Active Estrogen-Succinate Metabolism Promotes Heme Accumulation and Increases the Proliferative and Invasive Potential of Endometrial Cancer Cells.

All patients had undergone curettage or hysteroscopy previously and were confirmed pathologically as having grade I/II endometrioid carcinoma after receiving total hysterectomies. We ruled out participants who had other immune diseases, tumors, or a history of hormone drug use. The UEC tissues and paired adjacent healthy tissues were obtained from the same patients ( Table 1 ). The protocol for the present study was approved by the Human Research Ethics Committee of the Obstetrics and Gynecology Hospital, Fudan University (Shanghai, China), and written informed consent was obtained from all participants. The human uterine endometrial cancer cells (UECC) Ishikawa and RL95-2 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and both of them were cultured in DMEM/F12 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco Cell Culture, Carlsbad, CA, USA), 1% penicillin-streptomycin (HyClone, Logan, UT, USA). The NCOA1-silenced (si-NCOA1) plasmid was constructed by Genechem (Shanghai, China) and the si-NCOA1 Ishikawa and RL95-2 cells were obtained through transfection. An empty vector that did not target any gene was used as a negative control (NC). The transfections were performed using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. At 48 h post-transfection, we lysed the cells to extract the RNA and performed RT-qPCR. Paraffin sections (5 μm) of normal endometrial (n = 6) and UEC tissues (n = 6) from the patients were dehydrated in graded ethanol and incubated with 3% hydrogen peroxide (H 2 O 2 ) and 5% bovine serum albumin to block endogenous peroxidase activity. The samples were subsequently incubated with rabbit antihuman ALAS1 (1:200; cat. no. DF2900; Affinity, Cincinnati, OH, USA), antihuman SLC25A38 (1:200; cat. no. PA5-42472; Invitrogen), antihuman heme oxygenase 1 (1:100; cat. no. ab52947; Abcam, Cambridge, UK), or rabbit immunoglobulin G (IgG) isotype control (1:100; cat. no. ab125938; Abcam, negative control) overnight at 4 °C in a humid chamber. After washing three times with PBS, the sections were overlaid with peroxidase-conjugated goat antirabbit IgG, the reaction was developed using 3, 3-diaminobenzidine (DAB), and the sections were counterstained with hematoxylin. The immunohistochemical staining was quantified with integrated optical density values generated using Image-ProPlus 6.0 (Media Cybernetics, Rockville, MD, USA). The Ishikawa and RL95-2 cells were treated with estrogen (10 −7 M, HY-B070, MedChem Express, NJ, USA), succinate (5 mM, Sigma-Aldrich; Merck KGaA, SL, MO, USA), ERα antagonist (MPP, 10 −6 M, HY-103454, MedChem Express, Monmouth Junction, NJ, USA), ERβ antagonist (PHTPP, 10 −6 M, HY-103456, MedChem Express, Monmouth Junction, NJ, USA), total ER antagonist Fulvestrant (ICI182780, 10 −6 M, HY-13636, MedChem Express, Monmouth Junction, NJ, USA), and heme (0–100 μM, HY-19424, MedChem Express, Monmouth Junction, NJ, USA). Both cell lines were seeded in 12-well plates at a density of 2.5 × 10 5 cells/mL and cultured in DMEM/F12 medium (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS-charcoal-stripped (Gibco Cell Culture, Carlsbad, CA, USA) and 1% penicillin–streptomycin (HyClone, UT, USA). The relevant cells were cultured under 5% CO 2 in a 37 °C humidified incubator (Heal Force HF-90, Shanghai, China) for 48 h. The heme concentrations were measured using a heme assay kit (cat. No. MAK316; Sigma-Aldrich; Merck KgaA). A total of 250 μL of distilled water was added to a blank well while the standard well contained 50 μL of heme calibrator and 200 μL of distilled water. A total of 50 μL of cell sample supernatant was mixed with 200 μL of reagent. The absorbance of the well contents was measured at 400 nm after incubation at room temperature for 5 min. All the samples and standards were performed in duplicate. The total heme concentration of a sample was calculated with the following formula: (OD sample – OD blank )/(OD calibrator – OD blank ) × 62.5 (μmol/L). The total RNA was extracted using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA extraction was carried out on UEC tissues, paired adjacent healthy tissues, and two cell lines. The RNA was reverse-transcribed into cDNA using a Prime Script RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The qPCR was performed using an SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) and analyzed using an ABI Prism 7900 Fast Sequence Detection system (Thermo Fisher Scientific, Inc.). The primer sequences of these genes are provided in Table 2 . The fold change in the transcriptional expression of the above genes was calculated using the 2 −ΔΔCt method, and each sample was analyzed in three replicate wells. The relative mRNA expression levels were normalized to β-actin (ACTB). The STRING database ( http://string-db.org , accessed on 17 September 2022) was used for protein–protein interaction network prediction. After treatment with 0–100 μM of heme (HY-19424, MedChem Express, Monmouth Junction, NJ, USA) for 48 h, the viability and invasive ability of the Ishikawa and RL95-2 cells were analyzed via a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and a Matrigel (BD Biosciences, San Jose, CA, USA) invasion assay, respectively. We diluted the Matrigel to 1:30 and added 100 μL to each chamber. Then, we put the plate into a 37 °C incubator for 2 h, removed the excess liquid, and added 500 μL of serum-free culture into the lower chamber while adding the cells treated with heme for 48 h into the upper chamber. After another 48 h, we removed the excess liquid and wiped off the unpenetrated cells. We then fixed the cells with formaldehyde and stained them with crystal violet for 30 min, respectively. Finally, we observed the cells under a microscope and chose 4 fields of view for counting. The NC and si-NCOA1 cells were treated with or without 50 μM of heme for 48 h, and then the cell viability and invasiveness were analyzed again. The data are presented as means ± standard errors of the mean (means ± SEMs). Normality of the data was tested with a Shapiro–Wilk test, and the differences were statistically analyzed using Student’s t -tests or one-way ANOVA. Kruskal–Wallis tests were performed to assess the differences in the variables with skewed distribution between two groups or among the groups, respectively. Statistical analysis was performed using SPSS version 26.0, and a p value of < 0.05 was considered to indicate a statistically significant difference. All experiments were repeated at least three times.

Uterine endometrial cancer (UEC) is one of the most common malignancies for females, accounting for 30% of malignant tumors in the female reproductive system. Its incidence has gradually increased in recent years and has shown a trend of rejuvenation [ 1 , 2 ]. By the year 2030, the incidence of UEC is expected to reach 42.13/100,000 women, which is an increase of nearly 55% compared with that of 2010 [ 3 ]. According to the Bokhman classification, UEC can be divided into two types [ 4 ]. Type I is estrogen-dependent, where in the presence of chronic exposure to estrogen unopposed by progesterone, the endometrium undergoes a progression of simple hyperplasia, complex hyperplasia, atypical hyperplasia, and even carcinogenesis [ 5 ]. Type II is non-estrogen-dependent, and its pathogenesis is not clearly related to estrogen. Serous carcinoma, clear cell carcinoma, and some other rare, high-grade tumors belong to this type, and patients usually have poor prognoses [ 4 , 6 , 7 ]. Type I UEC accounts for 70–80% of all UEC cases. Among them, grade I/II endometrioid carcinoma is the most common histological type. Patients often present with concomitant obesity, hypertension, or diabetes or have a history of taking hormonal drugs such as tamoxifen [ 5 ]. It has been reported that by upregulating the c-MYC gene, estrogen upregulates the level of glutaminase in estrogen-sensitive uterine endometrial cancer cells (UECC), increasing cell vitality and decreasing autophagy [ 8 ]. Abnormal glycolipid metabolism is closely related to the occurrence