Expression, association with clinicopathological features and prognostic potential of FOXR2, mTOR, HIF-1α and PKM2 in high-grade serous carcinoma of the ovary

Expression, association with clinicopathological features and prognostic potential of FOXR2, mTOR, HIF-1α and PKM2 in high-grade serous carcinoma of the ovary | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression, association with clinicopathological features and prognostic potential of FOXR2, mTOR, HIF-1α and PKM2 in high-grade serous carcinoma of the ovary Lianguo Hou, Xinyi Dong, Min Li, Jiaxi Yang, Xueli Guo, Juan Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8479280/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background High-grade serous carcinoma (HGSC) of the ovary is the most common and deadly subtype of gynecological malignancy. More and more studies have shown that Forkhead Box R2 (FOXR2) is a carcinogenic driver that can enhance cell growth and tumor formation. However, the role of FOXR2 in the development of HGSC and its underlying molecular mechanisms remain unclear. This study investigated the role of FOXR2 in HGSC progression and its relationship with glycometabolic reprogramming, specifically the mTOR/HIF-1α signaling pathway and its downstream key glycolytic enzyme, PKM2. Methods Immunohistochemistry for FOXR2, mTOR, HIF-1α and PKM2 was performed on formalin-fixed paraffin-embedded HGSC tissue and normal fallopian tube tissue. Kaplan Meier analysis and Cox proportional hazards regression analysis were used to evaluate the overall survival curve and independent prognostic factors. Results In the present study, FOXR2, mTOR, HIF-1α and PKM2 proteins are highly expressed in HGSC, and are positively correlated with FIGO stage of ovarian cancer. In addition, these four proteins were positively correlated with each other. Overall survival analysis showed that the expression levels of FOXR2, mTOR, HIF-1α and PKM2 proteins, as well as FIGO stage, were correlated with patient prognosis. In addition, FIGO staging was an independent prognostic factor affecting the poor prognosis of patients. Conclusions Taken together, the present results suggested that FOXR2, mTOR, HIF-1α and PKM2 have crucial roles in the progression and prognosis of HGSC and may be potential therapeutic targets. High-grade serous carcinoma of the ovary FOXR2 glycometabolic reprogramming mTOR/HIF-1α signaling pathway PKM2 prognosis Figures Figure 1 Figure 2 Background Ovarian cancer is one of the most lethal gynecological malignancies. High-grade serous carcinoma (HGSC) is the most common type of ovarian cancer and most patients present with advanced stage disease (approximately 80%). Despite effective treatment, 75% of women with HGSC will develop recurrence and ultimately succumb to their disease, and the survival rate and prognosis are still poor [ 1 ]. Therefore, novel molecular markers or targets may contribute to the diagnosis and treatment of HGSC. Forkhead-Box (FOX) family members participate in the occurrence and development of tumors as tumor suppressor genes or proto-oncogenes [ 2 ]. Among them, Forkhead Box R2 (FOXR2), located on the X chromosome, has been implicated as an oncogene in a subset of cancers, which is normally only expressed in human testicular tissue [ 3 ]. In recent years, it has been found that it plays an important role in the occurrence and development of various malignant tumors (such as glioma [ 4 ], medulloblastoma [ 5 ], non-small cell lung cancer [ 6 ], and ovarian cancer [ 7 ]). On the one hand, FOXR2 can interact with MYC and regulate the transcriptional activity of MYC, thus promoting the proliferation of cancer cells [ 8 ]. On the other hand, the expression of FOXR2 is consistent with the regulation of ETS transcriptional circuits, which can activate the transcriptional activity of ETS family, and the two can synergistically drive tumor formation [ 3 ]. In 2022, Jessica et al. first reported in Cancer Research that that the FOXR2 gene, which is not normally expressed in most tissues of the body, was aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors [ 3 ]. It can be seen that FOXR2, as a kind of proto-oncogene discovered in recent years, is expected to become a new marker for tumor diagnosis and treatment. However, how FOXR2 plays a role in the occurrence and development of HGSC and its underlying molecular mechanisms remain unclear. Warburg et al. elaborated on the abnormal metabolic mode of tumor cells—Warburg effect, that is, under the condition of sufficient oxygen, malignant tumor cells will still carry out active glucose glycolysis to provide energy for cells [ 9 ]. As a key enzyme in the glycolytic pathway, PKM2 is abnormally expressed in many tumors (such as glioma [ 10 ], liver cancer [ 11 ], gastric cancer [ 12 ] and ovarian cancer [ 13 ]), and excess PKM2 can enter the nucleus to activate downstream gene expression, regulate glycolytic metabolism, and induce tumor occurrence [ 14 ]. mTOR is an atypical serine/threonine protein kinase [ 15 ]. As a key regulator of glucose metabolism, MTOR is involved in the regulation of cell growth, metabolism and survival, and plays a role by activating its downstream molecule HIF-1α [ 16 , 17 ]. HIF-1α promotes glucose uptake and increases the activity of glycolysis by regulating the expression of glycolytic enzyme. More and more research showed that the mTOR/HIF-1α signaling pathway interacts with PKM2, and then participates in the glucose metabolic reprogramming in tumor cells, providing energy for the growth and development of tumor cells, and thus promoting the occurrence and development of tumors. Studies have shown that in pancreatic cancer, mTOR/HIF-1α signaling pathway up-regulates the expression of PKM2, enhances the activity of cell glycolysis, and further promotes tumor growth [ 18 ]. However, whether the mTOR/HIF-1α/PKM2 signaling pathway plays a role in the occurrence and development of HGSC is still unclear. The purpose of this study was to compare and analyze the expression differences of FOXR2, mTOR, HIF-1α and PKM2 proteins in HGSC tissues and normal fallopian tube tissues, and to preliminatively explore the correlation between the expression of these four proteins and the occurrence of HGSC and the clinicopathological characteristics of patients. Methods Tissue specimens and clinical data The present study was a retrospective study. A total of 104 cases were included and their archival paraffin-embedded HGSC tissue and normal fallopian tube tissue specimens collected between January 2015 and December 2022 at the Department of Pathology of the Second Hospital of Hebei Medical University (Shijiazhuang, China) were obtained. The present study was approved by the Ethics Committees of the Second Hospital of Hebei Medical University (Shijiazhuang, China) and informed consent was obtained from each participant. According to the 2014 International Federation of Obstetrics and Gynecology (FIGO) criteria, these cases were classified as: Stage I 16, Stage II 18, Stage III 34 and Stage IV 6. At the same time, 30 normal fallopian tube tissues were also selected as controls. Among the 74 patients with HGSC, the age range was 38 to 86 years, with a mean age of 56.5 ± 10.0 years. The overall survival time was calculated from the date of diagnosis to the date of death or the date of the last follow-up or last time-point the patient was known to be alive, defined as censored (last evaluated January 31, 2024). Immunohistochemical staining Immunohistochemical staining for FOXR2, mTOR, HIF-1α and PKM2 proteins in HGSC sections was performed using a ready-to-use Biotin-Streptavidin/Horseradish peroxidase (HRP) Detection system (OriGene Technologies, Inc.). Paraffin sections (4 µm) were deparaffinized in xylene and then rehydrated with alcohol; heat-induced antigen recovery was performed in citrate buffer (pH 6.0) for 5 min at 120˚C or EDTA antigen retrieval solution (pH 8.0) for 5 min at 120˚C, followed by quenching of the endogenous peroxidase using 3% H 2 O 2 for 20 min at room temperature. Goat serum (OriGene Technologies, Inc.) was used for blocking nonspecific binding sites for 30 min at room temperature. Slides were then incubated with primary antibodies [anti-FOXR2 (1:200 dilution; Abcam; ab244513); anti-mTOR (1:500 dilution; HUABIO; ET1608-5); anti-HIF-1α (1:500 dilution; Gene Tex; GTX127309); anti-PKM2 (1:400 dilution; Cell Signaling; CST4053)] at 4˚C overnight. After washing with PBS, a Biotin-Streptavidin HRP Detection system (working solution; OriGene Technologies, Inc.; SP-9000/9001/9002) was used for detection of the antigen-antibody complex, which was visualized with diaminobenzidine as the chromogen. Finally, counterstaining was performed using Mayer's hematoxylin and the sections were dehydrated in alcohol prior to mounting. As a negative control, the primary antibody was omitted; no staining was observed. Scoring of staining Immunohistochemical staining was assessed using semi-quantitative scoring by two independent investigators (JW and YHL) who were blinded to the histopathological features and clinicopathological data of the samples. Before the scoring was performed, a set of criteria were established based on the literature with minor modifications in terms of the percentage scores of positive cells and the final score. The degree of staining was determined by the percentage of positive cells and the staining intensity. Cells that were stained with yellow or brown color were considered as positively-stained cells. Necrotic areas and areas with extensive bleeding were not