ERRγ, a Novel Biomarker, Associates with Pathoglycemia of Endometrial Cancer to Predict Myometrial Invasion

We aim to investigate the correlation between the expression of estrogen-related receptor γ (ERRγ) and endometrial cancer (EC) progression and to evaluate the potential of ERRγ as a new biomarker for EC diagnosis. We analyzed the ERRγ expression profile and the correlation with the corresponding clinical characteristics of EC samples from The Cancer Genome Atlas (TCGA), the Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases, and the International Cancer Genome Consortium (ICGC) databases. Immunohistochemical (IHC) analysis was conducted on tissue samples, and enzyme linked immunosorbent assay (ELISA) was used in serum samples to detect the levels of ERRγ. The diagnostic performance of ERRγ proteins was assessed using the receiver operating characteristic (ROC). ERRγ showed notably higher expression in EC tissues than in normal endometrium tissues (P < 0.001), which was consistent with the result of TCGA. Overexpression of ERRγ was significantly associated with deep myometrial invasion of EC (P=0.004), and fasting blood glucose (FBG) was higher in EC patients with deep myometrial invasion than in those with superficial myometrial invasion (P=0.040). Further analysis using ELISA showed that the serum ERRγ level was positively correlated with FBG (R = 0.355, P < 0.001). ERRγ is overexpressed in EC and may be involved in regulating glucose metabolism and promoting myometrial invasion of EC. In addition, the area under the ROC curve (AUC) for ERRγ was 0.834, in distinguishing EC patients from healthy individuals, presented 84.0% and 80.0% sensitivity and specificity, respectively, and serum ERRγ has a good diagnostic performance in distinguishing EC patients from healthy people and may be a promising noninvasive biomarker in EC.


Introduction
e incidence of endometrial cancer (EC) is increasing year by year, and it has become the first cancer of the female reproductive tract in the world [1]. Myometrial invasion in advanced EC is closely related to poor prognosis [2].
However, the mechanisms involved in the invasion and metastasis of malignant tumors are still unclear. Patients usually seek medical attention for abnormal uterine bleeding, which is found to be EC by pap-smear, curettage pathology, or hysteroscopy. Due to the lack of specific markers, it is of great significance to find reliable and valuable biomarkers for early identification and diagnosis of EC.
Abnormal glucose metabolism is a common feature often observed in patients with EC [3]. Accumulating evidence indicates that hyperglycemia is associated with poor prognosis of EC [4,5]. Recently, studies have found that the nuclear receptor estrogen-related receptor c (ERRc), as a metabolism-related gene, is widely involved in the regulation of several key enzymes in cell glucose metabolism, lipid metabolism, and amino acid metabolism [6]. ERRc is related to abnormal gluconeogenesis, insulin resistance, and other pathological states and participates in the occurrence and development of diseases with abnormal glucose metabolism [7]. ERRc is highly expressed in diabetic patients with poor blood glucose control [8] and is abnormally expressed in a variety of metabolismrelated diseases, including malignant tumors, and involved in the occurrence and development of cancer [9,10].
is study aimed to investigate the relationship between ERRc and EC and glucose metabolism to evaluate the role of ERRc as a biomarker for the diagnosis of EC.

Data Acquisition and Processing.
We explored mRNA expression of ERRc from e Cancer Genome Atlas (TCGA) database, including 546 EC and 35 normal endometrial tissue samples. We analyzed the expression of ERRc mRNA in patients with different histological subtypes and stages. Clinical Proteomic Tumor Analysis Consortium (CPTAC) analysis of the UALCAN portal (http://ualcan.path.uab.edu/ analysis-prot.html.) was used to identify the protein expression level of ERRc in UCEC (uterine corpus endometrial carcinoma). In addition, another dataset from the International Cancer Genome Consortium (ICGC) data portal (https://icgc.org/) was used to assess survival differences.

Patients.
e medical records of 525 EC patients treated in Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University from January 2012 to December 2018 were studied retrospectively. e exclusion criteria were as follows: (1) patients with a history of other malignancies; (2) patients with nonepithelial cancers of the uterus, such as carcinosarcoma; (3) patients treated with chemotherapy, radiotherapy, or hormone therapy before surgery; (4) patients missing clinical pathology data or with an unclear diagnosis; and (5) patients who did not agree for further analysis of their pathological tissue. Healthy controls were from those who had undergone curettage for other reasons such as endometrial polyps, adenomyosis, leiomyomas, hyperplasia, and hemorrhages due to congenital and acquired coagulopathies, ovarian dysfunction, and disorders of the local endometrial hemostasis mechanism with normal organ structure. Healthy controls received adequate screening and excluding for endometrial lesions or other types of malignancies and other disease during the same period. is study was approved by the Ethics Committee of Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University (No. YCXM20-01) and performed in compliance with the Declaration of Helsinki. Informed consent was obtained from all participants included in the study.