of UEC [ 9 , 10 ]. The tricarboxylic acid cycle (TCA cycle) is the main way for the body to obtain energy, and it is the final metabolic pathway of the three major nutrients (protein, carbohydrates, and fats) [ 11 , 12 ]. Succinate dehydrogenase B (SDHB) is a metabolic enzyme in the TCA cycle [ 13 ]. Many studies have shown that germline mutations of SDHB can cause the degradation of the SDHB protein, promoting tumorigenesis and the abnormal accumulation of succinate [ 11 , 12 ]. Our previous study proved that estrogen can inhibit the expression of SDHB through the PPARγ-SP1-UBC pathway, leading to the accumulation of succinate and accelerating the progression of UEC [ 14 ]. Heme has regulatory effects on cellular metabolism and is of great physiological significance in maintaining cellular homeostasis [ 15 ]. Aminolevulinate delta synthase 1 (ALAS1) is the key enzyme in heme biosynthesis and requires pyridoxal phosphate as a cofactor [ 16 ]. In the TCA cycle, α-ketoglutaric acid produces succinyl CoA under the action of dehydrogenase, and succinate can also be converted to succinyl CoA [ 17 ]. Glycine can be transported into mitochondria through the solute carrier family member 38 (SLC25A38) and bind to succinyl CoA via ALAS1. After a series of complex reactions, finally, heme is generated [ 18 , 19 ]. Mediated by heme oxygenase 1 (HMOX1), free heme can be degraded into iron, carbon monoxide, and biliverdin [ 19 , 20 ]. Our recent study showed that in patients with endometrial hyperplasia complicated by abnormal uterine bleeding, heme metabolism plays a regulatory role in the abnormal infiltration of immune cells [ 21 ]. However, the characteristics and mechanism of estrogen–succinate–heme metabolism in UEC has not yet been clarified. In this study, we focused on heme metabolism in the microenvironment of UEC. We explored the characteristics of estrogen–succinate–heme metabolism, aiming to provide a theoretical basis and potential target for the anticancer treatment of UEC.

To investigate the expression of heme metabolism-related molecules in UEC, we used The Cancer Genome Atlas (TCGA, https://cancergenome.nih.gov/ , accessed on 20 August 2022) and Clinical Proteomic Tumor Analysis Consortium (CPTAC, https://gdc.cancer.gov/ , accessed on 20 August 2022) public databases for our analysis. As shown, the expression of the key enzyme ALAS1 and the glycine mitochondrial transporter SLC25A38 in the primary tumors was significantly higher than that of the normal tissues ( Figure 1 A,B), though there was no difference in the expression of HMOX1, which mediates heme degradation ( Figure 1 C). Then, we collected six pairs of UEC samples and their adjacent healthy tissues, and RT-qPCR and immunohistochemistry were used to further verify the results of the public databases ( Figure 2 A,B). These results suggested that heme synthesis in UEC is extremely active, and it is apt to form a high-heme microenvironment. To explore the effect of estrogen on heme synthesis in UECC, we treated the Ishikawa and RL95-2 cells with or without 10 −7 M estrogen for 48 h, and then we detected the heme concentrations and the expression levels of ALAS1, SLC25A38, and HMOX1. As shown, we found that estrogen treatment significantly upregulated the expression of ALAS1 and SLC25A38, while there was no difference in the expression of HMOX1. As a result, we confirmed estrogen regulation of heme expression ( Figure 3 A). After adding 10 −6 M ERαi, ERβi, or total ER antagonist (ERαi + ERβi), the concentrations of heme in the cells were significantly reduced, and the blocking effect of ERβi appeared to be more significant and specific ( Figure 3 B). Subsequently, we treated the Ishikawa and RL95-2 cells with or without 5 mM succinate for 48 h, and measured the expression levels of ALAS1, SLC25A38, and HMOX1. Similarly, we found that succinate significantly upregulated the expression of ALAS1 and SLC25A38. As expected, heme levels were increased with the administration of succinate ( Figure 4 A). Further, ER expression was also significantly upregulated following succinate