Role of steroid receptor-associated and regulated protein in tumor progression and progesterone receptor signaling in endometrial cancer : Chinese Medical Journal

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Original Article

Role of steroid receptor-associated and regulated protein in tumor progression and progesterone receptor signaling in endometrial cancer

Liu, Jie1,2; Wang, Zhiqi1; Zhou, Jingyi1; Wang, Jiaqi1; He, Xiangjun2; Wang, Jianliu1

Editor(s): Guo, Lishao

Author Information

1Department of Obstetrics and Gynecology, Peking University People's Hospital, Beijing 100044, China

2Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, Beijing 100044, China.

Correspondence to: Dr. Xiangjun He, Central Laboratory and Institute of Clinical Molecular Biology, Peking University People's Hospital, No. 11, Xizhimen South Street, Beijing 100044, China E-Mail: [email protected] Dr. Jianliu Wang, Department of Obstetrics and Gynecology, Peking University People's Hospital, No.11, Xizhimen South Street, Beijing 100044, China E-Mail: [email protected]

How to cite this article: Liu J, Wang Z, Zhou J, Wang J, He X, Wang J. Role of steroid receptor-associated and regulated protein in tumor progression and progesterone receptor signaling in endometrial cancer. Chin Med J 2023;00:00–00. doi: 10.1097/CM9.0000000000002537

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Chinese Medical Journal ():10.1097/CM9.0000000000002537, May 05, 2023. | DOI: 10.1097/CM9.0000000000002537

Abstract

Introduction

Endometrial cancer (EC) is a complex and highly heterogeneous disease with different histological types (endometrioid, serous, etc), pathological grades (G1–G3), FIGO stages (stages I–IV), and molecular subtypes, including polymerase epsilon (POLE)-ultramutated, microsatellite instability (MSI), copy number low (CNL), and copy number high (CNH).[1,2] Patients with serous, high-grade, high-stage, and CNH types generally have a poor prognosis accompanied by low progesterone receptor (PR) expression.[1,3] High PR expression is associated with a favorable prognosis.[3,4] The human uterus is a hormone-responsive organ, and estrogen drives uterine cell proliferation via estrogen receptors (ER), whereas progesterone acts via PR, offsetting proliferation.[5] PR has three isoforms, PRA, PRB, and PRC, all of which are transcribed from a single gene, PGR.[6] After activation, PR binds to progesterone response elements (PRE) within the basal promoter regions, recruits coregulators to form a functional transcription factor complex, and regulates the expression of downstream target genes.[6] The PR signaling pathway is a key candidate therapeutic target for endometrioid EC.[7] Progesterone-based hormonal therapy has been used in clinical practice to treat well-differentiated and PR-positive early type I ECs in young patients who wish to preserve fertility.[7] However, progestin therapy is only effective in a subset of patients, suggesting that factors other than PR affect the response. In addition, many progestin-responsive patients exhibit progression with reduced PR expression and develop resistance during treatment, indicating the importance of the upstream regulators of PR.[7] A better understanding of the factors contributing to PR function and regulation is vital for improving the efficacy of progestin therapy.

Steroid receptor-associated and regulated protein (SRARP) is mainly expressed in the normal brain and steroid hormone-responsive tissues, including the breast, uterine endometrium, and cervix (see the Human Protein Atlas, https://www.proteinatlas.org). It was first reported in 2012 as a steroid receptor-associated gene in breast cancer and was named ER-related nuclear factor.[8] Higher SRARP expression is associated with a lower tumor stage and tumor grade, less lymph node metastasis, and a favorable prognosis in breast cancer.[8]SRARP interacts with ER or androgen receptors (AR) as a coregulator to modulate PR expression in cultured ER/PR-positive breast cancer cells.[9,10] However, the role of SRARP in tumor progression and PR signaling in EC is not defined. The aim of this study was to investigate the role of SRARP in tumor progression and PR signaling in EC.