Sample Preparation.
For the discovery phase, we collected endometrial cancer tissue from 525 EC patients who had retained tissue samples during the last 7 years. Excluding unqualified samples and those without informed consent, 79 cases of endometrial carcinoma were eventually included. Among them, stage I and II patients (n � 63) accounted for 78.5% of the total cases and endometrioid adenocarcinoma; nonendometrioid adenocarcinoma was about 4 : 1, consistent with the epidemiological distribution [11,12]. Patients with normal endometrial pathology (n � 32) were collected during the same period. All tissue samples were collected during the surgery. Immunohistochemistry (IHC) was used to detect the expression of ERRc in tissue chip samples. For the validation phase, serum samples from 50 EC patients from January 2021 to December 2021 were collected and paired with 50 healthy individuals. ere were 41 patients in the early stage, and the stage distribution was consistent with the epidemiological characteristics ( Figure 1). All serum samples were collected two days before surgery, and ERRc protein was evaluated by ELISA, which were entirely separated from the discovery set samples. All methods were carried out in accordance with relevant guidelines and regulations set out below.

Immunohistochemistry.
To examine the expression of ERRc in tissue, we performed a tissue microarray constructed by Shanghai Zhuoli Biotechnology Co., Ltd (Zhuoli Biotechnology Co., Shanghai, China). Rabbit polyclonal anti-ERRc (ab49129, Abcam) were used. Two pathologists independently evaluated the quantitation of immunostaining for ERRc, who were blinded to patient details. e expression of ERRc in tumor parenchyma was semiquantified by the immunoreactivity score (IR score) based on intensity and heterogeneity. e IR score was determined as the sum of heterogeneity and intensity. Intensity of staining was scored as 0 (negative), 1 (low), 2 (medium), and 3 (high). Area extent of staining was scored as 0 (0% stained), 1 (1-25% stained), 2 (26-50% stained), and 3 (51-100% stained). e final score was determined by multiplying the intensity scores with area extent and ranged from 0 to 9. Final scores (intensity score × percentage score) < 6 were considered as low and ≥6 were high expression.
2.5. Serum. ELISA. Blood samples were collected and centrifuged at 1500°g for 10 min. Serum samples were stored at − 70°C until the day of the analysis. e serum level of ERRc was assessed by using a solid phase sandwich enzyme linked immunosorbent assay (ELISA) kit (cat. #JL48961; Jianglai Inc., Shanghai, China), following the manufacturers' protocol. e optical density of each well was then read at 450 nm using a microplate reader. Serum levels of ERRc were calculated from a standard curve based on reference standards.

Statistical Analysis.
In this study, ERRc expression was compared to different groups using the chi-square or Fisher's exact test, when appropriate. Student's t-test was used to compare continuous variables in two groups. Pearson correlation analysis was used to evaluate the correlations between continuous variables. Survival rates were calculated using the Kaplan-Meier estimator. Receiveroperating characteristic (ROC) curves were used to evaluate the diagnostic value of ERRc. e ERRc cut-off value was calculated using the Youden index. All statistical analyses were performed using SPSS 22.0 (IBM, Chicago, IL, USA) and GraphPad Prism version 8.0 software (GraphPad Software, Inc., La Jolla, CA, USA). All P values in the statistical analysis were two-tailed. P < 0.05 was considered statistically significant.

Expression of ERRc in TCGA Database and the CPTAC
Database. As shown in Figures 2(a)-2(c), the mRNA expression profiles retrieved from TCGA revealed that the expression of ERRc was significantly higher in EC than the normal sample (P < 0.001). ere was no difference in both International Federation of Gynecology and Obstetrics (FIGO) stages and histological subtypes (P > 0.05). e CPTAC database showed that ERRc expression was higher in the deep myometrial invasion depth group than in the superficial myometrial invasion depth group, but the difference was not statistically significant (P > 0.05; Figure 2(d)). e expression of ERRc in G2-G3 group was higher than that in G1 group (P < 0.05; Figure 2(e)), while there was no statistical significance in ERRc expression in different stage groups (P > 0.05; Figure 2(f )).

ERRc Is Highly Expressed in EC Tissue.
We analyzed the expression of ERRc in 79 cases of EC tissue samples and 32 cases of healthy controls by IHC (Figure 2(g)). ERRc was more highly expressed in EC tissues than in normal endometrial tissue (P < 0.001). Among 79 cases of EC samples, 59 samples showed high ERRc expression (Figure 2(h)). In addition, we analyzed the differences in the expression of      m)). We observed that high ERRc expression is associated with deep myometrial invasion (P � 0.004), and there was no statistical significance in the expression of ERRc among different clinical stages, pathological types, and lymph node metastatic status groups (P > 0.05).

