, and phase contrast images were taken after 6, 9, and 12 h. Right: the values of wound areas in 0 h were set as 1. The fold changes in wound areas are presented as means±SDs (lower). (D) Migration rate measured using the transwell assay. Left: the OVISE cells with or without 20 g/ml FN treatment were seeded in 24-well transwell plates and incubated for 24 h in medium without serum. The cells were stained by HE and counted using light microscope. Right: numbers of migrated cells are presented as means±SDs (right). (E) The OVISE cells with or without 20 g/ml FN treatment were seeded at low density. The cell numbers are presented as means±SDs. P0, P3, P6, and P8 are 0, 3, 6, and 8 days after cell seeding, respectively.","description":"","filename":"Figure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Figure 6.tif"},{"id":791932,"identity":"55453aaf-6803-4dc7-9449-593f85f1eea8","added_by":"auto","created_at":"2020-03-31 03:21:12","extension":"tif","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":8971354,"visible":true,"origin":"","legend":"IHC findings in serial sections of OCCCa and OHGSeCa tumors (A) Staining by HE and IHC staining for the indicated proteins in OCCCa and OHGSeCa. Original magnification, x100. (B) FN/p53 IHC scores in the immunoprofile groups (IPGs) of OECa including OCCCa, OHGSeCa, OLGSeCa, OEmCa, and OMuCa. OMuCa are excluded from IPG VII (w/o OMuCa). The data shown are as means±SDs.","description":"","filename":"Figure7.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Figure 7.tif"},{"id":791933,"identity":"1af7440f-1d26-4d85-8432-04823422526a","added_by":"auto","created_at":"2020-03-31 03:21:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":89533,"visible":true,"origin":"","legend":"Relationship between FN and p53 expression and prognosis in OCCCa/OHGSeCa. OS (left) and PFS (right) relative to FN (A), p53 (B), and combined FN and p53 expression (C) in OCCCa/OHGSeCa. N, number of cases.","description":"","filename":"8.PNG","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/8.PNG"},{"id":791934,"identity":"878b33d7-a526-4240-a2e4-8df6be4bb9f1","added_by":"auto","created_at":"2020-03-31 03:21:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":39191,"visible":true,"origin":"","legend":"Schematic representation of the interplay between the p53 pathway, FN expression, and the Akt/GSK-3/Snail axis in the aggressive OCCCa/OHGSeCa.","description":"","filename":"9.PNG","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/9.PNG"},{"id":13496159,"identity":"77e68d29-4885-4da3-8566-7bce7a492fba","added_by":"auto","created_at":"2021-09-16 22:48:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":41247183,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/c6174958-e83b-404c-a78e-773bd8e990b3.pdf"},{"id":791930,"identity":"d33105bf-4f3f-46a4-bf91-2afdbe414006","added_by":"auto","created_at":"2020-03-31 03:21:11","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1075260,"visible":true,"origin":"","legend":"","description":"","filename":"SupFigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Sup Figure S3.tif"},{"id":791928,"identity":"afe7a3c1-0935-4fc7-ae47-596ab877d0cf","added_by":"auto","created_at":"2020-03-31 03:21:11","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":828176,"visible":true,"origin":"","legend":"","description":"","filename":"SupFigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Sup Figure S2.tif"},{"id":791926,"identity":"3b42ffad-bbe3-4d75-a3b7-f6824023786f","added_by":"auto","created_at":"2020-03-31 03:21:10","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1957064,"visible":true,"origin":"","legend":"","description":"","filename":"SupFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Sup Figure S1.tif"},{"id":791924,"identity":"5e711c22-e176-443c-a5b7-30089ad9c8f7","added_by":"auto","created_at":"2020-03-31 03:21:10","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13214,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Table 1.xlsx"},{"id":791922,"identity":"a507295a-394e-45c7-89f9-ed81c7e0b677","added_by":"auto","created_at":"2020-03-31 03:21:09","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":12399,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Revised Table 3.xlsx"},{"id":791920,"identity":"de751b3b-fb10-4b08-ae66-1927165aa0c7","added_by":"auto","created_at":"2020-03-31 03:21:09","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":12475,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-13200/v2/Revised Table 2.xlsx"}],"financialInterests":"","formattedTitle":"Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype","fulltext":[{"header":"Background","content":"\u003cp\u003e\u0026nbsp;\u0026nbsp; Ovarian epithelial carcinomas (OECa) are among the most aggressive tumors and the leading cause of mortality among all types of malignancies in the female reproductive system [1]. Since the ovaries have a relatively inaccessible location and ovarian carcinoma patients very often lack symptoms in the early neoplastic stage, more than 75% of the patients are diagnosed with advanced stage disease that is characterized by metastasis to the peritoneal cavity [2]. In addition, approximately 80% of advanced stage patients have residual disease after surgery and receive front-line platinum-based combination chemotherapy; these individuals have a median progression-free survival (PFS) of 18 months [3]. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; p53 is widely acknowledged as the most frequently mutated gene in human malignancy and its mutational status is a prognostic marker in several tumor types [4]. Activated wild-type p53 (p53wt) acts as a checkpoint control for recognizing damaged DNA, allowing DNA repair and delayed entrance into the DNA replication phase of the cell cycle; together with observations that the incidence of tumorigenesis increases in p53 null or mutant tissues, these data confirm that p53 is a bona fide tumor suppressor [5]. Mutations in the \u003cem\u003eTP53\u003c/em\u003e gene are found in more than 50% of human malignancies and its inactivation can occur at various stages depending on the tissue that gives rise to the tumor. Therefore, loss of p53 function can promote neoplastic transformation as well as progression of established tumors to a more aggressive disease stage [6,7]. In OECa, and particularly in ovarian high-grade serous carcinomas (OHGSeCa), mutant p53 (p53mt) missense mutations are frequently found in the hotspot codon R175, R248, and R273 (\u003ca href=\"http://www-p53.iarc.fr/\"\u003ehttp://www-p53.iarc.fr/\u003c/a\u003e) that are critical contact residues in the p53 DNA-binding domain. The mutations occur early during tumorigenesis, most likely in precursor lesions of OECa, highlighting the importance of p53mt as a driver of the malignancy [8-11].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; We previously developed an effective immunoprofiling classification system for OECa using only 4 immunohistochemical markers (HNF-1b, p53, ARID1A, and WT1) [12]. Using this system, we demonstrated that tumors with an HNF-1b+/p53+/ARID1A+ immunophenotype including OHGSeCa and ovarian clear cell carcinomas (OCCCa) were associated with the most unfavorable prognosis. In this study, we hypothesized that alterations in the p53 signaling pathway may play a key role in determining phenotypic characteristics in OECa with the HNF-1b+/p53+/ARID1A+ immunophenotype. To test this, we set out to first examine the effects of knocking down p53wt (p53-KD) in OCCCa cells expressing endogenous HNF-1b and ARID1A. Next, we applied a next generation sequencing (NGS) assay to identify the molecules associated with loss of p53 function. Finally, we examined associations between molecules that were differentially expressed following p53-KD, tumor phenotypic characteristics and prognostic significance in OHGSeCa and OCCCa.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003ePlasmids and cell lines\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; The p53-specific short hairpin RNA (shRNA) oligonucleotides were designed as described previously [13]. Single-stranded p53 oligonucleotides were annealed and then cloned into \u003cem\u003eBamH\u003c/em\u003e1-\u003cem\u003eEcoR\u003c/em\u003eV sites of RNAi-Ready pSIREN-RetroQ vector (Takara, Shiga, Japan), according to the manufacturer\u0026rsquo;s instructions. The p53mt (R248Q) was generated by PCR-based methods using a pCMV-p53wt construct. The human\u003cem\u003e Fibronectin 1\u003c/em\u003e promoter (UCSC genome browser, https://genome.ucsc.edu/) between -2028 and -23 (where +1 represents the transcription start site) was also generated by PCR and was cloned into the pGL3B vector (Promega, Madison, WT, USA). The primer sequences for the PCR reaction used in this study are listed in Table 1. pCMV-p53wt, pGL3B-(-1109/+36) Snail luc, pGL3B-(-899/+47) HNF-1b luc, and pGL3B-(-140/+216) HNF-1b luc were also used as described previously [14-16].\u003c/p\u003e\n\u003cp\u003eFour OCCCa cell lines, OVISE, ES2, OVTOKO, and TOV-21G were used as described previously [13,16,17], and two OHGSeCa cell lines, OVSAHO and OVCAR-3, were obtained from the National Institute of Biomedical Innovation (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA), respectively. p53 shRNA knockdown cells were established using OVISE cells, which have a wild-type \u003cem\u003ep53\u003c/em\u003e gene and abundant expression of endogenous HNF-1b and ARID1A (Supplementary Figure S1), as described previously [13,17]. In addition, spindle-shaped cells were defined as those that showed narrow and elongated phenotypes, along with weak or absent adhesions between cells, as described previously [17].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAntibodies and reagents\u003c/p\u003e\n\u003cp\u003eAnti-p53, anti-p21\u003csup\u003ewaf1\u003c/sup\u003e, anti-cyclin D1, and anti-bcl2 antibodies were purchased from Dako (Copenhagen, Denmark). Anti-HNF-1b, anti-GSK-3b, anti-Rb, anti-p27\u003csup\u003ekip1\u003c/sup\u003e, anti-XIAP, anti-bax, and anti-integrin b1 antibodies were obtained from BD Biosciences (San Jose, CA, USA). Anti-ARID1A, anti-cyclin B1, and anti-MDM2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail, anti-Akt, anti-phospho(p)Akt Serine473, anti-pGSK-3b Serine9, anti-pRb Serine807/811, anti-cleaved caspase-3, and anti-integrin b3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-fibronectin (FN), anti-E-cadherin, and anti-b-actin antibodies were obtained from Abcam (Cambridge, MA, USA), Takara (Shiga, Japan), and Sigma-Aldrich Chemicals (St. Louis, MO, USA), respectively. Anti-cyclin A2 and anti-integrin b2 antibodies were from Novocastra (Newcastle, UK) and Merck KGaA (Darmstadt, Germany), respectively. FN (catalog number #F2006) and cisplatin (CDDP: #479306) were purchased from Sigma-Aldrich Chemicals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTransfection\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Transfection was carried out using LipofectAMINE PLUS (Invitrogen, Carlsbad, CA, USA) as described previously [14-16]. Luciferase activity was assayed as described previously [14-16].