Methods

Ethical approval

The study was approved by the Ethics Committee of Peking University People's Hospital (No. IRB00001052-19142). All patients agreed and signed written informed consents.

Data resources

Three public databases, including The Cancer Genome Atlas (TCGA, https://xenabrowser.net/datapages/),[11] Clinical Proteomic Tumor Analysis Consortium (CPTAC, https://cptac-data-portal.georgetown.edu/cptacPublic/),[12] and Gene Expression Omnibus (GEO, GSE17025, http://www.ncbi.nlm.nih.gov/geo),[13] were used for analysis of the association between gene expression and clinicopathologic characteristics of patients with EC. RNA sequencing data, clinical information and survival data of EC were downloaded and analyzed. cBioPortal (www.cbioportal.org) was used to retrieve information regarding the molecular subtype classification (POLE, MSI, CNL, and CNH), and mutation and gene co-expression data.[14] After excluding normal solid tissue samples, and repeated and recurrent samples, 543 cases from TCGA were included in the analyses. CPTAC and GEO datasets contain 96 and 91 cases, respectively. Clinical features and demographic characteristics of the above datasets are summarized in Supplementary Table 1, https://links.lww.com/CM9/B425. RNA sequencing and mutation data of 28 EC cell lines were obtained from the website of Cancer Cell Line Encyclopedia (CCLE; https://sites.broadinstitute.org/ccle/datasets).[15]

Cell culture and tissue samples

The Ishikawa cell line was obtained from the Chinese National Infrastructure of Cell Line Resource (Beijing, China) and cultured in Dulbecco's Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12, Gibco-BRL, Gaithersburg, MD, USA) with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). The HEC50B cell line was obtained from the Japanese Collection of Research Bioresources (https://cellbank.nibiohn.go.jp/english/) and cultured in Minimum Essential Medium (MEM) (Omacgene, Beijing, China) with 15% FBS. All cells were cultivated in a 37°C, 5% CO2 incubator and subcultured every 2 days.

Tissue samples were obtained from patients with EC treated at the Department of Obstetrics and Gynecology of Peking University People's Hospital (Beijing, China). The pathological grade and immunohistochemical staining results for PR were determined by the pathologists in our hospital. Twenty-five EC samples with PR staining results were selected, including 17 PR-positive and eight PR-negative samples. The study was approved by the Ethics Committee of Peking University People's Hospital (No. IRB00001052-19142).

Construction of lentiviral vectors

SRARP cDNA was cloned into the plasmid GV367. The recombinant lentivirus containing SRARP cDNA (lenti-SRARP) and negative control lentivirus (lenti-NC) were produced by GeneChem Technologies (Shanghai, China). The viral titer was 2.5 × 108 TU/mL. The multiplicity of infection (MOI), defined as the ratio of infectious virions to susceptible cells,[16] was determined by serial dilution. According to the manufacturer's instructions, EC cells were seeded at a concentration of 1 × 105 cells/well in 6-well culture plates and cultivated for 24 h to reach approximately 30% to 50% confluence. Ishikawa and HEC50B cells were infected with MOI values of 100 and 30, respectively. MEM or DMEM/F12 containing HitransG A/P (GeneChem Technologies, Shanghai, China) was used to dilute the recombinant lentivirus to enhance the efficiency of lentivirus infection. Cells were harvested at 72 h after infection for further analysis.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. qRT-PCR was performed using SYBR™ Select Master Mix (Transgene, Shenzhen, China, cat# AH311-02) and the BIO-RAD CFX96TM Real-Time System (Hercules, CA, USA). The specificity was confirmed by a melting curve analysis of the amplified products. Relative mRNA levels of each gene were normalized to GAPDH. Primers are listed in Supplementary Table 2, https://links.lww.com/CM9/B425.

