Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer

Background:The cellular tumor protein p53(TP53)is a tumor suppressor gene that is frequently mutated in human cancers.Among various cancer types,the very aggressive high-grade serous ovarian carcinoma(HGSOC)exhibits the high-est prevalence of TP53 mutations,present in>96%of cases.Despite intensive efforts to reactivate p53,no clinical drug has been approved to rescue p53 func-tion.In this study,our primary objective was to administer in vitro-transcribed(IVT)wild-type(WT)p53-mRNA to HGSOC cell lines,primary cells,and ortho-topic mouse models,with the aim of exploring its impact on inhibiting tumor growth and dissemination,both in vitro and in vivo.Methods:To restore the activity of p53,WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system.Moreover,IVT WT p53 mRNA was delivered into different HGSOC model systems(primary cells and patient-derived organoids)using liposomes and studied for proliferation,cell cycle progression,apoptosis,colony formation,and chromosomal instabil-ity.Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells,followed by ingenuity path-way analysis.In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models.Results:Reactivation of the TP53 tumor suppressor gene was explored in differ-ent HGSOC model systems using newly designed IVT mRNA-based methods.The introduction of WT p53 mRNA triggered dose-dependent apoptosis,cell cycle arrest,and potent long-lasting inhibition of HGSOC cell proliferation.Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling,such as apoptosis,cell cycle regulation,and DNA damage.Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells,under-scoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress.Furthermore,in various mouse models,treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner.Conclusions:The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC,providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

Characterization of candidate factors associated with the metastasis and progression of high-grade serous ovarian cancer

Background:High-grade serous ovarian cancer(HGSOC)is the biggest cause of gynecological cancer-related mortality because of its extremely metastatic nature.This study aimed to explore and evaluate the characteristics of candidate factors associated with the metastasis and progression of HGSOC.Methods:Transcriptomic data of HGSOC patients’samples collected from primary tumors and matched omental metastatic tumors were obtained from three independent studies in the National Center for Biotechnology Information(NCBI)Gene Expression Omnibus(GEO)database.Differentially expressed genes(DEGs)were selected to evaluate the effects on the prognosis and progression of ovarian cancer using data from The Cancer Genome Atlas(TCGA)database.Hub genes’immune landscapes were estimated by the Tumor Immune Estimation Resource(TIMER)database.Finally,using 25 HGSOC patients'cancer tissues and 10 normal fallopian tube tissues,immunohistochemistry(IHC)was performed to quantify the expression levels of hub genes associated with International Federation of Gynecology and Obstetrics(FIGO)stages.Results:Fourteen DEGs,ADIPOQ,ALPK2,BARX1,CD37,CNR2,COL5A3,FABP4,FAP,GPR68,ITGBL1,MOXD1,PODNL1,SFRP2,and TRAF3IP3,were upregulated in metastatic tumors in every database while CADPS,GATA4,STAR,and TSPAN8 were downregulated.ALPK2,FAP,SFRP2,GATA4,STAR,and TSPAN8 were selected as hub genes significantly associated with survival and recurrence.All hub genes were correlated with tumor microenvironment infiltration,especially cancer-associated fibroblasts and natural killer(NK)cells.Furthermore,the expression of FAP and SFRP2 was positively correlated with the International Federation of Gynecology and Obstetrics(FIGO)stage,and their increased protein expression levels in metastatic samples compared with primary tumor samples and normal tissues were confirmed by IHC(P=0.0002 and P=0.0001,respectively).Conclusions:This study describes screening for DEGs in HGSOC primary tumors and matched metastasis tumors using integrated bioinformatics analyses.We identified six hub genes that were correlated with the progression of HGSOC,particularly FAP and SFRP2,which might provide effective targets to predict prognosis and provide novel insights into individual therapeutic strategies for HGSOC.