included. FOXR2 protein was localized in the nucleus. Percentage scoring for FOXR2 protein was as follows: 1, ≤ 10%; 2, 11–50%; 3, 51–75%; and 4, > 75%; while the intensity scoring was as follows: 0, no staining; 1, light yellow; 2, brownish-yellow; and 3, brown-brown. The staining degree was calculated as follows: Overall score = percentage score × intensity score. Overall scores of 0–3 indicated low expression, whereas overall scores of 4 or more indicated high expression [ 19 ]. Positive expression of mTOR protein mainly localized in the cytoplasm. Percentage scoring for mTOR protein was as follows: 0, 50%. while the intensity scoring was as follows: 0, no staining; 1, light yellow; 2, brownish-yellow; and 3, brown-brown. The staining degree was calculated as follows: Overall score = percentage score × intensity score. Overall scores of 0–3 indicated low expression, whereas overall scores of 4 or more indicated high expression [ 20 ]. Positive expression of HIF-1α protein was identified in the cytoplasm or nucleus. Percentage scoring for HIF-1α protein was as follows: 0, ≤ 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, > 75%; while the intensity scoring was as follows: 0, no staining; 1,yellow; 2, brown; and 3, brownish-brown. The staining degree was calculated as follows: Overall score = percentage score × intensity score. Overall scores of 0–4 indicated low expression, whereas overall scores of 5 or more indicated high expression [ 21 , 22 ]. Positive expression of PKM2 protein was identified in the cytoplasm and nucleus. Percentage scoring for PKM2 protein was as follows: 1, 67%; while the intensity scoring was as follows: 1, weak expression; 2, moderate expression; and 3, strong expression. The staining degree was calculated as follows: Overall score = percentage score × intensity score. Overall scores of 1–3 indicated low expression, whereas overall scores of 4 or more indicated high expression [ 23 ]. Based on this, the patients we divided into low and high expression groups. Statistical analysis All statistical analyses were performed using SPSS 21.0 software (IBM Corp.). χ 2 tests were used to compare the staining results for target proteins in different groups and for comparisons with clinicopathological characteristics. The correlations between FOXR2 and other proteins were determined using nonparametric Spearman's correlation tests. Kaplan-Meier analysis was performed to draw overall survival curves and statistical significance was assessed using log-rank tests. Univariate and multivariate analysis were performed using the Cox proportional hazard regression model. P < 0.05 was considered to indicate statistical significance. Results FOXR2, mTOR, HIF-1α and PKM2 levels in HGSC tissues Immunohistochemical analyses were performed to determine the expression levels and distributions of target proteins. Representative images and the results are provided in Fig. 1 and Table 1 , respectively. For the 30 normal fallopian tube tissue, high expression of FOXR2, mTOR, HIF-1α and PKM2 was observed in 0 (0.0%), 3 (10.0%), 0 (0.0%) and 4 (13.3%) cases, respectively. For the HGSC tissues, high expression of these proteins was observed in 55 (74.3%), 57 (77%), 53 (71.6%) and 58 (78.4%) of cases, respectively. Thus, high expression of these markers was significantly more frequent in HGSC than in normal fallopian tube tissue (P < 0.05). Table 1 The expression of FOXR2, mTOR, HIF-1α and PKM2 in normal fallopian tubes and high-grade serous ovarian cancer Tissue type n FOXR2 P mTOR P HIF−1α P PKM2 P 0.000 0.000 0.000 0.000 Fallopian tube 30 0(0.0%) 3(10.0%) 0(0.0%) 4(13.3%) High-grade serous ovarian cancer 74 55(74.3%) 57(77.0%) 53(71.6%) 58(78.4%) Clinicopathological characteristics of FOXR2, mTOR, HIF-1α and PKM2 in HGSC tissues Next, the associations of protein levels and clinicopathological features in patients with HGSC were assessed by X 2 tests and Spearman correlation analysis. As presented in Table 2 , the expression levels of FOXR2, mTOR, HIF-1α and PKM2 were significantly associated with FIGO grade (P < 0.05). Moreover, high levels of FOXR2 were associated with age in patients (P 0.05; Table 2 ). Furthermore, Spearman correlation analysis indicated that the levels of FOXR2, mTOR, HIF-1α and PKM2 were correlated with the FIGO grade (r FOXR2 =0.243, P < 0.05; r mTOR =0.311, P < 0.05; r HIF−1α =0.285, P < 0.05; r PKM2 =0.326, P < 0.05), while the levels of FOXR2 were correlated with the patient age (r FOXR2 =-0.311, P < 0.05). Taken together, these results indicated that FOXR2, mTOR, HIF-1α and PKM2 may have roles in HGSC progression. Table 2 Relationship among the expression of FOXR2, mTOR, HIF-1α and PKM2 with clinical pathological characteristics in high-grade serous ovarian cancer Pathological Characteristics FORX2 P mTOR P HIF−1α P PKM2 P High Low High Low High Low High Low Age <56 years 34 5 33 6 30 9 34 5 0.007 0.104 0.292 0.053 ≥ 56 years 21 14 24 11 23 12 24 11 FIGO grade I 11 5 12 4 12 4 11 5 II 10 8 8 10 6 12 10 8 III 28 6 0.037 31 3 0.007 29 5 0.014 31 3 0.005 IV 6 0 6 0 6 0 6 0 Correlations among FOXR2, mTOR, HIF-1α and PKM2 in HGSC tissues As presented in Table 3 , Spearman's rank correlation analyses revealed that the correlation among FOXR2, mTOR, HIF-1α and PKM2 was significant in HGSC (P < 0.05). The expression of mTOR, HIF-1α and PKM2 is positively correlated with FOXR2 (r mTOR =0.635, r HIF−1α =0.659, r PKM2 =0.743, P < 0.05). And the expression of HIF-1α and PKM2 is positively correlated with mTOR (r HIF−1α =0.796, r PKM2 =0.806, P < 0.05). Moreover, the expression of HIF-1α is positively correlated with PKM2 expression (r = 0.762, P < 0.05). These results suggested that FOXR2 may interact with the other proteins to mediate invasive growth of HGSC. Table 3 Correlations among FOXR2 and mTOR, HIF-1α, PKM2 in high-grade serous ovarian cancer mTOR HIF−1α PKM2 protein High Low r P High Low r P High Low r P FOXR2 0.635 0.000 0.659 0.000 0.743 0.000 High 51 4 49 6 53 2 Low 6 13 4 15 5 14 mTOR 0.796 0.000 0.806 0.000 High 52 5 55 2 Low 1 16 3 14 HIF−1α 0.762 0.000 High 52 1 Low 6 15 Prognostic value of FOXR2, mTOR, HIF-1α and PKM2 in patients with HGSC tissues As presented in Fig. 2 , overall survival analysis indicated that higher expression levels of FOXR2, mTOR, HIF-1α and PKM2 were associated with significantly poorer overall survival than that for patients with lower levels of these proteins. Indeed, FOXR2, mTOR, HIF-1α and PKM2 levels were correlated with patient prognosis (P < 0.05, Fig. 2 A-D). Other conventional prognostic factors, including FIGO grade, were correlated with patient prognosis (P < 0.05, Fig. 2 E). The median survival time in patients with FIGO grade I was 79 months, while the median survival time in patients with FIGO grade IV was only 21 months. However, age did not influence the clinical prognosis (P > 0.05; Fig. 2 F). Cox proportional hazards models was further used to conduct univariate and multivariate analysis, and the correlation among the expression of FOXR2, mTOR, HIF-1α and PKM2 proteins and overall survival were investigated (Table 4 ). The results of univariate analysis showed that the expression levels of FOXR2, mTOR, HIF-1α and PKM2 proteins and FIGO grade were the factors affecting the risk of poor prognosis in FOXR2, mTOR, HIF-1α and PKM2 (P < 0.05). Multivariate analysis showed that FIGO grade was an independent prognostic factor for HGSC (P < 0.05). Table 4 Univariate and multivariate analysis of the prognostic factors of patients with high-grade serous ovarian cancer Variable Univariate analysis Multivariate analysis HR(95% CI) P HR(95% CI) P FOXR2 (High vs Low expression) 2.588 (1.010–6.632) 0.048 0.455 (0.105–1.981) 0.294 mTOR (High vs Low expression) 4.395 (1.350−14.307) 0.014 1.521 (0.256–9.047) 0.645 HIF−1α (High vs Low expression) 3.200 (1.249–8.196) 0.015 1.678 (0.349–8.076) 0.518 PKM2 (High vs Low expression) 7.109 (1.703–29.679) 0.007 4.339 (0.584–32.214) 0.151 FIGO grade (II /III/IVvs I) 0.001 0.033 Age (≥ 56years vs <56years) 0.822 (0.436–1.550) 0.544 These results indicate that the expression levels of FOXR2, mTOR, HIF-1α and PKM2 proteins and FIGO stage are factors affecting the risk of poor prognosis of HGSC. Among them, FIGO stage is an independent predictor of prognosis of HGSC. Discussion Ovarian cancer is the main cause of death of female reproductive system malignant tumors [ 24 ]. Among them, HGSC is the histological subtype with the highest incidence and the most aggressive among ovarian cancers, often accompanied by large-scale pelvic and abdominal metastasis. More than 75% of patients are diagnosed with advanced stage, which is the main cause of death of ovarian cancer [ 25 ]. Although certain progress has been made in treatment in recent years, it is prone to recurrence after initial treatment. After relapse, the treatment effect is not good, the overall survival rate is still low, and the prognosis is not optimistic. Therefore, it is necessary to further study the molecular mechanism of the occurrence and development of HGSC. FOXR2 is a member of the forkhead frame transcription factor family located on the X chromosome. It is expressed in normal tissues and only in testis, but it is highly expressed in a variety of malignant tumors, and promotes the proliferation of cancer cells, and plays a certain regulatory role in the occurrence, development and prognosis of tumors. In studies of prostate cancer [ 26 ], non-small cell lung cancer [ 6 ] and colorectal cancer [ 27 ], FOXR2 plays a key role in tumor cell proliferation and invasion by