Western blotting analysis

Cells and tissues were lysed in RIPA lysis buffer (Beyotime, Haimen, China) containing phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail (NCM Biotech, Suzhou, China). Cell pellets were removed by centrifugation at 12,000 r/min for 20 min at 4°C. The supernatant was supplemented with 5× sodium dodecylsulfate (SDS) sample buffer and boiled at 95°C for 10 min. Equal protein amounts were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein bands were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing Tween 20 (TBST) for 60 min with gentle shaking, followed by incubation with the primary antibodies (anti-SRARP, Sigma-Aldrich, St. Louis, MO, USA, HPA026676; anti-E-cadherin, Cell Signaling Technology, Danvers, MA, USA, 14472S; anti-N-cadherin, Cell Signaling Technology, 13116T; anti-Wnt family member 7A [WNT7A], Abcam, Cambridge, UK, ab274321; anti-PR, Thermo, Waltham, MA, USA, MA112626; anti-GADPH, Proteintech, Rosemont, IL, USA, 10494-1-AP) at 4°C overnight. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein signals were visualized by enhanced chemiluminescence (ChemiDoc MP Imaging System, BIO-RAD, Hercules, CA, USA).

In vitro cell proliferation assay

Cells were seeded on a 96-well plate (Corning, Inc., Corning, NY, USA) at a density of 1000 cells/100 μL/well in triplicate and evaluated after 0, 24, 48, 72, and 96 h of incubation. After removing the medium, 100 μL of complete medium containing 10% Cell Counting Kit-8 (CCK-8, Yisean, Shanghai, China) was added to each well and incubated for an additional 2 h at 37°C according to the manufacturer's instructions. Subsequently, cell viability was determined at 450 nm using a spectrophotometer (NanoQuant, infinite M200, Tecan, Männedorf, Switzerland).

Wound healing assays

Cells were seeded onto 6-well plates and cultured overnight at 37°C to form a confluent monolayer. The monolayers were artificially scraped with a 200 μL pipette tip and washed with PBS. Then, a new medium was added to each well. Images were obtained at 0 and 24 h, and the width of each scratch was compared.

Cell migration and invasion assays

Cell invasion potential was evaluated in 24-well Transwell plates (Corning, Inc., Corning, NY, USA). The upper chamber of each insert was coated with 50 μL of Matrigel (Corning, Inc., Corning, NY, USA) (1:5 diluted in serum-free DMEM/F12 or MEM) and incubated at 37°C for 4 h. Cells were trypsinized and resuspended in 100 μL (3 × 104/mL) of culture medium without serum and seeded in the upper chamber. The lower chamber was supplemented with 500 μL of culture medium supplemented with 10% FBS. After cultivation for 24 h, cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet before microscopic visualization. Migration assays were conducted similarly, except that the Transwell inserts were not coated with Matrigel.

Cell cycle analysis by flow cytometry

Cells infected with lenti-SRARP or lenti-NC were trypsinized and washed with ice-cold PBS, fixed with 75% ethanol, and stained with propidium Iodide (PI) staining buffer (BD, Franklin Lakes, NJ, USA) for 30 min at room temperature in the dark. A cell cycle analysis was performed using a flow cytometer (FACS Calibur, BD Biosciences, San Jose, CA, USA). FlowJo (FlowJo, LLC, Ashland, OR, USA) was used to acquire cell cycle distribution curves.

Luciferase assay

PRE was inserted into plasmid pGM-Luc upstream of a luciferase reporter gene. At 80% confluence, cells were transfected with 2 μg of recombinant plasmid (pGM-PRE-Luc) using Lipofectamine® 3000 (Invitrogen, Thermo Fisher Scientific, USA) following the manufacturer's protocol. At 48 h post-transfection, transfected cells were treated with 400 μg/mL gentamycin (G418, MedChemExpress, Monmouth Junction, NJ, USA) for 2 weeks to select stably transfected cells. An equal number of cells were seeded and incubated with 5 mmol/L medroxyprogesterone acetate (MPA) for 48 h. Control group was treated with dimethyl sulfoxide. Luciferase activity was measured using the Luciferase Reporter Assay System (Promega, Madison, WI, USA) and a luminometer. Results are representative of at least three independent experiments performed in triplicate.