Overexpression of ERRc Significantly Correlates with Deep
Myometrial Invasion. We analyzed the correlation between the expression of ERRc and immunohistochemical markers in EC tissues (Table 1). e results showed that the high expression rate of ERRc in the vimentin-positive group was higher than that in the vimentin-negative group (76.8% vs. 23.2%), and Spearman correlation analysis showed a significant positive correlation between ERRc and vimentin (R � 0.368, P � 0.001). ere was no statistical correlation between ERRc and ER, PR, PTEN, P53, and Ki67 (P > 0.05).
In addition, the expressions of vimentin and Ki67 were different in different myometrial invasion groups (P � 0.018, P � 0.042), and there was no significant difference in the expressions of ER, PR, PTEN, and P53 in different myometrial invasion groups (P > 0.05).

Risk Factors for Deep Myometrial Invasion in EC.
According to the depth of myometrial invasion, patients with EC were divided into two groups (  Table S1). In addition, our results showed that the high expression of ERRc in EC tissue was associated with higher FBG and CA125 (P˂0.001, P � 0.004; Figures 3(a) and 3(b)), while there was no statistical correlation with TC, TG, CA15-3, and CA19-9 (all P > 0.05; Figures 3(c)-3(f )). e level of FBG in the deep myometrial invasion group was higher than that in the superficial myometrial invasion group (P � 0.040; Figure 3(g)), while the difference of CA125 in different myometrial invasion groups was not statistically significant in the 79 EC patients (P � 0.177; Figure 3(h)).

Prognostic Value of the ERRc Expression Level in EC
Tissue.
e ICGC database showed that there was no statistical difference in overall survival and disease-free survival between donors with and without ERRc mutations (P > 0.05; Figures 4(a) and 4(b)). We followed up 79 cases of endometrial cancer with cancer tissue for nearly 7 years, including 1 case of loss to follow-up, and only 4 cases of death among 78 patients.
ere was no statistically significant difference in the survival rate between patients with high and low expression of ERRc protein in tissues (P > 0.05; Figure 4(c)).

3.6.
e Expression Levels of ERRc in Serum. Next, the serum ERRc level was determined in the cohort of 50 EC patients and 50 control samples from healthy people by ELISA. e results showed that serum levels of ERRc was significantly higher in EC patients (2.156 ± 1.254 ng/mL) than in healthy controls (0.994 ± 0.879 ng/mL, P < 0.001; Figure 5(a)). No significant association was shown between serum ERRc levels and depth of myometrial invasion (P � 0.954; Figure 5(b)).

Diagnostic Value of the Serum ERRc Level in EC.
We further sought to evaluate the diagnostic ability of serum ERRc in EC. A receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of the serum ERRc level to further determine whether ERRc could serve as a noninvasive biomarker ( Figure 6; Table S2). e area under the ROC curve (AUC) for ERRc, CA125, and FBG was 0.834, 0.648, and 0.601, respectively, in distinguishing EC patients from healthy individuals, and the ERRc cutoff value was 1.050 ng/ml with a sensitivity of 84.0% and a specificity of 80.0%. Moreover, in the stratified study of patients with

Correlation between Serum Levels of ERRc and FBG.
Our results showed that serum ERRc levels in subjects with FBG ≥5.56 mmol/L were significantly higher than those with FBG <5.56 mmol/L (P � 0.006; Figure 5(c)). en, we observed a significant positive correlation between serum ERRc levels and FBG in EC patients and healthy controls, with a correlation coefficient of 0.355 (P < 0.001; Figure 5(d)). However, there was no significant correlation between serum ERRc levels and CA125 and age (P � 0.135, P � 0.602; Figures 5(e) and 5(f )). ese findings provided further evidence to support that the serum ERRc levels were associated with FBG levels.