treatment ( Figure 4 B). Together, these data indicated that the administration of estrogen or succinate significantly upregulated the expression of heme metabolism-related molecules and increased the concentrations of heme in UECC. To explore the possible molecular mechanisms in estrogen-regulated heme synthesis, a protein–protein interaction network analysis was performed. As shown in Figure 5 A, nuclear receptor coactivator 1 (NCOA1) may be a key molecule between ER and heme synthesis-related molecules (ALAS1 and SLC25A38) ( Figure 5 A). It has been reported that NCOA1 overexpression can be observed in a variety of pathological conditions, such as diabetes, obesity, and tumors [ 22 , 23 ]. Subsequently, we used RT-qPCR to verify the that the expression of NCOA1 in UEC was significantly higher than that in normal tissues ( Figure 5 B). We treated Ishikawa and RL95-2 cells with 10 −7 M estrogen or 10 −6 M ERi for 48 h and quantified the relative mRNA expression of NCOA1. We found that estrogen treatment increased the expression of NCOA1 whereas 10 −6 M ERαi, ERβi, or nonselective ERi significantly reduced the expression of NCOA1 ( Figure 5 C), suggesting that the NCOA1 gene is regulated by the estrogen pathway. Therefore, we speculated that there may be an interactive regulatory relationship between estrogen, succinate, and NCOA1 in UEC. To clarify whether NCOA1 has a regulatory effect on the proliferation and invasion of UECC, we constructed a negative control (NC) and NCOA1-silenced (si-NCOA1) plasmids, and transfected the Ishikawa and RL95-2 cells with them, respectively. We detected that the cell viability at 24, 48, and 72 h was significantly reduced following the silencing of NCOA1 ( Figure 6 A). Next, 10 −7 M estrogen, 10 −6 M Eri, or 5 mM of succinate was used to treat the NC or the si-NCOA1 Ishikawa and RL95-2 cells for 48 h. We detected the heme concentrations and the expression levels of the heme metabolism-related molecules. As shown, we found that compared with the NC, the heme concentration and the expression of ALAS1 were significantly decreased in the si-NCOA1 cells while there were no differences in the expression levels of SLC25A38 and HMOX1. Moreover, although estrogen and succinate could increase heme synthesis in UECC, silencing NCOA1 partially or completely eliminated this effect ( Figure 6 B,C). To further analyze the mechanism of heme in accelerating the progression of UEC, we treated Ishikawa and RL95-2 cells with different concentrations of heme (0, 0.1, 1, 10, 30, 50, and100 μM) for 48 h and detected cell viability using a CCK8 assay. We found that cell viability was significantly increased with the administration of heme of up to 50 μM ( Figure 7 A). Through destroying the basement membrane of cells, matrix metalloproteinases (MMPs) allow tumor cells to enter the blood and lymphatic vessels, leading to the invasion and metastasis of tumors [ 24 , 25 ]. Ki-67 is present in the G1, S, and G2 phases of the cell cycle rather than in the G0 phase, where cells stop dividing [ 26 ]. Therefore, Ki-67 has been widely used as a proliferative marker to assess the proliferative activity of cells. We treated Ishikawa and RL95-2 cells with 50 μM of heme for 48 h. Invasiveness was analyzed using a Matrigel invasion assay, and the expression levels of MMP2, MMP9, and Ki-67 were analyzed using RT-qPCR. We determined that the invasiveness of UECC was increased with the administration of heme, and the expression levels of MMP2, MMP9, and Ki-67 were also upregulated ( Figure 7 B,C). In summary, exposure to heme promoted the proliferation and invasiveness of UECC. We treated NC or si-NCOA1 Ishikawa and RL95-2 cells with or without 50 μM heme for 48 h, and cell viability and invasiveness were detected using CCK8 and Matrigel invasion assays. We found that, compared with the NC, silencing NCOA1 significantly reduced the cell viability and invasiveness of UECC. In addition, although heme could promote the cell viability and invasiveness of UECC, this effect could be partially or completely eliminated in the si-NCOA1 cells ( Figure 8 A,B).