Co-immunoprecipitation (Co-IP) analysis

For protein interaction, Co-IP experiments were performed according to the kit instructions (88804, Thermo Fisher Scientific, USA). A small amount of lysate was collected before each IP to evaluate input. Following IP assays with an SRARP antibody (Sigma-Aldrich, HPA026676), supernatants were collected and applied for immunoblotting analysis to detect the pull-down of SRARP and PR proteins. Control assays were conducted with a non-specific rabbit IgG.

Statistical analyses

Statistical analyses were performed using GraphPad Prism (v.8.0, La Jolla, CA, USA) and SPSS (v.23.0, Chicago, IL, USA). Student's t-tests were used for comparing normally distributed continuous data between two groups. Mann–Whitney U-tests were used to compare mean values between two groups for non-normally distributed continuous data. For the survival analysis, patients were divided into high- and low-expression groups using the optimal cutoff point determined using X-tile program (v.3.6.1, Yale University School of Medicine, New Haven, CT, USA).[17] Overall survival (OS) time was defined as the number of days from the date of diagnosis to the date of death for any reason or the last follow-up. Disease-free survival (DFS) time referred to the number of days from the date of diagnosis to the date of an event including recurrence, metastasis, and deaths from any cause. The distributions of OS and DFS were estimated for each group using the Kaplan–Meier method. The log-rank test was used to examine statistical significance. P < 0.05 indicated statistical significance.

Results

Down-regulation of SRARP was associated with more aggressive cancer types and poor prognosis in EC

Using TCGA data, we analyzed the frequency of SRARP mutations in EC. Among 530 EC samples with mutation data, there were five missense mutations (A143V, A61T, P90S, P126L, and T40M) in five samples and two synonymous mutations (L57L and P126P) in two samples. Among 28 EC cell lines with mutation data available in the CCLE database, only HEC108 cell line had one missense mutation (V117M). These results suggested that the SRARP mutation frequency is low in EC. Therefore, our study focused on alterations in the expression levels of SRARP that might lead to functional changes.

We analyzed the associations of SRARP expression with clinicopathologic characteristics in 543 EC samples from TCGA. As shown in Figures 1A–F, the expression levels of SRARP were significantly lower in more aggressive EC, such as CNH subtype, serous type, advanced stage, higher grade, samples with positive lymph nodes, and samples with positive peritoneal lavage cytology, than in less aggressive types.

F1
Figure 1:
Down-regulation of SRARP was significantly associated with more aggressive cancer types. SRARP expression in different molecular subtypes (A), histological types (B), grades (C), clinical stages (D), lymph node metastasis status (E), and peritoneal washing cytology status (F) in TCGA samples. SRARP expression in different molecular subtypes (G), histological types (H), stages (I), and grades (J) in samples from CPTAC dataset. SRARP expression in different histological types (K) and grades (L) in samples from GEO dataset. Data are shown as the mean ± standard error of mean. ∗ P ≤ 0.05; † P ≤ 0.01; ‡ P ≤ 0.001. CPTAC: Clinical Proteomic Tumor Analysis Consortium; CNH: Copy number high; CNL: Copy number low; E: Endometriod EC; EC: Endometrial cancer; MSI: Microsatellite instability; S: Serous EC; SRARP: Steroid receptor associated and regulated protein; TCGA: The Cancer Genome Atlas.

Similar results were observed in analyses of CPTAC [Figure 1G–J] and GEO datasets [Figure 1K, L]; however, SRARP expression was not lower in stage IV cases than that in lower stages in the CPTAC dataset because only two samples were included in this group.

For survival analysis, patients were stratified into high or low SRARP expression groups by the X-tile method [Figure 2A]. Kaplan–Meier plots showed that patients with high SRARP expression had a significantly better OS [Figure 2B] and DFS [Figure 2C] (P < 0.0001) than those with low SRARP expression.