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReverse transcription (RT)-PCR\u0026nbsp; \u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; cDNA was synthesized from 2 mg of total RNA. Amplification by RT-PCR was carried out in the exponential phase to allow comparison among cDNA synthesized from identical reactions using specific primers (Table 1). Primers for the \u003cem\u003eHNF-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e, \u003cem\u003eSnail\u003c/em\u003e, and \u003cem\u003eGAPDH\u003c/em\u003e genes were also applied, as described previously [14-16]. The signal intensity was analyzed by ImageJ software version 1.41 (NIH, Bethesda, MD, USA).\u003c/p\u003e\n\u003cp\u003eFor quantitative analysis, real-time RT-PCR was also conducted using a Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Fluorescent signals were detected using the ABI 7500 Real-time PCR System SDS Software (Applied Biosystems). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWestern blot assay\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Total cellular proteins were isolated using RIPA buffer [20 mM Tris-HCl (pH7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate]. Aliquots of the proteins were resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies, coupled with the ECL detection system (Amersham Pharmacia Biotechnology, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFlow cytometry and Aldefluor assay\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Cells were fixed using 70% alcohol and stained with propidium iodide (Sigma) for cell cycle analysis. Aldehyde dehydrogenase 1 (ALDH1) enzyme activity in viable cells was determined using a fluorogenic dye-based Aldefluor assay (Stem Cell Technologies, Grenoble, France) according to the manufacturer\u0026rsquo;s instructions. The prepared cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences) and CellQuest Pro software version 3.3 (BD Biosciences).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCell Counting Kit-8 assay\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; The quantitation of viable cell number in proliferation after CDDP treatment was carried out using a Cell Counting Kit-8 (CCK-8; Dojindo Lab, Kumamoto, Japan), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWound healing assay\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; \u0026nbsp;Cells were seeded into 24-well tissue culture plates, and grown to reach almost total confluence. After a cell monolayer formed, a wound was scratched with a sterile 200-ml tip. The area of the wound was analyzed by ImageJ software version 1.41 (NIH). Cell migration parameters were calculated in pixels as wound closure.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMigration assay\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Cell migration was determined using 24-well Transwell chambers with an 8-mm pore size (Corning, NY, USA). The lower chamber was filled with medium containing 10% serum. Cell were suspended in serum-free medium with or without FN and transferred into the upper chamber. After 24 h, the number of cells stained by hematoxylin-eosin (HE) on the bottom surface of the polycarbonate membranes was counted visually using a light microscope.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eApoptotic index\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Apoptotic cells were identified in HE-stained sections, according to the criteria of Kerr et al. [18]. A total 10 fields were randomly selected, and the number of apoptotic cells was calculated by counting the mean number of apoptotic figures per high power field (HPF).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNGS assay\u003c/p\u003e\n\u003cp\u003eTotal RNAs were extracted from OV-shp53 and mock cells using the NucleoSpin RNA system (Takara). The concentration and quality of the RNA was verified with the Quantus Fluorometer (Promega) and Agilent 2100 Bioanalyzer, respectively. All the samples showed RIN values over 9. Total RNA (500 ng) was used for RNA library preparation, according to the instructions of the Quant Seq 3\u0026rsquo; mRNA-seq library preparation kit FWD from Illumina (Lexogen, Vienna, Austria). The libraries were PCR-amplified for 12 cycles.\u003c/p\u003e\n\u003cp\u003eSequencing of the libraries (via single-end 75-bp reads) was conducted on the Illumina NextSeq500 system. All data analyses were conducted using Strand NGS (v3.2, Agilent Technologies). The adapter sequences were removed from the raw reads, and base trimming was performed from the 3\u0026rsquo; end of each read to remove bases with quality below Q10 up to a minimum length of 25 bp. Each read was mapped to the reference human genome hg38 with default settings. Expression patterns of transcripts were compared after normalization of DESeq [19] using default settings.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTCGA data analysis\u003c/p\u003e\n\u003cp\u003eThe Cancer Genome Atlas (TCGA) OHGSeCa annotated \u003cem\u003eTP53\u003c/em\u003e gene alteration and mRNA expression data (RNA Seq V2 PSEM) for HNF-1b and ARID1A were extracted from cBioportal for Cancer Genomics (\u003ca href=\"http://www.cbioportal.org/\"\u003ehttp://www.cbioportal.org/\u003c/a\u003e) for 398 OHGSeCa cases.