Heme is a complex of iron and protoporphyrin IX. It plays a crucial role in biosynthesis in the form of covalent or noncovalent binding [ 18 , 27 ]. Heme b participates in the synthesis of hemoglobin and myoglobin in a noncovalent binding form, which is of great importance for the reversible binding and transportation of oxygen. Heme c forms covalent binding with cytochrome c and is involved in electron transfer in the mitochondrial respiratory chain [ 28 ]. Heme is also part of the prosthetic group for many key enzymes in maintaining cellular homeostasis, such as inducible nitric oxide synthase (iNOS), cytochrome P450, and soluble guanylate cyclase (sGC) [ 29 ]. In addition, there are small amounts of free heme in cells, which are known as unstable heme pools, and which also have a regulatory effect on cellular metabolism. For example, they act as important signaling molecules that regulate circadian rhythms [ 30 ]. Due to the important role heme plays in cell metabolism, maintaining heme homeostasis is of great physiological significance. The levels of free heme in cells depend on the expression of heme metabolism-related molecules [ 15 ]. ALAS1 is a key enzyme in heme biosynthesis [ 16 ]. As a housekeeping gene, ALAS1 is widely present in almost all tissues. Impaired heme synthesis can lead to the accumulation of the toxic heme intermediate δ-amino-γ-ketovaleric acid (ALA), which can eventually lead to porphyria [ 17 ]. Solute carrier family 25 A38 (SLC25A38), which is also known as a glycine mitochondrial transporter, can transport exogenous glycine into mitochondria for biosynthesis [ 17 ]. In the TCA cycle, α-ketoglutaric acid produces succinyl CoA under the action of dehydrogenase while glycine is transported into mitochondria through SLC25A38. Succinyl CoA binds to glycine via ALAS1, and after a series of complex reactions, finally, heme is generated [ 18 , 19 ]. HMOX1 is a key enzyme in mediating heme degradation, and it exists in almost all mammalian tissues. Under normal physiological circumstances, free heme can be degraded by HMOX1 into iron, carbon monoxide, and biliverdin [ 19 , 20 ]. Through analyzing the TCGA and CPTAC public databases, we found that the expression levels of ALAS1 and SLC25A38 in primary tumors are significantly higher than those in normal tissues, and there were no significant differences in the expression levels of HMOX1 in the two types of tissues. The results of the clinical samples were also consistent with the public databases. Estrogen can regulate the activity of various tumor-associated enzymes such as liver ribonucleases and glutaminase (GLS). Estrogen also plays a crucial role in the progression of type I UEC [ 6 , 31 ]. Studies have shown that by upregulating the expression of the proto-oncogene c-MYC, estrogen can significantly increase the level of GLS, resulting in increased cell viability and decreased autophagy in estrogen-sensitive UEC [ 8 ]. In our study, we found that heme concentrations and the expression levels of metabolism-related molecules in UECC are significantly increased with the administration of succinate or estrogen. We also observed that succinate can upregulate the expression of two ERs, especially that of ERβ. Not surprisingly, adding ERi reduced the concentrations of heme in the cells while the blocking effect of ERβi was more significant and specific. The expression of genes is precisely regulated by transcriptional complexes that consist of transcription factors and transcriptional regulators [ 32 ]. Rather than regulating the target gene directly, transcriptional regulators must rely on a corresponding transcription factor to play a role [ 33 , 34 ]. NCOA1, also known as steroid receptor coactivator 1, can stimulate transcription in a hormone-dependent manner by binding to nuclear receptors [ 23 , 35 ]. We used a protein–protein interaction network analysis and found that NCOA1 may be a key molecule in the process in which estrogen regulates succinate–heme metabolism. It has been reported that NCOA1 overexpression has been observed in a variety of pathological conditions, such as diabetes, obesity, and carcinogenesis. The increased expression of NCOA1 in bladder cancer patients is positively correlated with the tumor clinical stage, the pathological grade, and lymph node