Discussion
In this study, we found ERRc is overexpressed in both tissues and serum of EC patients. e expression level of ERRc in tissues was significantly correlated with myometrial invasion in EC patients, and the level of ERRc was positively correlated with the FBG level. In addition, ROC analysis showed that serum ERRc has a good diagnostic performance in distinguishing EC patients from healthy people. ese results suggest that ERRc may be involved in regulating glucose metabolism and promoting myometrial invasion of EC and may be a noninvasive biomarker source for endometrial cancer detection and progression monitoring. e prognostic factors of EC have been studied in detail. e most important factors include FIGO stage, myometrial invasion, histological subtypes and grades, and lymphatic invasion [2]. Among them, myometrial invasion is an important manifestation of invasion and metastasis of malignant   Journal of Oncology tumor, while two recent systematic reviews and a metaanalysis showed that both deep myometrial invasion and lymphovascular space invasion have prognostic value independent of TCGA signature, as well as age and adjuvant treatment [13,14]. ERRc is one of the members of the orphan nuclear receptor [15]. With the in-depth research in recent years, it has been found that the expression of ERRc is abnormal in a variety of malignant tumors and plays a role in the development of tumors [9]. In breast cancer, ERRc is usually overexpressed and upregulated after acquisition of tamoxifen resistance, suggesting that ERRc plays a promoting role in cancer. In prostate cancer [16], selective ERRɑ/c reverse agonist SLU-PP-1072 can inhibit the Warburg effect and induce apoptosis of prostate cancer cells [17]. Sun Y et al.
found that ERRc was positively expressed in EC cells, and ERRc could promote the proliferation of estrogen-dependent EC cells by activating the AKT-ERK1/2 signal pathway [18]. Hua T et al. reported that ERRc could promote the expression of E-cadherin and participate in the migration and metastasis of EC cells [19]. e results showed that ERRc was closely related to the progress of EC. In this study, bioinformatics analysis was carried out based on TCGA database, and IHC results confirmed that ERRc was highly expressed in EC, and our data indicated that ERRc was closely related to the deep myometrial invasion of EC, which was an invasion-related indicator with potential prognostic value. However, the prognostic relationship between ERRc and different pathological and molecular types remains to be further studied. In recent years, various clinical studies have found that EC is associated with metabolic disorders, including obesity, diabetes, and metabolic syndrome [20]. EC patients are often accompanied by systemic metabolic disorders, and hyperglycemia is the main clinical feature, which is related to poor outcome [4,21,22]. Our clinical data analysis found that the depth of myometrial invasion of EC was correlated with FBG, suggesting that poor blood glucose status is closely related to the development of EC. At present, more and more evidence confirms that ERRc plays a central role in metabolic genes and the regulation of cellular energy metabolism [23]. Previous studies have shown that ERRc can bind and regulate a variety of glycolytic gene promoters such as hexokinase 2 (Hk2), Aldolase C (Aldo-C) enolase 1, and lactate dehydrogenase A (LDHA) [24]. O-GlcNAcylation of ERRc serves as a major signal to promote hepatic gluconeogenesis [25].
ese results indicate that ERRc is involved in maintaining glucose homeostasis in vivo, and the imbalance of glucose metabolism-high level of glycolysis-is one of the characteristics of tumor cell metabolism [26,27]. However, there are few studies on the relationship between ERRc and abnormal glucose metabolism in tumor. Our results suggest that ERRc is significantly correlated with blood glucose in EC, and it is likely that ERRc is involved in regulating blood glucose in EC and promoting myometrial invasion.
Current biomarkers for EC metastasis, such as immunohistochemical markers ER and PR, have great limitations and lack specificity. With the development of molecular typing of endometrial cancer, these old markers are no longer clinically useful; it is of great significance to find a new biomarker. Raffone A et al. reported that metabolomics may be suitable for a noninvasive diagnosis and screening of EC, offering the possibility to predict tumor behavior and pathological characteristics [28]. Several metabolites such as homocysteine, phospholipase-  Journal of Oncology A2, and lysophospholipase-D, may be useful for diagnosis, screening, and prediction of tumor histotype, myometrial invasion, lymph vascular invasion, and cancer progression in patients with EC [28]. It is noteworthy that ERRc, as a metabolism-related gene, is closely associated with tumor glucose metabolism and may be added to the list. ERRc has good diagnostic performance in distinguishing EC patients from healthy people, with high sensitivity and specificity. ERRc detection is not only suitable for tissue but also for serum, and with the increase of the expression level of tumor progression, it has the characteristics of tumor markers, which could have an extraordinary impact on the management of EC in the future. Some shortcomings of this study should be acknowledged. First, the sample size in this study is relatively small, which might raise the bias of analysis. Second, it is necessary to further explore the internal mechanism of ERRc regulating glucose metabolism and promoting myometrial invasion of EC. In addition, we only measured serum ERRc levels in the validation phase; however, the comparative information of ERRc expression in and out of cells could not be determined.

Conclusion
Collectively, ERRc is overexpressed in EC and may be involved in regulating glucose metabolism and promoting myometrial invasion of EC. In addition, serum ERRc has a good diagnostic performance in distinguishing EC patients from healthy people and may be a promising noninvasive biomarker in EC.

Data Availability
Data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.