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClinical cases\u003c/p\u003e\n\u003cp\u003eA total of 199 cases of OECa, surgically resected at Kitasato University Hospital between 2006 and 2017, were selected from our patient records according to the criteria of the 2014 World Health Organization classification [20]. All patients underwent oophorectomy with or without hysterectomy. None of the patients had received chemotherapy or any other preoperative treatment, while most patients had received Paclitaxel/Carboplatin-based chemotherapy after surgical treatment. Of these, 99 cases including 41 OHGSeCa and 58 OCCCa showed complete resection of the tumors, while 28 cases including 17 OHGSeCa and 11 OCCCa had residual tumors after debulking surgery.\u003c/p\u003e\n\u003cp\u003eEvaluation of relapse and disease progression was conducted on the basis of radiologic image findings. The tumor cases investigated were comprised of 58 OHGSeCa, 9 ovarian low grade serous carcinoma, 29 ovarian endometroid carcinomas, 71 OCCCa, and 30 ovarian mucinous carcinomas. All tissues were routinely fixed in 10% formalin and processed for embedding in paraffin wax. Approval for this study was given by the Ethics Committee of the Kitasato University School of Medicine (B16-10).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry (IHC)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; IHC was performed using a combination of the microwave-oven heating and polymer immunocomplex (Envision, Dako) methods, as described previously [14-16].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; For evaluation of IHC findings, scoring of nuclear/cytoplasmic immunoreactivity was performed, on the basis of the percentage of immunopositive cells and the immunointensity, with multiplication of values of the two parameters, as described previously [14-16]. In addition, FN/p53 score was generated by multiplication of the values of the two scores.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; To evaluate the prognostic significance of FN and p53 expression, the scores were divided into two categories (high and low) with the mean values as the cut-off in each category (Table 2). With regard to p53, cases that were completely negative for p53 immunoreactivity were categorized into the high p53 score group (score = 12), since combining 2 IHC labeling patterns associated with p53 mutations (0% and 60-100% positive cells) correctly identified a mutation in 94% of cases [ 21].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStatistics\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Comparative data were analyzed using the Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test. Overall survival (OS) was calculated as the time between onset and death or the date of the last follow-up evaluation. PFS was also examined from the onset of treatment until relapse, disease progression, or last follow-up evaluation. OS and PFS were estimated using the Kaplan-Meier methods, and the statistical comparisons were made using the log rank test. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. The cut-off for statistical significance was set as p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eLoss of p53 function leads to induction of EMT features\u003c/em\u003e\u003cem\u003e\u0026nbsp; \u003cbr /\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; To examine the phenotypic characteristics of OECa cells with the HNF-1b+/p53+/ARID1A+ immunophenotype, we first established two independent cell lines in which p53 expression was blocked by a p53-specific shRNA (OV-shp53#2 and #8) using OVISE (OV) cells. The OV-p53-KD cells showed increased expression of both HNF-1b and ARID1A, in contrast to decreased expression of MDM2 and p21\u003csup\u003ewaf1\u003c/sup\u003e, which are p53 target genes (Figure 1A). \u003cem\u003eHNF-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e mRNA expression was also increased in the OV-p53-KD cells as compared to the mock cells (Figure 1B), in line with the observation of dose-dependent repression of \u003cem\u003eHNF-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e promoter activity following transfection of p53wt. In contrast, the repressive effects were not evident when p53mt was transfected (Figure 1C).\u003c/p\u003e\n\u003cp\u003eThe OV-p53-KD cells also demonstrated a significant switch towards a fibroblastic morphology (Figure 2A), along with increased expression of Snail, pAkt, and pGSK-3b, and decreased E-cadherin expression (Figure 2B). Although \u003cem\u003eSnail\u003c/em\u003e promoter activity was inhibited by p53wt, but not p53mt (Figure 2C), changes in mRNA expression were relatively minor in OV-p53-KD cells as compared to the mock cells (Figure 2D). These findings suggest that loss of p53 function contributes to increased expression of HNF-1b and ARID1A, leading to induction of epithelial-mesenchymal transition (EMT) features, probably through post-translational regulation of Snail expression.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLoss of p53 function is associated with CSC features and acceleration of cell mobility\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; To examine whether p53-KD affects cell proliferation, the two independent OV-p53-KD cell lines were seeded at low density. OV-p53-KD cells tended to proliferate more slowly, particularly in the exponential growth phase, along with an increased proportion of cells in G2/M phase of the cell cycle (Figure 3A). To further examine alterations in expression of several cell cycle-related molecules during cell growth, the OV-p53-KD cells were rendered quiescent by serum starvation and were subsequently stimulated with serum. At 6, 12, and 24 h after release into the cell cycle, p27\u003csup\u003ekip1\u003c/sup\u003e expression was substantially increased in OV-p53-KD cells relative to the mock cells, in contrast to the progressive reduction of cyclin B1 expression in the former (Figure 3B).