inhibiting the activation of Wnt/β-catenin and Shh signaling pathways. In addition, in glioma, FOXR2 promotes the proliferation, migration and invasion of glioma cells by regulating various cell cycle regulatory genes and the PI3K-Akt-p27 signaling pathway [ 4 ]. On the one hand, the increased expression of FOXR2 in ovarian cancer is related to cell growth, migration and epithelial-mesenchymal transformation. On the other hand, FOXR2 is abnormally high expressed in ovarian cancer and regulates the malignant behavior of ovarian cancer cells by stimulating angiogenesis and activating Hedgehog signaling pathway [ 7 ]. As a novel protomogenic gene discovered in recent years, the study of FOXR2 provides a new idea for tumor diagnosis and treatment. However, how FOXR2 plays a role in the occurrence and development of HGSC and its potential molecular mechanism remain to be studied. In order to further understand the expression of FOXR2 protein in HGSC, immunohistochemistry was used to detect FOXR2 protein in 74 cases of HGSC and 30 cases of normal fallopian tube tissue. The results showed that the positive expression level of FOXR2 in HGSC was significantly higher than that in normal fallopian tube tissue. Moreover, the expression level of FOXR2 was positively correlated with FIGO stage of HGSC, suggesting that FOXR2 protein may be involved in the occurrence and development of HGSC, and is closely related to its malignant process. In recent years, a series of studies have shown that the reprogramming of energy metabolism has become one of the core characteristics of tumors [ 28 ]. Among them, glucose metabolic reprogramming, characterized by increased proportion of glycolytic pathways, is one of the most prominent metabolic changes in tumor cells [ 29 ]. Pyruvate kinase plays an important role in the glycolysis pathway by catalyzing the last step of glycolysis, which promotes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP, providing energy substances for cell metabolism [ 30 ]. There are four types of pyruvate kinase subtypes (M1, M2, L and R) in mammals, among which PKM2 can promote the transformation of more polysaccharide glycolysis intermediates, and mainly exists in anabolic active cells such as embryonic cells, stem cells and tumor cells [ 31 ]. More and more studies have shown that PKM2 is highly expressed in a variety of tumor cells and is closely related to malignant behaviors such as proliferation, migration and invasion of tumor cells. For example, in malignant tumors such as glioma [ 11 ] and gastric cancer [ 12 ], glycolysis is enhanced through up-regulation of PKM2 expression level, which further promotes tumor occurrence and development. In addition, PKM2 is highly expressed in ovarian cancer and produces high concentration of lactic acid through regulation of glycolytic metabolism, which promotes the proliferation and metastasis of ovarian cancer and is closely related to its poor prognosis and low survival rate [ 13 ]. mTOR, located in the short arm of human chromosome 1, is an evolutionarily conserved atypical protein kinase. It regulates the metabolic pathways of cell proliferation, growth and metabolism by sensing the nutritional state and energy level of cells to maintain normal physiological functions. However, in tumor cells, the mTOR signaling pathway is over-activated, which not only promotes the proliferation, invasion and metastasis of tumor cells, but also inhibits the apoptosis and autophagy of tumor cells, which is the key to promoting the growth and development of tumor cells [ 32 ]. By regulating its downstream transcription factors (such as HIF-1), mTOR plays a central role in glucose metabolism, protein and lipid synthesis, mitochondrial and lysosomal biosynthesis, thereby promoting cell growth and proliferation. HIF-1 is a transcription factor that plays an important role in a variety of physiological and pathological processes. HIF-1α is the active subunit of HIF-1, and its expression is closely related to the tension of oxygen in the environment. It is a key transcription factor regulating hypoxic stress response in tumor cells, and is widely involved in the malignant proliferation and invasive progression of tumors [ 33 ]. More and more studies have shown that mTOR is involved in malignant processes such as tumor growth and metastasis by activating its downstream transcription factor HIF-1α. For example, in prostate cancer, mTOR plays a key role in tumor growth and metastasis by regulating HIF-1α protein accumulation and transcriptional activity in tumor cells [ 34 ]. In thyroid cancer, activated mTOR/HIF-1α signaling pathway can enhance cellular glycolysis and further promote tumor cell proliferation and metastasis [ 35 ]. In addition, a series of studies have shown that the interaction between mTOR/HIF-1α signaling pathway and PKM2 enhances the glycolytic metabolism of tumor cells and further promotes the occurrence and development of tumors. Studies have found that mTOR signaling pathway can affect the expression level of PKM2 in human pancreatic cancer (PANC-1), prostate cancer (PC3) and liver cancer (HepG2) cell lines, thereby activating glycolytic metabolism and promoting the growth of tumor cells [ 36 ]. Xiaoyu H et al. found that in esophageal squamous cell carcinoma, the mTOR/HIF-1α signaling pathway was activated, which promoted the increase of PKM2 expression, thus promoting the glycolytic metabolism of tumor cells, converting glucose into lactic acid to produce energy, and further affecting the energy metabolism and growth of tumor cells. It plays an important regulatory role in the development of esophageal squamous cell carcinoma [ 37 ]. Immunohistochemical results of this study showed that the expression of mTOR, HIF-1α and PKM2 in HGSC was significantly higher than that in normal fallopian tube tissue. Statistical analysis of clinicopathological features showed that the expression levels of mTOR, HIF-1α and PKM2 proteins in HGSC were positively correlated with their FIGO stage, suggesting that these three proteins may be involved in the occurrence and development of HGSC. In addition, the correlation analysis results with the age of patients showed that the expression level of FOXR2 protein was negatively correlated with the age of patients, while the expression of the other three proteins was not significantly correlated with the age of patients. Further correlation analysis showed that FOXR2, mTOR, HIF-1α and PKM2 were all positively correlated, suggesting that FOXR2 may have synergistic effect with mTOR/HIF-1α/PKM2 signaling pathway. We speculate that FOXR2 may promote the occurrence and development of HGSC by regulating glucose metabolic reprogramming through mTOR/HIF-1α/PKM2 signaling pathway. More and more studies have shown that FOXR2, mTOR, HIF-1α and PKM2 proteins are abnormally expressed in a variety of malignant tumors, and are closely related to the poor prognosis of tumors. For example, in breast cancer and endometrioid carcinoma, the high expression of FOXR2 is an independent factor affecting the poor prognosis of tumor patients [ 38 , 39 ]. In gastric cancer, mTOR is highly expressed, which regulates malignant behaviors such as proliferation and metastasis of tumor cells, and is also an independent factor affecting the prognosis of gastric cancer [ 40 ]. HIF-1α plays a crucial role in promoting cervical cancer cell migration and invasion, and is a prognostic biomarker of cervical cancer [ 41 ]. Wang et al. found that high expression of PKM2 is associated with poor prognosis of breast cancer patients, and PKM2 is an independent prognostic predictor of surgically resected breast cancer patients and neoadjuvant chemotherapy patients [ 42 ]. The results of overall survival analysis in this study showed that the expression of FOXR2, mTOR, HIF-1α and PKM2 and FIGO stage of HGSC were factors affecting the risk of poor prognosis of HGSC. Further multifactor analysis showed that FIGO stage was an independent predictor of prognosis of HGSC. Conclusion In summary, FOXR2, mTOR, HIF-1α and PKM2 proteins are highly expressed in HGSC, and are positively correlated with FIGO stage of ovarian cancer. In addition, these four proteins were positively correlated with each other. Therefore, we speculated that FOXR2 might enhance the glycolysis of tumor cells and regulate the reprogramming of glucose metabolism through the mTOR/HIF-1α/PKM2 signaling pathway, and thus participate in the occurrence and development of high-grade ovarian serous cancer. Overall survival analysis showed that the expression levels of FOXR2, mTOR, HIF-1α and PKM2 proteins, as well as FIGO stage, were correlated with patient prognosis. In addition, FIGO staging was an independent prognostic factor affecting the poor prognosis of patients. These results indicate that the high expression of FOXR2, mTOR, HIF-1α and PKM2 play an important role in the occurrence, development and prognosis of HGSC, and may become a new direction for the prevention and treatment of HGSC in the future. Abbreviations HGSC High-grade serous carcinoma FOX Forkhead Box FOXR2 Forkhead Box R2 HRP Horseradish peroxidase Declarations Consent for publication Not applicable. Competing interests The authors declare no competing interests. Conflict of interest The authors declare that they have no competing interests. Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the second hospital of Hebei Medical University (Approval No.: 2024-R614). All experiments were performed in accordance with relevant guidelines and regulations such as the Declaration of Helsinki and the patients signed the informed consent form and agreed to be published. Funding This work was supported by the Government-funded Program for Clinical Medicine Talents‌‌ (Grant No. ZF2025214), the Medical Science Research Project of Hebei Province (No. 20230811), the China Anti-Cancer Association Regional Cancer Research Cultivation Fund (No. CESTDQLCORP300-09), the Beijing Top-Doctors Charity Foundation and China Medical and Health Development Foundation and Hebei Natural Science Foundation (Grant No. H2025206715). Author Contribution Lianguo Hou: Conceptualization; writing original draft. Xinyi Dong: Resources; conceptualization. Min Li: Resources. Jiaxi Yang: Resources. Xueli Guo: Resources. Juan Wang: Supervision; visualization. Jun Zhang: Supervision; visualization; writing review and editing. Acknowledgements Not applicable. Data Availability All data were presented in this paper and there were no additional supporting files. References Prat J, D'Angelo E, Espinosa I. 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Criteria of immunohistochemical reaction results[J]. CHINA Oncol. 1996;6:229–31. Lu J, Pan Y, Xia X, Gu Y, Lei Y. Prognostic Significance of mTOR and PTEN in Patients with Esophageal Squamous Cell Carcinoma. Biomed Res Int. 2015; 2015: 417210. Yin X, Xia J, Sun Y, Zhang Z. CHCHD2 is a potential prognostic factor for NSCLC and is associated with HIF-1a expression. BMC Pulm Med. 2020;20:40. Li W, Zheng G, Xia J, Yang G, Sun J, Wang X, Wen M, Sun Y, Zhang Z, Jin F. Cell cycle-related and expression-elevated protein in tumor overexpression is associated with proliferation behaviors and poor prognosis in non-small-cell lung cancer. Cancer Sci. 2018;109:1012–23. Mohammad GH, Olde Damink SW, Malago M, Dhar DK, Pereira SP. Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome. PLoS ONE. 2016;11:e0151635. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71:209–49. Kurman RJ, Shih Ie M. The Dualistic Model of Ovarian Carcinogenesis: Revisited, Revised, and Expanded. Am J Pathol. 2016;186:733–47. Xu W, Chang J, Liu G, Du X, Li X. Knockdown of FOXR2 suppresses the tumorigenesis, growth and metastasis of prostate cancer. Biomed Pharmacother. 2017;87:471–5. Lu SQ, Qiu Y, Dai WJ, Zhang XY. FOXR2 Promotes the Proliferation, Invasion, and Epithelial-Mesenchymal Transition in Human Colorectal Cancer Cells. Oncol Res. 2017;25:681–9. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74. Li M, Bu X, Cai B, Liang P, Li K, Qu X, Shen L. Biological role of metabolic reprogramming of cancer cells during epithelial–mesenchymal transition (Review). Oncol Rep. 2019;41:727–41. Israelsen WJ, Dayton TL, Davidson SM, Fiske BP, Hosios AM, Bellinger G, Li J, Yu Y, Sasaki M, Horner JW, Burga LN, Xie J, Jurczak MJ, DePinho RA, Clish CB, Jacks T, Kibbey RG, Wulf GM, Di Vizio D, Mills GB. Cantley LC and Vander Heiden MG. PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells. Cell. 2013;155:397–409. Mazurek S, Boschek CB, Hugo F, Eigenbrodt E. Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin Cancer Biol. 2005;15:300–8. Murugan AK, mTOR. Role in cancer, metastasis and drug resistance. Semin Cancer Biol. 2019;59:92–111. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol (1985). 2000; 88: 1474-80. Hudson CC, Liu M, Chiang GG, Otterness DM, Loomis DC, Kaper F, Giaccia AJ, Abraham RT. Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin. Mol Cell Biol. 2002;22:7004–14. Liu Y, Liao L, An C, Wang X, Li Z, Xu Z, Liu J, Liu S. alpha-Enolase Lies Downstream of mTOR/HIF1alpha and Promotes Thyroid Carcinoma Progression by Regulating CST1. Front Cell Dev Biol. 2021;9:670019. Sun Q, Chen X, Ma J, Peng H, Wang F, Zha X, Wang Y, Jing Y, Yang H, Chen R, Chang L, Zhang Y, Goto J, Onda H, Chen T, Wang MR, Lu Y, You H, Kwiatkowski D, Zhang H. Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth. Proc Natl Acad Sci U S A. 2011;108:4129–34. Xiaoyu H, Yiru Y, Shuisheng S, Keyan C, Zixing Y, Shanglin C, Yuan W, Dongming C, Wangliang Z, Xudong B, Jie M. The mTOR Pathway Regulates PKM2 to Affect Glycolysis in Esophageal Squamous Cell Carcinoma. Technol Cancer Res Treat. 2018;17:1533033818780063. Song H, He W, Huang X, Zhang H, Huang T. High expression of FOXR2 in breast cancer correlates with poor prognosis. Tumour Biol. 2016;37:5991–7. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8479280","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":580338111,"identity":"9aa047ae-068c-4000-bdee-009a04ec9c49","order_by":0,"name":"Lianguo Hou","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lianguo","middleName":"","lastName":"Hou","suffix":""},{"id":580338112,"identity":"f6946337-6395-47ad-8e59-0e603b5e6a32","order_by":1,"name":"Xinyi Dong","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Dong","suffix":""},{"id":580338113,"identity":"d724b918-1dcb-43b2-b228-7b091e931175","order_by":2,"name":"Min Li","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Li","suffix":""},{"id":580338114,"identity":"0914ecdf-0c27-42fb-b070-68a32a0cda7c","order_by":3,"name":"Jiaxi Yang","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiaxi","middleName":"","lastName":"Yang","suffix":""},{"id":580338116,"identity":"8de29d78-c641-4cad-b95c-b6821adae36f","order_by":4,"name":"Xueli Guo","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xueli","middleName":"","lastName":"Guo","suffix":""},{"id":580338117,"identity":"6b2c5898-7a5c-47c7-a1f2-ed03f220e35d","order_by":5,"name":"Juan Wang","email":"","orcid":"","institution":"Second Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Wang","suffix":""},{"id":580338118,"identity":"033f161b-8c04-46aa-8638-69fa1d4ef43e","order_by":6,"name":"Jun Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIie3OMUvDQBTA8XccXBBejeByIZV8hRyB4CD4VdIlUwdBEIcMB4F0Kq7xY7jEjpHATQeuQhwChUwOzSI4VGy7lsR263D/4Q0PfrwHYDKdYB61u2b1mKDtLRUQuVv6g0TMIBC5VmNHxvFhBN4hvDzL6I1fTsPDCEkhpsAY+qX+brpFMZFWWnBIPnuJRUEt73CMztv8VTzreiJRPXBQ7cAVMgtyzvC8GhXuKNsQPg05kVX/ZxVlLvoUQWHrrrfE+/qPsA2JKF5oZC7ZXcFhIlKkIi8VOjkLnbmugwzj++tI9RPP1qTpfpNbm9OW/yzqqyerevlYJQOP7cW2IzoCmEwmk2m/P2xNVLf4y40vAAAAAElFTkSuQmCC","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-12-30 08:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8479280/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8479280/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101274426,"identity":"4600ecf0-95f5-4d69-a7c4-b78645635492","added_by":"auto","created_at":"2026-01-28 03:10:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5394961,"visible":true,"origin":"","legend":"\u003cp\u003eFOXR2, mTOR, HIF-1α and PKM2proteins in High-grade serous carcinoma were detected by immunohistochemistry. FOXR2 was localized in the nuclei of tumor cells. mTOR was localized in the cytoplasm of tumor cells. HIF-1α and PKM2protein were expressed in the cytoplasm and the cell nuclei. Low expression of FOXR2, mTOR, HIF-1α and PKM2 was present in fallopian tube, while high expression was observed in High-grade serous carcinoma (magnification, x200).\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8479280/v1/5c15dfb90dfbefc7d96c34eb.png"},{"id":101274416,"identity":"bccce6bc-cb0f-4a5a-9b66-2a5eeaca941a","added_by":"auto","created_at":"2026-01-28 03:10:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":451910,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier analysis of overall survival in High-grade serous carcinoma. (A-D) The survival of those patients with with high levels of (A) FOXR2, (B) mTOR, (C) HIF-1α and (D) PKM2 was significantly worse than that of patients with low levels (P\u0026lt;0.05). (E) With the increase of the tumor Figo-grade of high-grade serous ovarian cancer, the survival time of patients was shorter (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). (F) The age of patients was not associated with overall survival (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05).\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8479280/v1/e2cb53df3dc2422341be7de2.png"},{"id":101274454,"identity":"880b4607-e182-46d8-9c10-a2e755e980d1","added_by":"auto","created_at":"2026-01-28 03:10:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6790746,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8479280/v1/5cf3c212-673b-49d4-a626-ed4e8d0cae04.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression, association with clinicopathological features and prognostic potential of FOXR2, mTOR, HIF-1α and PKM2 in high-grade serous carcinoma of the ovary","fulltext":[{"header":"Background","content":"\u003cp\u003eOvarian cancer is one of the most lethal gynecological malignancies. High-grade serous carcinoma (HGSC) is the most common type of ovarian cancer and most patients present with advanced stage disease (approximately 80%). Despite effective treatment, 75% of women with HGSC will develop recurrence and ultimately succumb to their disease, and the survival rate and prognosis are still poor [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, novel molecular markers or targets may contribute to the diagnosis and treatment of HGSC.