Prognostic Significance of Hormonal Receptor Status of Malignant Ovarian Tumors

The objective of this study is to investigate hormonal receptor status of MOT （malignant ovarian tumor） and to evaluate its clinical and prognostic significance. Retrospective analysis of the case reports of 284 patients with MOT of different histogenesis, stages I-IV, and immunohistochemical study of paraffin-embedded tissues were performed. Hormonal receptor status of tumors with different morphology genesis was studied and hormonal receptor phenotype of serous OC （ovarian cancer） was determined. The analysis of correlation between the expression of steroid hormone receptors （receptors to estrogens （ER）, progesterone （PR） and testosterone （TR）） in ovarian tumors, histological type of tumors and clinical morphological parameters were performed. Overall and relapse-free survival rates of the patients with serous OC depending on the hormonal receptor phenotype of the tumor were assessed. Presence of positive expression of steroid hormone receptors in serous OC （ER-66.4%, PR^53.4%, TR-53.0%）, mucinous OC （ER-88.0%, PR-84.0%, TR-60.0%） and in sex cord stromal tumors （ER-74.1%, PR and TR-77.8%） is proved by correlation of all steroid receptors expression with morphology type of ovarian tumors （ER - r = 0.4; PR - r = 0.4; TR - r = 0.3; p 〈 0.05）. Direct correlation between hormonal receptor phenotype of serous OC and the age period of the patients was established （r = 0.5; p = 0.002）： postmenopausal women patients reported the most increased frequency of serous OC with positive hormonal receptor tumor phenotypes （52.4%）, in particular during their late post-menopausal period （39.0%）. Significantly low overall survival among the patients with positive hormonal receptor phenotype of serous OC was recorded （29.5±3.4%） in comparison with the same score in the patients with negative phenotype of tumors （44.5±3.7%） （p 〈 0.05）. Multifactor analysis of Cox-regression model has defined that positive hormonal receptor phenotype of serous OC increases the risk of disease relapse （HR 1.4; 95.0% CI 1.1-1.7）, significantly decreases overall survival rates in the patients （HR 1.4; 95.0% CI 1.1-1.8）. Positive hormonal receptor status of MOT is an independent factor of unfavorable clinical progress of tumor process which can be regarded as the criterion for development of the methods of hormonal therapy application in complex treatment of the patients, and demands further large-scale multi-center studies in that direction.

Shortening of the 3＇ untranslated region： an important mechanism leading to overexpression of HMGA2 in serous ovarian cancer

Background Oncofetal protein high-mobility-group AT-hook protein 2 （HMGA2） is reactivated in serous ovarian cancer （SOC） and its overexpression correlates with poor prognosis.To explore the mechanism,we investigated whether HMGA2 could avoid microRNA regulation due to gene truncation or 3＇ UTR shortening by alternative polyadenylation.Methods Real-time reverse transcription polymerase chain reaction （RT-PCR） was used to evaluate the abundance of different regions of HMGA2 mRNA in 46 SOC samples.Rapid amplification of cDNA 3＇ ends （3＇ RACE） and Southern blotting were used to confirm the shortening of 3＇ untranslated region （UTR）.5＇ RACE and Southern blotting were used to prove the mRNA decay.Results No significant difference in the ratio of the stable coding region to the fragile region was observed between SOC and control normal fallopian tubes,indicating that the HMGA2 gene is not truncated in SOC.Varying degrees of 3＇ UTR shortening in SOC samples were observed by comparing the abundance of the proximal region and distal region of the HMGA2 3＇ UTR.The ratio of the proximal to the distal region of the 3＇ UTR correlated significantly with expression of the HMGA2 coding region in SOC （r=0.579,P ＜0.01）.Moreover,although the abundance of the HMGA2 coding region varied,all samples,including the very low expressed samples,exhibit relatively high levels of the proximal 3＇ UTR region,suggesting a dynamic decay of HMGA2 mRNA from the 5＇ end.The shortening of 3＇ UTR and the decay from the 5＇ end were confirmed by 3＇ RACE,5＇ RACE and subsequent Southern blotting.Conclusion Heterogeneous 3＇ UTR lengths render HMGA2 susceptible to different levels of negative regulation by microRNAs,which represents an important mechanism of HMGA2 reactivation in SOC.