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Next, we examined the association between loss of p53 function and apoptotic features in response to cytotoxic effects. Treatment of OV-p53-KD cells with CDDP showed decreased apoptotic cells as compared to the mock (Figure 3C), in line with the results of increased cell viability during CDDP treatment (Figure 3D). The expression of cleaved caspase-3, as well as bax and bcl-2, were also apparently decreased in the OV-p53-KD cells as compared to mock cells, in contrast to increased pAkt, but not XIAP, expression (Figure 3E).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Since EMT promotes stem cell properties and further generates cells with cancer stem cell (CSC)-like features [22], we examined the association between loss of p53 function and CSC properties. As shown in Figure 4A, there was a significant increase in the ALDH\u003csup\u003ehigh\u003c/sup\u003e population, which includes a high percentage of CSC-like cells, in the p53-KD cells compared to the mock cells.\u003c/p\u003e\n\u003cp\u003eTo further examine whether loss of p53 function contributes to cell motility, we carried out scratch and migration assays. The OV-p53-KD cells refilled wounded empty spaces more rapidly (Figure 4B), in line with the significantly increased migration rates as compared to the mock cells (Figure 4C). These findings suggest that loss of p53 function also engenders CSC features and accelerates cell motility in OVISE cells; these changes are accompanied by inhibition of cell proliferation and susceptibility to apoptosis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eUpregulation of FN expression by loss of p53 function \u003c/em\u003e\u003cem\u003e\u0026nbsp; \u003cbr /\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; To identify genes that are differentially expressed following p53-KD, NGS assays were carried out using total RNAs extracted from OV-p53-KD cells. A total of 12051 and 13094 genes in OV-shp53#2 and OV-shp53#8 cells were dysregulated, respectively. Of these, 57 and 83 genes were upregulated or downregulated over 5-fold, respectively, in the p53-KD cells as compared to the mock cells. As shown Figure 5A, hierarchical clustering revealed that the genes could be readily categorized into eleven groups, and we focused on the \u003cem\u003eFN1\u003c/em\u003e gene in group IV that was overexpressed by 17-fold. \u003cem\u003eFN1\u003c/em\u003e mRNA and protein expression were apparently increased in OV-p53-KD cells, along with increased expression of integrin b1, b3, and b3 (Figure 5B, C). Moreover, \u003cem\u003eFN1\u003c/em\u003e promoter activity was repressed by transfection of p53wt, but not p53mt (Figure 5D).\u003c/p\u003e\n\u003cp\u003eSince FN is an EMT-related marker [23,24], we asked whether there was an association of FN with either EMT or cell motility. OVISE cells treated with FN did not show any changes in cell morphology or expression of E-cadherin, Snail, Akt and GSK-3b (Figure 6A); the expression of apoptosis-related molecules was also unchanged (Figure 6B). In contrast, both scratch and migration assays revealed that FN treatment resulted in a significant increase in migration capacity (Figure 6C,D), along with a decrease in proliferation at later stages (Figure 6E). These findings suggest that FN is an important determinant of cellular function in p53-KD cells due to its effects on cell mobility and proliferation, rather than via modulation of EMT or apoptosis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrognostic significances of FN and p53 expression in OCCCa/OHGSeCa\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative IHC findings for FN and p53 in OCCCa and OHGSeCa are illustrated in Figure 7A, demonstrating cytoplasmic immunostaining for FN and nuclear staining for p53. FN score was significantly higher in OCCCa as compared to that of OHGSeCa, in contrast to a significantly higher p53 score in the latter (Supplementary Figure S2A). Previously, we used hierarchical clustering analysis to identify seven immunopurified groups (IPGs) in OECa including OCCCa, OHGSeCa, OLGSeCa, OEmCa, and OMuCa [12]. Here, we observed that average FN/p53 scores were significantly higher in IPG VII, which includes OCCCa/OHGSeCa with the HNF-1b+/p53+/ARID1A immunophenotype, and lower in the IPGs IV, V, and VI (Figure 7B). Similar findings were also observed in p53, but not FN, scores among IPGs including the five OECa histotypes (Supplementary Figure S2B).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eThe FN scores were also significantly associated with tumor histotype, tumor size, and lymph node metastasis in OCCCa; p53 score was also significantly correlated with clinical stage, histotype, tumor size, and distant metastasis in OHGSeCa (Table 2).\u003c/p\u003e\n\u003cp\u003eThe Kaplan-Meier curves showed that patients with high FN and p53 scores had poorer OS and PFS when compared to patients with low scores in the OCCCa/OHGSeCa category (Figure 8A, B), although such associations were not observed in p53 scores in OHGSeCa (Supplementary Figure S3). Patients with a combination of high FN and high p53 scores also had the worst OS and PFS in OCCCa/OHGSeCa, whereas patients with low values of both scores had the best prognosis (Figure 8C).