\u003c/p\u003e \u003cp\u003eForkhead-Box (FOX) family members participate in the occurrence and development of tumors as tumor suppressor genes or proto-oncogenes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among them, Forkhead Box R2 (FOXR2), located on the X chromosome, has been implicated as an oncogene in a subset of cancers, which is normally only expressed in human testicular tissue [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In recent years, it has been found that it plays an important role in the occurrence and development of various malignant tumors (such as glioma [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], medulloblastoma [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], non-small cell lung cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and ovarian cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]). On the one hand, FOXR2 can interact with MYC and regulate the transcriptional activity of MYC, thus promoting the proliferation of cancer cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. On the other hand, the expression of FOXR2 is consistent with the regulation of ETS transcriptional circuits, which can activate the transcriptional activity of ETS family, and the two can synergistically drive tumor formation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In 2022, Jessica et al. first reported in Cancer Research that that the FOXR2 gene, which is not normally expressed in most tissues of the body, was aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It can be seen that FOXR2, as a kind of proto-oncogene discovered in recent years, is expected to become a new marker for tumor diagnosis and treatment. However, how FOXR2 plays a role in the occurrence and development of HGSC and its underlying molecular mechanisms remain unclear.\u003c/p\u003e \u003cp\u003eWarburg et al. elaborated on the abnormal metabolic mode of tumor cells\u0026mdash;Warburg effect, that is, under the condition of sufficient oxygen, malignant tumor cells will still carry out active glucose glycolysis to provide energy for cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As a key enzyme in the glycolytic pathway, PKM2 is abnormally expressed in many tumors (such as glioma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], liver cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], gastric cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and ovarian cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]), and excess PKM2 can enter the nucleus to activate downstream gene expression, regulate glycolytic metabolism, and induce tumor occurrence [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. mTOR is an atypical serine/threonine protein kinase [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As a key regulator of glucose metabolism, MTOR is involved in the regulation of cell growth, metabolism and survival, and plays a role by activating its downstream molecule HIF-1α [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. HIF-1α promotes glucose uptake and increases the activity of glycolysis by regulating the expression of glycolytic enzyme. More and more research showed that the mTOR/HIF-1α signaling pathway interacts with PKM2, and then participates in the glucose metabolic reprogramming in tumor cells, providing energy for the growth and development of tumor cells, and thus promoting the occurrence and development of tumors. Studies have shown that in pancreatic cancer, mTOR/HIF-1α signaling pathway up-regulates the expression of PKM2, enhances the activity of cell glycolysis, and further promotes tumor growth [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, whether the mTOR/HIF-1α/PKM2 signaling pathway plays a role in the occurrence and development of HGSC is still unclear.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to compare and analyze the expression differences of FOXR2, mTOR, HIF-1α and PKM2 proteins in HGSC tissues and normal fallopian tube tissues, and to preliminatively explore the correlation between the expression of these four proteins and the occurrence of HGSC and the clinicopathological characteristics of patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue specimens and clinical data\u003c/h2\u003e \u003cp\u003eThe present study was a retrospective study. A total of 104 cases were included and their archival paraffin-embedded HGSC tissue and normal fallopian tube tissue specimens collected between January 2015 and December 2022 at the Department of Pathology of the Second Hospital of Hebei Medical University (Shijiazhuang, China) were obtained. The present study was approved by the Ethics Committees of the Second Hospital of Hebei Medical University (Shijiazhuang, China) and informed consent was obtained from each participant. According to the 2014 International Federation of Obstetrics and Gynecology (FIGO) criteria, these cases were classified as: Stage I 16, Stage II 18, Stage III 34 and Stage IV 6. At the same time, 30 normal fallopian tube tissues were also selected as controls. Among the 74 patients with HGSC, the age range was 38 to 86 years, with a mean age of 56.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0 years. The overall survival time was calculated from the date of diagnosis to the date of death or the date of the last follow-up or last time-point the patient was known to be alive, defined as censored (last evaluated January 31, 2024).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemical staining\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical staining for FOXR2, mTOR, HIF-1α and PKM2 proteins in HGSC sections was performed using a ready-to-use Biotin-Streptavidin/Horseradish peroxidase (HRP) Detection system (OriGene Technologies, Inc.). Paraffin sections (4 \u0026micro;m) were deparaffinized in xylene and then rehydrated with alcohol; heat-induced antigen recovery was performed in citrate buffer (pH 6.0) for 5 min at 120˚C or EDTA antigen retrieval solution (pH 8.0) for 5 min at 120˚C, followed by quenching of the endogenous peroxidase using 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 20 min at room temperature. Goat serum (OriGene Technologies, Inc.) was used for blocking nonspecific binding sites for 30 min at room temperature. Slides were then incubated with primary antibodies [anti-FOXR2 (1:200 dilution; Abcam; ab244513); anti-mTOR (1:500 dilution; HUABIO; ET1608-5); anti-HIF-1α (1:500 dilution; Gene Tex; GTX127309); anti-PKM2 (1:400 dilution; Cell Signaling; CST4053)] at 4˚C overnight. After washing with PBS, a Biotin-Streptavidin HRP Detection system (working solution; OriGene Technologies, Inc.; SP-9000/9001/9002) was used for detection of the antigen-antibody complex, which was visualized with diaminobenzidine as the chromogen. Finally, counterstaining was performed using Mayer's hematoxylin and the sections were dehydrated in alcohol prior to mounting. As a negative control, the primary antibody was omitted; no staining was observed.\u003c/p\u003e\n\u003ch3\u003eScoring of staining\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical staining was assessed using semi-quantitative scoring by two independent investigators (JW and YHL) who were blinded to the histopathological features and clinicopathological data of the samples. Before the scoring was performed, a set of criteria were established based on the literature with minor modifications in terms of the percentage scores of positive cells and the final score. The degree of staining was determined by the percentage of positive cells and the staining intensity. Cells that were stained with yellow or brown color were considered as positively-stained cells. Necrotic areas and areas with extensive bleeding were not included.\u003c/p\u003e \u003cp\u003eFOXR2 protein was localized in the nucleus. Percentage scoring for FOXR2 protein was as follows: 1, \u0026le;\u0026thinsp;10%; 2, 11\u0026ndash;50%; 3, 51\u0026ndash;75%; and 4, \u0026gt;\u0026thinsp;75%; while the intensity scoring was as follows: 0, no staining; 1, light yellow; 2, brownish-yellow; and 3, brown-brown. The staining degree was calculated as follows: Overall score\u0026thinsp;=\u0026thinsp;percentage score \u0026times; intensity score. Overall scores of 0\u0026ndash;3 indicated low expression, whereas overall scores of 4 or more indicated high expression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePositive expression of mTOR protein mainly localized in the cytoplasm. Percentage scoring for mTOR protein was as follows: 0, \u0026lt;\u0026thinsp;5%; 1, 5%-25%; 2, 25%-50% ; 3, \u0026gt;\u0026thinsp;50%. while the intensity scoring was as follows: 0, no staining; 1, light yellow; 2, brownish-yellow; and 3, brown-brown. The staining degree was calculated as follows: Overall score\u0026thinsp;=\u0026thinsp;percentage score \u0026times; intensity score. Overall scores of 0\u0026ndash;3 indicated low expression, whereas overall scores of 4 or more indicated high expression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePositive expression of HIF-1α protein was identified in the cytoplasm or nucleus. Percentage scoring for HIF-1α protein was as follows: 0, \u0026le;\u0026thinsp;5%; 1, 6\u0026ndash;25%; 2, 26\u0026ndash;50%; 3, 51\u0026ndash;75%; and 4, \u0026gt;\u0026thinsp;75%; while the intensity scoring was as follows: 0, no staining; 1,yellow; 2, brown; and 3, brownish-brown. The staining degree was calculated as follows: Overall score\u0026thinsp;=\u0026thinsp;percentage score \u0026times; intensity score. Overall scores of 0\u0026ndash;4 indicated low expression, whereas overall scores of 5 or more indicated high expression [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePositive expression of PKM2 protein was identified in the cytoplasm and nucleus. Percentage scoring for PKM2 protein was as follows: 1, \u0026lt;33%; 2, 33\u0026ndash;67%; 3, \u0026gt;\u0026thinsp;67%; while the intensity scoring was as follows: 1, weak expression; 2, moderate expression; and 3, strong expression. The staining degree was calculated as follows: Overall score\u0026thinsp;=\u0026thinsp;percentage score \u0026times; intensity score. Overall scores of 1\u0026ndash;3 indicated low expression, whereas overall scores of 4 or more indicated high expression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Based on this, the patients we divided into low and high expression groups.