Aberrant Alternative Polyadenylation is Responsible for Survivin Up-regulation in Ovarian Cancer

Background： Survivin is an oncoprotein silenced in normal mature tissues but reactivated in serous ovarian cancer （SOC）. Although transcriptional activation is assumed for its overexpression, the long 3＇-untranslated region （3＇-UTR） in survivin gene, which contains many alternate polyadenylation （APA） sites, implies a propensity for posttranscriptional control and therefore was the aim of our study. Methods： The abundance of the coding region, the proximal and the distal region of survivin mRNA 3＇-UTR, was evaluated by real-time polymerase chain reaction （PCR） in SOC samples, cell lines, and normal fallopian tube （NFT） tissues. The APA sites were confirmed by rapid amplification ofcDNA 3＇ ends and DNA sequencing. Real-time PCR were used to screen survivin-targeting microRNAs （miRNAs） that were inversely correlated with survivin. The expression of an inversely correlated miRNA was restored by pre-miRNA transfection or induction with a genotoxic agent to test its inhibitory effect on survivin overexpression. Results： Varying degrees of APA were observed in SOC by comparing the abundance of the proximal and the distal region of survivin 3＇-UTR, and changes of 3＇-UTR correlated significantly with survivin expression （r = 0.708, P 〈 0.01）. The main APA sites are proved at 1197 and 1673 of survivin 3＇-UTR by DNA sequencing. Higher level of 3＇-UTR proximal region than coding region was observed in NFT, as well as in SOC and cell lines. Among the survivin-targeting miRNAs, only a few highly expressed miRNAs were inversely correlated with survivin levels, and they mainly targeted the distal part of the 3＇-UTR. However, in ovarian cancer cells, restoration of an inversely correlated miRNA （miR-34c） showed little effect on survivin expression. Conclusions： In NFT tissues, survivin is not transcriptionally silenced but regulate posttranscriptionally. In SOC, aberrant APA leads to the shortening of survivin 3＇-UTR which enables it to escape the negative regulation of miRNAs and is responsible for survivin up-regulation.

Efficacy of Postoperative Hormone Replacement Therapy on Prognosis of Patients with Serous Ovarian Carcinoma

Background： Ovarian cancer is the most common cause of gynecological cancer-associated death. Iatrogenic menopause might adversely affect the quality of life and health outcomes in young female cancer survivors. We evaluated whether postoperative hormone replacement therapy （HRT） had a negative influence on the progression-free survival （PFS） of patients with papillary serous ovarian cancer （SOC）. Methods： We retrospectively reviewed the medical records of patients with papillary SOC, treated from January 1980 to December 2009, who suffered from menopause with or without HRT. Clinical characteristics of patients were compared between the two groups （HRT and non-HRT）. Blood samples were collected from all the participants to detect serum cancer antigen （CA） 125. Hazard ratios with 95% confidential intervals for each variable were calculated by univariable and multivariable conditional Logistic regression analyses. Results： Among 112 identified patients, 31 were HRT users and 81 were not. The two groups did not significantly differ in median age at diagnosis （t = 0.652, P = 0.513）, International Federation of Gynecology and Obstetrics （FIGO） stage （X2 = 0.565, P = 0.754）, differentiation （X2 = 1.728, P = 0.422）, resection status （X2 = 0.070, P = 0.791）, relapse （X2 = 0.109, P = 0.741）, chemotherapy course （t = -1.079, P - 0.282）, follow-up interval （t = 0.878, P = 0.382）, or PFS （t = 0.580, P = 0.562）. Median Kupperman score at the onset of HRT was 30.81 and 12.19 after the therapy （t： 3.302, P = 0.001）. According to the analysis, the strongest independent variables in predicting PFS were FIGO stage and disease that was not optimally debulked. Conclusions： Postoperative HRT is not a prognostic factor for PFS of patients with papillary SOC. However, multicenter studies are needed to verify and extend our findings.