metastasis. Poorer outcomes may be due to NCOA1 promotion of cell proliferation through upregulating the level of androgen receptors [ 36 , 37 ]. We found that silencing NCOA1 decreased cell viability and downregulated the expression of ALAS1, suggesting that estrogen may promote heme biosynthesis by activating the expression of NOCA1 and increasing ALAS1 synthesis. In addition, although estrogen and succinate can increase the levels of heme in Ishikawa and RL95-2 cells, silencing NCOA1 can partially or even completely eliminate this effect. This further confirmed that NCOA1 may be an important regulatory molecule in estrogen–succinate–heme metabolism. The occurrence of breast cancer is also closely related to estrogen. Studies have verified that estrogen can upregulate the expression of NCOA1 through ER. The combination of NCOA1 and the transcription factor HOXC11 can activate the transcription of downstream genes, including integrin α5, ADAM22, and Myc, and can thus promote the metastasis and progression of breast cancer [ 38 ]. The mitogen-activated protein kinase (MAPK) signal pathway is a key kinase that participates in the regulation of cell proliferation and apoptosis. It has been confirmed that the activation of the MAPK pathway can lead to the translocation of nuclear transcription factor κB (NF-κB) from the cytoplasm to the nucleus and upregulate the expression of NF-κB downstream molecules, such as interleukin-1β (IL1β), IL6, and tumor necrosis factor-α [ 39 ]. Peng et al. found that the levels of NCOA1 in patients with myocardial dysfunction were significantly increased. Further studies showed that NCOA1 can form a transcriptional complex with NF-κB, and thus activates the expression of proinflammatory-related cytokines, such as in the myocardium [ 22 ]. However, the exact regulatory mechanisms of estrogen on NCOA1 and of the ERβ/NCOA1 axis on ALAS1 in UEC need to be explored further. A growing volume of studies have suggested that heme metabolism plays a key role in the pathogenesis of various diseases. Some scholars have already proposed that heme-targeted drugs may be used as potential angiogenesis inhibitors and are expected to be new treatment strategies for many diseases in the future [ 40 ]. Heme-regulated inhibitor (HRI), also known as eIF2AK1, is the member of eukaryotic translation initiation factor 2 alpha kinase (eIF2AK) family. It has been reported that heme could affect protein biosynthesis via phosphorylating α subunit of eIF2 on serine 51 residue [ 41 ]. HRI has been proven to increase fetal hemoglobin production via diminishing the expression of BCL11A, and this may be a potential therapeutic target for sickle cell disease [ 42 ]. Moreover, mifepristone activates cytotoxic effects through the eIF2AK1-mediated signal transduction pathway, promoting the drug synergies in ovarian cancer therapy [ 43 ]. In our study, we treated UECC with different concentrations of heme and found that heme increased the cell viability and invasiveness of UECC while, as expected, silencing NCOA1 could partially or even completely eliminate this effect of heme. In addition, our previous study confirmed that excess heme impairs the phagocytosis of peritoneal macrophages, promoting the progression of endometriosis [ 44 ]. We speculated that the large amounts of heme present in UEC may also have similar functions. However, follow-up in vivo experiments still need to be conducted. Moreover, the underlying molecular mechanisms of how heme regulates cell biological behavior also deserve to be further studied.

In this study, as shown in Figure 9 , we found that there are high levels of heme in UECC. By activating the expression of NCOA1 through ERs, estrogen increases the expression of ALAS1 and promotes heme synthesis in UECC. Meanwhile, the low levels of SDHB in UEC lead to the abnormal accumulation of succinate, which also promotes heme synthesis and upregulates ER levels. Estrogen and succinate synergistically promote the accumulation of free heme, increasing the proliferative and invasive potential of UECC. In summary, our study tried to clarify the characteristics of estrogen–succinate–heme metabolism in UEC progression. We believe this study will provide a theoretical basis and potential target for the anticancer treatment of UEC in the future.