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using SPSS 21.0 software (IBM Corp.). χ\u003csup\u003e2\u003c/sup\u003e tests were used to compare the staining results for target proteins in different groups and for comparisons with clinicopathological characteristics. The correlations between FOXR2 and other proteins were determined using nonparametric Spearman's correlation tests. Kaplan-Meier analysis was performed to draw overall survival curves and statistical significance was assessed using log-rank tests. Univariate and multivariate analysis were performed using the Cox proportional hazard regression model. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFOXR2, mTOR, HIF-1α and PKM2 levels in HGSC tissues\u003c/h2\u003e \u003cp\u003eImmunohistochemical analyses were performed to determine the expression levels and distributions of target proteins. Representative images and the results are provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, respectively. For the 30 normal fallopian tube tissue, high expression of FOXR2, mTOR, HIF-1α and PKM2 was observed in 0 (0.0%), 3 (10.0%), 0 (0.0%) and 4 (13.3%) cases, respectively. For the HGSC tissues, high expression of these proteins was observed in 55 (74.3%), 57 (77%), 53 (71.6%) and 58 (78.4%) of cases, respectively. Thus, high expression of these markers was significantly more frequent in HGSC than in normal fallopian tube tissue (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe expression of FOXR2, mTOR, HIF-1α and PKM2 in normal fallopian tubes and high-grade serous ovarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTissue type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOXR2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003emTOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHIF\u0026minus;1α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePKM2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c10\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFallopian tube\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0(0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3(10.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0(0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4(13.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-grade serous\u003c/p\u003e \u003cp\u003eovarian cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55(74.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e57(77.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e53(71.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58(78.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClinicopathological characteristics of FOXR2, mTOR, HIF-1α and PKM2 in HGSC tissues\u003c/h3\u003e\n\u003cp\u003eNext, the associations of protein levels and clinicopathological features in patients with HGSC were assessed by X\u003csup\u003e2\u003c/sup\u003e tests and Spearman correlation analysis. As presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the expression levels of FOXR2, mTOR, HIF-1α and PKM2 were significantly associated with FIGO grade (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, high levels of FOXR2 were associated with age in patients (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, patient age were not significantly associated with the expression levels of the three proteins (mTOR, HIF-1α and PKM2; P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, Spearman correlation analysis indicated that the levels of FOXR2, mTOR, HIF-1α and PKM2 were correlated with the FIGO grade (r\u003csub\u003eFOXR2\u003c/sub\u003e=0.243, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; r\u003csub\u003emTOR\u003c/sub\u003e=0.311, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; r\u003csub\u003eHIF\u0026minus;1α\u003c/sub\u003e=0.285, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; r\u003csub\u003ePKM2\u003c/sub\u003e=0.326, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the levels of FOXR2 were correlated with the patient age (r\u003csub\u003eFOXR2\u003c/sub\u003e=-0.311, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Taken together, these results indicated that FOXR2, mTOR, HIF-1α and PKM2 may have roles in HGSC progression.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelationship among the expression of FOXR2, mTOR, HIF-1α and PKM2 with clinical pathological characteristics in high-grade serous ovarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePathological\u003c/p\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eFORX2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003emTOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eHIF\u0026minus;1α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003ePKM2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;56 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;56 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFIGO grade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCorrelations among FOXR2, mTOR, HIF-1α and PKM2 in HGSC tissues\u003c/h3\u003e\n\u003cp\u003eAs presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Spearman's rank correlation analyses revealed that the correlation among FOXR2, mTOR, HIF-1α and PKM2 was significant in HGSC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The expression of mTOR, HIF-1α and PKM2 is positively correlated with FOXR2 (r\u003csub\u003emTOR\u003c/sub\u003e=0.635, r\u003csub\u003eHIF\u0026minus;1α\u003c/sub\u003e=0.659, r\u003csub\u003ePKM2\u003c/sub\u003e=0.743, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). And the expression of HIF-1α and PKM2 is positively correlated with mTOR (r\u003csub\u003eHIF\u0026minus;1α\u003c/sub\u003e=0.796, r\u003csub\u003ePKM2\u003c/sub\u003e=0.806, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the expression of HIF-1α is positively correlated with PKM2 expression (r\u0026thinsp;=\u0026thinsp;0.762, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results suggested that FOXR2 may interact with the other proteins to mediate invasive growth of HGSC.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations among FOXR2 and mTOR, HIF-1α, PKM2 in high-grade serous ovarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003emTOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eHIF\u0026minus;1α\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003ePKM2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eprotein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFOXR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.635\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.659\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.743\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emTOR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.806\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIF\u0026minus;1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePrognostic value of FOXR2, mTOR, HIF-1α and PKM2 in patients with HGSC tissues\u003c/h2\u003e \u003cp\u003eAs presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, overall survival analysis indicated that higher expression levels of FOXR2, mTOR, HIF-1α and PKM2 were associated with significantly poorer overall survival than that for patients with lower levels of these proteins. Indeed, FOXR2, mTOR, HIF-1α and PKM2 levels were correlated with patient prognosis (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Other conventional prognostic factors, including FIGO grade, were correlated with patient prognosis (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The median survival time in patients with FIGO grade I was 79 months, while the median survival time in patients with FIGO grade IV was only 21 months. However, age did not influence the clinical prognosis (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCox proportional hazards models was further used to conduct univariate and multivariate analysis, and the correlation among the expression of FOXR2, mTOR, HIF-1α and PKM2 proteins and overall survival were investigated (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results of univariate analysis showed that the expression levels of FOXR2, mTOR, HIF-1α and PKM2 proteins and FIGO grade were the factors affecting the risk of poor prognosis in FOXR2, mTOR, HIF-1α and PKM2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Multivariate analysis showed that FIGO grade was an independent prognostic factor for HGSC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUnivariate and multivariate analysis of the prognostic factors of patients with high-grade serous ovarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eUnivariate analysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eMultivariate analysis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR(95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eHR(95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFOXR2\u003c/p\u003e \u003cp\u003e(High vs Low expression)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.588\u003c/p\u003e \u003cp\u003e(1.010\u0026ndash;6.632)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.455\u003c/p\u003e \u003cp\u003e(0.105\u0026ndash;1.981)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.294\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emTOR\u003c/p\u003e \u003cp\u003e(High vs Low expression)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.395\u003c/p\u003e \u003cp\u003e(1.350\u0026minus;14.307)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.521\u003c/p\u003e \u003cp\u003e(0.256\u0026ndash;9.047)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.645\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIF\u0026minus;1α\u003c/p\u003e \u003cp\u003e(High vs Low expression)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.200\u003c/p\u003e \u003cp\u003e(1.249\u0026ndash;8.196)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.678\u003c/p\u003e \u003cp\u003e(0.349\u0026ndash;8.076)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.518\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePKM2\u003c/p\u003e \u003cp\u003e(High vs Low expression)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.109\u003c/p\u003e \u003cp\u003e(1.703\u0026ndash;29.679)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e4.339\u003c/p\u003e \u003cp\u003e(0.584\u0026ndash;32.214)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.151\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFIGO grade\u003c/p\u003e \u003cp\u003e(II /III/IVvs I)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.033\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003cp\u003e(\u0026ge;\u0026thinsp;56years vs \u0026lt;56years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.822\u003c/p\u003e \u003cp\u003e(0.436\u0026ndash;1.550)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese results indicate that the expression levels of FOXR2, mTOR, HIF-1α and PKM2 proteins and FIGO stage are factors affecting the risk of poor prognosis of HGSC. Among them, FIGO stage is an independent predictor of prognosis of HGSC.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOvarian cancer is the main cause of death of female reproductive system malignant tumors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Among them, HGSC is the histological subtype with the highest incidence and the most aggressive among ovarian cancers, often accompanied by large-scale pelvic and abdominal metastasis. More than 75% of patients are diagnosed with advanced stage, which is the main cause of death of ovarian cancer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Although certain progress has been made in treatment in recent years, it is prone to recurrence after initial treatment. After relapse, the treatment effect is not good, the overall survival rate is still low, and the prognosis is not optimistic. Therefore, it is necessary to further study the molecular mechanism of the occurrence and development of HGSC.\u003c/p\u003e \u003cp\u003eFOXR2 is a member of the forkhead frame transcription factor family located on the X chromosome. It is expressed in normal tissues and only in testis, but it is highly expressed in a variety of malignant tumors, and promotes the proliferation of cancer cells, and plays a certain regulatory role in the occurrence, development and prognosis of tumors. In studies of prostate cancer [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], non-small cell lung cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and colorectal cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], FOXR2 plays a key role in tumor cell proliferation and invasion by inhibiting the activation of Wnt/β-catenin and Shh signaling pathways. In addition, in glioma, FOXR2 promotes the proliferation, migration and invasion of glioma cells by regulating various cell cycle regulatory genes and the PI3K-Akt-p27 signaling pathway [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. On the one hand, the increased expression of FOXR2 in ovarian cancer is related to cell growth, migration and epithelial-mesenchymal transformation. On the other hand, FOXR2 is abnormally high expressed in ovarian cancer and regulates the malignant behavior of ovarian cancer cells by stimulating angiogenesis and activating Hedgehog signaling pathway [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As a novel protomogenic gene discovered in recent years, the study of FOXR2 provides a new idea for tumor diagnosis and treatment. However, how FOXR2 plays a role in the occurrence and development of HGSC and its potential molecular mechanism remain to be studied.\u003c/p\u003e \u003cp\u003eIn order to further understand the expression of FOXR2 protein in HGSC, immunohistochemistry was used to detect FOXR2 protein in 74 cases of HGSC and 30 cases of normal fallopian tube tissue. The results showed that the positive expression level of FOXR2 in HGSC was significantly higher than that in normal fallopian tube tissue. Moreover, the expression level of FOXR2 was positively correlated with FIGO stage of HGSC, suggesting that FOXR2 protein may be involved in the occurrence and development of HGSC, and is closely related to its malignant process.\u003c/p\u003e \u003cp\u003eIn recent years, a series of studies have shown that the reprogramming of energy metabolism has become one of the core characteristics of tumors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Among them, glucose metabolic reprogramming, characterized by increased proportion of glycolytic pathways, is one of the most prominent metabolic changes in tumor cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Pyruvate kinase plays an important role in the glycolysis pathway by catalyzing the last step of glycolysis, which promotes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP, providing energy substances for cell metabolism [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. There are four types of pyruvate kinase subtypes (M1, M2, L and R) in mammals, among which PKM2 can promote the transformation of more polysaccharide glycolysis intermediates, and mainly exists in anabolic active cells such as embryonic cells, stem cells and tumor cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. More and more studies have shown that PKM2 is highly expressed in a variety of tumor cells and is closely related to malignant behaviors such as proliferation, migration and invasion of tumor cells. For example, in malignant tumors such as glioma [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and gastric cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], glycolysis is enhanced through up-regulation of PKM2 expression level, which further promotes tumor occurrence and development. In addition, PKM2 is highly expressed in ovarian cancer and produces high concentration of lactic acid through regulation of glycolytic metabolism, which promotes the proliferation and metastasis of ovarian cancer and is closely related to its poor prognosis and low survival rate [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003emTOR, located in the short arm of human chromosome 1, is an evolutionarily conserved atypical protein kinase. It regulates the metabolic pathways of cell proliferation, growth and metabolism by sensing the nutritional state and energy level of cells to maintain normal physiological functions. However, in tumor cells, the mTOR signaling pathway is over-activated, which not only promotes the proliferation, invasion and metastasis of tumor cells, but also inhibits the apoptosis and autophagy of tumor cells, which is the key to promoting the growth and development of tumor cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. By regulating its downstream transcription factors (such as HIF-1), mTOR plays a central role in glucose metabolism, protein and lipid synthesis, mitochondrial and lysosomal biosynthesis, thereby promoting cell growth and proliferation. HIF-1 is a transcription factor that plays an important role in a variety of physiological and pathological processes. HIF-1α is the active subunit of HIF-1, and its expression is closely related to the tension of oxygen in the environment. It is a key transcription factor regulating hypoxic stress response in tumor cells, and is widely involved in the malignant proliferation and invasive progression of tumors [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. More and more studies have shown that mTOR is involved in malignant processes such as tumor growth and metastasis by activating its downstream transcription factor HIF-1α. For example, in prostate cancer, mTOR plays a key role in tumor growth and metastasis by regulating HIF-1α protein accumulation and transcriptional activity in tumor cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In thyroid cancer, activated mTOR/HIF-1α signaling pathway can enhance cellular glycolysis and further promote tumor cell proliferation and metastasis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, a series of studies have shown that the interaction between mTOR/HIF-1α signaling pathway and PKM2 enhances the glycolytic metabolism of tumor cells and further promotes the occurrence and development of tumors. Studies have found that mTOR signaling pathway can affect the expression level of PKM2 in human pancreatic cancer (PANC-1), prostate cancer (PC3) and liver cancer (HepG2) cell lines, thereby activating glycolytic metabolism and promoting the growth of tumor cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Xiaoyu H et al. found that in esophageal squamous cell carcinoma, the mTOR/HIF-1α signaling pathway was activated, which promoted the increase of PKM2 expression, thus promoting the glycolytic metabolism of tumor cells, converting glucose into lactic acid to produce energy, and further affecting the energy metabolism and growth of tumor cells. It plays an important regulatory role in the development of esophageal squamous cell carcinoma [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImmunohistochemical results of this study showed that the expression of mTOR, HIF-1α and PKM2 in HGSC was significantly higher than that in normal fallopian tube tissue. Statistical analysis of clinicopathological features showed that the expression levels of mTOR, HIF-1α and PKM2 proteins in HGSC were positively correlated with their FIGO stage, suggesting that these three proteins may be involved in the occurrence and development of HGSC. In addition, the correlation analysis results with the age of patients showed that the expression level of FOXR2 protein was negatively correlated with the age of patients, while the expression of the other three proteins was not significantly correlated with the age of patients. Further correlation analysis showed that FOXR2, mTOR, HIF-1α and PKM2 were all positively correlated, suggesting that FOXR2 may have synergistic effect with mTOR/HIF-1α/PKM2 signaling pathway. We speculate that FOXR2 may promote the occurrence and development of HGSC by regulating glucose metabolic reprogramming through mTOR/HIF-1α/PKM2 signaling pathway.