\u003c/p\u003e\n\u003cp\u003eUnivariate Cox progression hazards regression revealed that FN, p53, age, tumor histotype, FIGO stage, lymph node metastasis, distant metastasis, and residual tumors after surgical treatment were significant prognostic factors for OS or PFS in OCCCa/OHGSeCa. In addition, multivariate Cox regression analysis showed that FN, FIGO stage, and distant metastasis were significant and independent prognostic factors for OS or PFS (Table 3). These findings suggest that a combined IHC analysis of FN and p53 expression is useful for prognostic prediction of OCCCa/OHGSeCa. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough extensive studies on the gain-of-function (GOF) effects of p53mt have been conducted using\u003cem\u003e in vitro\u003c/em\u003e cell culture systems, several \u003cem\u003ein vivo\u003c/em\u003e models have indicated that the primary effect of p53 mutation is the loss of p53wt function, with little or no GOF effect on tumorigenesis. Thus, the GOF of a particularly p53mt is likely to be determined by tissue- and tumor type-specific factors. For example, MMTV-\u003cem\u003eHrasTP53\u003csup\u003eR172H/R172H\u003c/sup\u003e\u003c/em\u003e and MMTV-\u003cem\u003eHras/TP53\u003csup\u003e-/- \u003c/sup\u003e\u003c/em\u003emice were very similar with regard to age of salivary tumor onset, tumor growth rate, tumor histopathological features, and response to a DNA-damaging agent [25]. This was also the case in a \u003cem\u003eK-ras\u003c/em\u003e-driven lung cancer model, as well as in the context of \u003cem\u003eWAP-Cre\u003c/em\u003e-induced expression of the p53R270H mutant in p53-null-mouse mammary glands, and in p53R172H homozygous knock-in mouse models [26-28]. In addition, the GOF activity of any particular p53mt is largely dependent on multiple signals required for its post-translational stabilization: the presence of such signals is likely to vary among both normal and tumor cells [29]. Based on the above evidence, we examined alterations in the p53 signaling pathway using cells following shRNA-mediated knockdown of p53wt.\u003c/p\u003e\n\u003cp\u003eHere, we provide clear evidence that p53 loss leads to upregulation of HNF-1b, as well as ARID1A, at both mRNA and protein levels in OCCCa cells. Moreover, transfection of p53wt, but not p53mt, represses\u003cem\u003e HNF-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e promoter activity, suggesting that alterations in the \u003cem\u003ep53\u003c/em\u003e gene may play an important role in development of OCCCa that have the HNF-1b+/p53+ /ARID1A+ immunophenotype. However, analysis of the TCGA database revealed that expression of \u003cem\u003eHNF1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e and \u003cem\u003eARID1A\u003c/em\u003e mRNA was not correlated with p53 status in OHGSeCa. Given the evidence that HNF-1b is a sensitive and specific marker for OCCCa and is not expressed in OHGSeCa with clear cell changes [30], it appears that some cell type-specific factors may also be required for establishment of OHGSeCa with the immunophenotypic features we specify above.\u003c/p\u003e\n\u003cp\u003eWe also found that p53-KD cells have dramatically altered cell morphology and are more fibroblast-like in appearance; these changes are accompanied by increased expression of E-cadherin-repressor Snail, as well as pAkt and pGSK-3b, and decreased E-cadherin expression. Although \u003cem\u003eSnail \u003c/em\u003epromoter activity was specifically repressed by p53wt, we did not observe differences in \u003cem\u003eSnail\u003c/em\u003e mRNA levels between p53-KD and the mock-transfected cells. In general, Snail expression is decreased through GSK-3b-mediated phosphorylation/degradation [31], while GSK-3b activity is inhibited following activation of Akt [32]. Given the mutual antagonism between the p53 and Akt networks [33-35], it is suggested that loss of p53 function leads to post-translational upregulation of Snail through activation of the Akt/GSK-3b axis, which in turn leads to induction of EMT. Interestingly, p53mt can directly bind and trans-repress the promoter of \u003cem\u003emiR-130b\u003c/em\u003e, a microRNA that specifically downregulates ZEB1, leading to the upregulation of BMI-1 and Snail [36]. In addition, p53wt can induce MDM2 mediated degradation of Snail [37].\u003c/p\u003e\n\u003cp\u003eHere, we found that p53-KD cells had a reduced proliferative rate and enhanced migration capability, along with enhanced G2/M arrest and increased p27\u003csup\u003ekip1\u003c/sup\u003e expression. Moreover, cyclin B1 expression also decreased progressively after serum stimulation in p53-KD cells. Our findings are consistent with those of previous reports. First, p27\u003csup\u003e kip1\u003c/sup\u003e is important for the initial activation of G2/M checkpoint in response to low-dose ionizing radiation [38], in line with evidence that Cdc2, which is essential for entry into mitosis, physically interacts with, and is inhibited by p27\u003csup\u003e kip1 \u003c/sup\u003e[39,40]. In addition, binding of Cdc2 to cyclin B1 is required for its activity and repression of cyclin B1 contributes to blocking entry into mitosis [C]. Second, hematopoietic cells expressing p53wt arrest in both G1 and G2 phase of the cell cycle, while p53-null cells or cells overexpressing p53mt exhibited only G2 arrest [42]. Third, reduced levels of p53 correlate with increased G2/M phase arrest in response to paclitaxel treatment in normal human fibroblast depleted of functional p53 [43]. Finally, migratory cells have a lower proliferation rate in comparison with cells in the tumor core, indicating an inverse correlation between cell proliferation and mobility [44-46].