\u003c/p\u003e \u003cp\u003eMore and more studies have shown that FOXR2, mTOR, HIF-1α and PKM2 proteins are abnormally expressed in a variety of malignant tumors, and are closely related to the poor prognosis of tumors. For example, in breast cancer and endometrioid carcinoma, the high expression of FOXR2 is an independent factor affecting the poor prognosis of tumor patients [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In gastric cancer, mTOR is highly expressed, which regulates malignant behaviors such as proliferation and metastasis of tumor cells, and is also an independent factor affecting the prognosis of gastric cancer [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. HIF-1α plays a crucial role in promoting cervical cancer cell migration and invasion, and is a prognostic biomarker of cervical cancer [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Wang et al. found that high expression of PKM2 is associated with poor prognosis of breast cancer patients, and PKM2 is an independent prognostic predictor of surgically resected breast cancer patients and neoadjuvant chemotherapy patients [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The results of overall survival analysis in this study showed that the expression of FOXR2, mTOR, HIF-1α and PKM2 and FIGO stage of HGSC were factors affecting the risk of poor prognosis of HGSC. Further multifactor analysis showed that FIGO stage was an independent predictor of prognosis of HGSC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, FOXR2, mTOR, HIF-1α and PKM2 proteins are highly expressed in HGSC, and are positively correlated with FIGO stage of ovarian cancer. In addition, these four proteins were positively correlated with each other. Therefore, we speculated that FOXR2 might enhance the glycolysis of tumor cells and regulate the reprogramming of glucose metabolism through the mTOR/HIF-1α/PKM2 signaling pathway, and thus participate in the occurrence and development of high-grade ovarian serous cancer. Overall survival analysis showed that the expression levels of FOXR2, mTOR, HIF-1α and PKM2 proteins, as well as FIGO stage, were correlated with patient prognosis. In addition, FIGO staging was an independent prognostic factor affecting the poor prognosis of patients. These results indicate that the high expression of FOXR2, mTOR, HIF-1α and PKM2 play an important role in the occurrence, development and prognosis of HGSC, and may become a new direction for the prevention and treatment of HGSC in the future.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHGSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-grade serous carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFOX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eForkhead Box\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFOXR2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eForkhead Box R2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the second hospital of Hebei Medical University (Approval No.: 2024-R614). All experiments were performed in accordance with relevant guidelines and regulations such as the Declaration of Helsinki and the patients signed the informed consent form and agreed to be published.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Government-funded Program for Clinical Medicine Talents\u0026zwnj;\u0026zwnj; (Grant No. ZF2025214), the Medical Science Research Project of Hebei Province (No. 20230811), the China Anti-Cancer Association Regional Cancer Research Cultivation Fund (No. CESTDQLCORP300-09), the Beijing Top-Doctors Charity Foundation and China Medical and Health Development Foundation and Hebei Natural Science Foundation (Grant No. H2025206715).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLianguo Hou: Conceptualization; writing original draft. Xinyi Dong: Resources; conceptualization. Min Li: Resources. Jiaxi Yang: Resources. Xueli Guo: Resources. Juan Wang: Supervision; visualization. Jun Zhang: Supervision; visualization; writing review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data were presented in this paper and there were no additional supporting files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePrat J, D'Angelo E, Espinosa I. Ovarian carcinomas: at least five different diseases with distinct histological features and molecular genetics. Hum Pathol. 2018;80:11\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh M, Igarashi M, Fukuda H, Nakagama H, Katoh M. Cancer genetics and genomics of human FOX family genes. 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Cancer Biomark. 2021;32:221\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"diagnostic-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dpat","sideBox":"Learn more about [Diagnostic Pathology](http://diagnosticpathology.biomedcentral.com)","snPcode":"13000","submissionUrl":"https://submission.nature.com/new-submission/13000/3","title":"Diagnostic Pathology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"High-grade serous carcinoma of the ovary, FOXR2, glycometabolic reprogramming, mTOR/HIF-1α signaling pathway, PKM2, prognosis","lastPublishedDoi":"10.21203/rs.3.rs-8479280/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8479280/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHigh-grade serous carcinoma (HGSC) of the ovary is the most common and deadly subtype of gynecological malignancy. More and more studies have shown that Forkhead Box R2 (FOXR2) is a carcinogenic driver that can enhance cell growth and tumor formation. However, the role of FOXR2 in the development of HGSC and its underlying molecular mechanisms remain unclear. This study investigated the role of FOXR2 in HGSC progression and its relationship with glycometabolic reprogramming, specifically the mTOR/HIF-1α signaling pathway and its downstream key glycolytic enzyme, PKM2.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eImmunohistochemistry for FOXR2, mTOR, HIF-1α and PKM2 was performed on formalin-fixed paraffin-embedded HGSC tissue and normal fallopian tube tissue. Kaplan Meier analysis and Cox proportional hazards regression analysis were used to evaluate the overall survival curve and independent prognostic factors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the present study, FOXR2, mTOR, HIF-1α and PKM2 proteins are highly expressed in HGSC, and are positively correlated with FIGO stage of ovarian cancer. In addition, these four proteins were positively correlated with each other. Overall survival analysis showed that the expression levels of FOXR2, mTOR, HIF-1α and PKM2 proteins, as well as FIGO stage, were correlated with patient prognosis. In addition, FIGO staging was an independent prognostic factor affecting the poor prognosis of patients.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTaken together, the present results suggested that FOXR2, mTOR, HIF-1α and PKM2 have crucial roles in the progression and prognosis of HGSC and may be potential therapeutic targets.\u003c/p\u003e","manuscriptTitle":"Expression, association with clinicopathological features and prognostic potential of FOXR2, mTOR, HIF-1α and PKM2 in high-grade serous carcinoma of the ovary","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 03:09:14","doi":"10.21203/rs.3.rs-8479280/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-01-22T15:06:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T13:48:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T07:27:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Diagnostic Pathology","date":"2026-01-13T01:05:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"diagnostic-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dpat","sideBox":"Learn more about [Diagnostic Pathology](http://diagnosticpathology.biomedcentral.com)","snPcode":"13000","submissionUrl":"https://submission.nature.com/new-submission/13000/3","title":"Diagnostic Pathology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d70e0d7-c9a4-43da-aa49-07f702fc0a28","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-28T03:09:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 03:09:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8479280","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8479280","identity":"rs-8479280","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}