\u003c/p\u003e\n\u003cp\u003eOur findings also revealed that susceptibility to apoptosis in response to CDDP treatment was significantly inhibited in p53-KD cells. This may be explained by the prolonged high levels of pAkt in response to loss of p53 function, because there is an \u0026lsquo;all-or-none\u0026rsquo; switching behavior between a pro-survival cellular state (low p53 and high Akt levels) and a pro-apoptotic state (high p53 and low Akt levels) [33], as well as decreased expression of the pro-apoptotic protein, bax [47].\u003c/p\u003e\n\u003cp\u003eAn important finding in this study was that \u003cem\u003eFN1\u003c/em\u003e mRNA and protein expression were significantly increased in p53-KD cells, while \u003cem\u003eFN1\u003c/em\u003e promoter activity was repressed by p53wt, but not p53mt, in line with other studies [48,49]. Moreover, treatment of OCCCa cells with FN resulted in an enhanced migration capability and a reduced proliferation rate. In contrast, the effects of FN on cell morphology, expression of EMT-related molecules, and susceptibility to apoptosis were minimal. Taken together with our results that p53-KD increases the expression of the FN receptors integrins b1, b2, and b3 [50], we conclude that FN upregulation due to loss of p53 function is closely associated with enhancement of cell mobility, but not induction of EMT and apoptotic features. However, we could not demonstrate a direct correlation between FN and p53 scores in the OCCCa/OHGSeCa category, indicating that a p53-independent pathway must regulate FN expression in the tumors, particularly in OCCCa. In fact, FN expression is upregulated through the PI3K/Akt pathway in tamoxifen-resistant breast cancer cells [51].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Finally, patients with a combination of high FN and high p53 IHC scores had significantly worse OS and PFS than did patients with low values for both scores in OCCCa/OHGSeCa. Both FN and p53 scores were also significantly associated with several unfavorable clinicopathological factors in the tumors. Moreover, multivariate Cox regression analysis also showed that FN, but not p53, was a significant and independent unfavorable prognostic factor for OS and PFS. Furthermore, there was a positive association between high FN score, enlarged tumor size and nodal metastasis, suggesting that combined IHC analysis for FN and p53 expression may have great utility in OCCCa/OHGSeCa prediction and prognosis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eUpregulation of FN following loss of p53 function may influence the malignant properties of OCCCa/OHGSeCa, particularly in those tumors with an HNF-1b+/p53+/ARID1A+ immunophenotype. The accompanying induction of EMT/CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics (Figure 9). \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003cp\u003eOECa: ovarian epithelial carcinomas; OHGSeCa: ovarian high grade serous carcinoma; OCCCa: ovarian clear cell carcinoma; KD: knockdown; p53wt: wild-type p53; p53mt: mutant type p53; IHC; immunohistochemistry; EMT: epithelial-mesenchymal transition; FN: fibronectin; OS: overall survival; PFS: progression-free survival; IPG; immunoprofile group\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproval for this study was given by the Ethics Committee of the Kitasato University School of Medicine (B16-10).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; Not applicable\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Data and materials will be shared.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; The authors declare that they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from JSPS KAKENHI Grant Number 17K08703.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAY, TM, and MS carried out the majority of the experiments, analyzed the data, and wrote the manuscript. They were helped by YO, YH, MT, and MN. All authors reviewed and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin. 2007;57:43-63.\u003c/li\u003e\n\u003cli\u003eWang Y, Helland A, Holm R, Skomedal H, Abeler VM, Danielsen HE, Trope CG, Borresen-Dale A-L, Kristensen GB. TP53 mutations in early-stage ovarian carcinoma, relation to long-term survival. Br J Cancer. 2004;90:678-85.\u003c/li\u003e\n\u003cli\u003eSalani R, Backes FJ, Fung MF, Holschneider CH, Parker LP, Bristow RE, Goff BA. Posttreatment surveillance and diagnosis of recurrence in women with gynecologic malignancies: Society of Gynecologic Oncologists recommendations. Am J Obstet Gynecol. 2001;204:466-78.\u003c/li\u003e\n\u003cli\u003eHollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. 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Cancer Res. 1991;51:6304-11.\u003c/li\u003e\n\u003cli\u003eWahl AF, Donaldson KL, Faircnild C, Lee FYF, Foster SA, Demers GW, Galloway DA. Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis. Nat Med. 1996;2:72-9.\u003c/li\u003e\n\u003cli\u003eGiese A, Loo MA, Tran N, Haskett D, Coons SW, Berens ME. Dichotomy of astrocytoma migration and proliferation. Int J Cancer. 1996;67:275-82.\u003c/li\u003e\n\u003cli\u003eGiese A, Bjerkvig R, Berens ME, Westphal M. Cost of migration: invasion of malignant glioma and implications for treatment. J Clin Oncol. 2003;21:1624-36.\u003c/li\u003e\n\u003cli\u003eMerzak A, McCrea S, Koocheckpour S, Pilkington GJ. Control of human glioma cell growth, migration and invasion in vitro by transforming growth factor beta 1. Br J Cancer. 1994;70:199-203.\u003c/li\u003e\n\u003cli\u003ePawloaski J, Kraft AS. Bax-induced apoptotic cell death. Proc Natl Acad Sci USA. 2000;97:529-31.\u003c/li\u003e\n\u003cli\u003eIotsova V, Stehelin D. Down-regulation of fibronectin gene expression by the p53 tumor suppressor protein. Cell Growth Differ. 1996;7:629-34.\u003c/li\u003e\n\u003cli\u003eYou D, Jung SP, Jeong Y, Bae SY, Kim S. Wild-type p53 controls the level of fibronectin expression in breast cancer cells. Oncol Rep. 2017;38:2551-7.\u003c/li\u003e\n\u003cli\u003eWang JP, Hielscher A. Fibronectin: how its aberrant expression in tumors may improve therapeutic targeting. J Cancer. 2017;8:674-82.\u003c/li\u003e\n\u003cli\u003eYou D, Jung SP, Jeong Y, Bae SY, Lee JE, Kim S. Fibronectin expression is upregulated by PI-3K/Akt activation in tamoxifen-resistant breast cancer cells. BMB Rep. 2017;50:615-20.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"Due to technical limitations, tables are only available as a download in the supplemental files section\n"},{"header":"Additional File Information","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1. p53, HNF-1\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e, and ARID1A expression in OCCCa cells.\u003c/strong\u003e (A) Western blot analysis for the indicated proteins in total lysates from four OCCCa cell lines. Note p53 mutation was only presented in ES-2 cells. wt, wild-type. (B) Analysis of TCGA data for associations between \u003cem\u003ep53\u003c/em\u003e gene abnormalities with expression of\u003cem\u003e HNF-1\u003c/em\u003e\u003cem\u003eb\u003c/em\u003e and \u003cem\u003eARID1A\u003c/em\u003e mRNAs (left and right, respectively).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure S2. FN and p53 expression in OECa.\u003c/strong\u003e (A) FN and p53 scores in OECa. (B) FN/p53 IHC scores in the immunoprofile groups (IPGs) of OECa including OCCCa, OHGSeCa, OLGSeCa, OEmCa, and OMuCa. OMuCa are excluded from IPG VII (w/o OMuCa). The data shown are as means\u0026plusmn;SDs.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure S3.\u003c/strong\u003e \u003cstrong\u003eRelationship between FN and p53 expression and prognosis in OCCCa or OHGSeCa. \u003c/strong\u003e(A) OS (left) and PFS (right) relative to FN and p53 expression (upper and lower, respectively) in OCCCa. B) OS (left) and PFS (right) relative to FN and p53 expression (upper and lower, respectively) in OHGSeCa. N, number of cases.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ovarian carcinoma, p53, HNF-1, ARID1A, fibronectin, prognosis, immunophenotype, cell proliferation, cell mobility, apoptosis","lastPublishedDoi":"10.21203/rs.2.22626/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.22626/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground We previously demonstrated that ovarian high grade serous carcinomas (OHGSeCa) and ovarian clear cell carcinomas (OCCCa) with an HNF-1β+/p53+/ARID1A+ immunophenotype were associated with the worst unfavorable prognosis. To clarify the molecular mechanisms underlying this finding, we focused on alterations in the p53 signaling pathway in these tumors. \u003c/p\u003e\u003cp\u003eMethods Changes in cell phenotype and function following knockdown of wild-type p53 (p53-KD) were assessed using OCCCa cells expressing endogenous HNF-1β and ARID1A. The prognostic significance of molecules that were deregulated following p53-KD was also examined using 129 OCCCa/OHGSeCa cases. \u003c/p\u003e\u003cp\u003eResults p53-KD cells had increased expression of Snail, phospho-Akt (pAkt), and pGSK3β, and decreased E-cadherin expression, leading to epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) features. The cells also exhibited acceleration of cell motility and inhibition of cell proliferation and apoptosis. Next generation sequencing revealed that fibronectin (FN) expression was significantly increased in the p53 KD-cells, in line with our observation that wild-type p53 (but not mutant p53) repressed FN1 promoter activity. In addition, treatment of OCCCa cells with FN significantly increased cell migration capacity and decreased cell proliferation rate, independent of induction of EMT features. In clinical samples, FN/p53 scores were significantly higher in OCCCa/OHGSeCa with the HNF-1β+/p53+/ARID1A+ immunophenotype when compared to others. Moreover, high FN/high p53 expression was associated with the worst overall survival and progression-free survival in OCCCa/OHGSeCa patients. \u003c/p\u003e\u003cp\u003eConclusion These findings suggest that upregulation of FN following loss of p53 function may impact the biological behavior of OCCCa/OHGSeCa, particularly in tumors with an HNF-1β+/p53+/ARID1A+ immunophenotype, through alterations in cell mobility and cell proliferation. The accompanying induction of EMT/CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics.\u003c/p\u003e","manuscriptTitle":"Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-03-31 03:21:07","doi":"10.21203/rs.2.22626/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-04-10T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-04-09T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's 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