F2
Figure 2:
Down-regulation of SRARP was associated with poor prognosis in endometrial cancer. (A) Number of TCGA patients with high- or low-SRARP expression, death, and recurrence. (B, C) Kaplan–Meier plot of the distribution of overall survival and disease-free survival with high- or low-SRARP expression samples group. SRARP: Steroid receptor associated and regulated protein; TCGA: The Cancer Genome Atlas.

SRARP overexpression reduced cell proliferation by inducing G1 phase arrest and suppressed migration and invasion

To verify the tumor-suppressive effect of SRARP, we investigated phenotypic changes in SRARP-overexpressing cells. First of all, SRARP expression level in EC cell lines was investigated. Using the CCLE website, we found that all 28 EC cell lines with RNA-sequencing data showed very low SRARP mRNA levels. Then, two cell lines, Ishikawa and HEC50B, were selected for analysis. The qRT-PCR results showed that although Ishikawa displayed higher SRARP expression than other cell lines, its expression levels in EC cell lines were generally very low, compared with that in non-cancerous uterine endometrium and EC tissues with different grades [Supplementary Figure 1, https://links.lww.com/CM9/B425]. Therefore, we overexpressed SRARP in EC cell lines using a recombinant lentivirus carrying SRARP cDNA.

The lentivirus-mediated overexpression of SRARP in Ishikawa and HEC50B cells was confirmed by Western blotting and qRT-PCR [Figure 3A]. Cell proliferation was evaluated by a CCK-8 assay. Ishikawa and HEC50B cells infected with lenti-SRARP exhibited markedly lower cell proliferation than that of cells infected with lenti-NC [Figure 3B]. Then, the cell cycle was analyzed by flow cytometry. The percentage of cells in the G0/G1 phase increased significantly and cells in the S and G2/M phases decreased significantly in SRARP-overexpressing Ishikawa [Figure 3C] and HEC50B cells [Figure 3D] compared with that in the control group.

F3
Figure 3:
Overexpression of SRARP suppressed the growth of endometrial cancer cells by inducing G1 phase cycle arrest. (A) The overexpression of SRARP in Ishikawa and HEC50B cells were detected by Western blotting and quantitative real-time polymerase chain reaction. (B) Proliferation ability was detected by Cell Counting Kit-8 assay in Ishikawa and HEC50B cells infected with lenti-SRARP. (C,D) Cells cycle result displayed by flowcytometry in cells infected with lenti-SRARP and those infected with negative control lentivirus in Ishikawa (C) and HEC50B (D). Data are based on at least three independent experiments, and are shown as the mean ± standard deviation. ∗ P < 0.05; † P < 0.01; ‡ P ≤ 0.001. 50B-NC: HEC50B infected with control lentivirus; 50B-SRARP: HEC50B infected with lenti-SRARP. ISK-NC: Ishikawa infected with control lentivirus; ISK-SRARP: Ishikawa infected with lenti-SRARP; lenti-SRARP: Lentivirus containing SRARP cDNA; SRARP: Steroid receptor associated and regulated protein.

Other tumor progression-related phenotypic changes, such as migration and invasion ability, were evaluated in SRARP-overexpressing cells. Wound healing assays showed delayed closure of scratches in SRARP-overexpressing Ishikawa [Figure 4A, E] and HEC50B [Figure 4B, E] cells compared to cells infected with the control lentivirus at 24 h, indicating reductions in cell proliferation and migration. A transwell assay showed that rates of migration [Figure 4C, F] and invasion [Figure 4D, F] were significantly lower in cells with SRARP overexpression than in the control group. As the results of wound healing assay and transwell migration/invasion assays reflect not only the migration and invasion of cells but also the excessive proliferation of cells, we detected the expression of actin-binding genes involved in cell deformation and movement, including FLNA[18] and FSCN1.[19] The expression levels of these genes were significantly lower in SRARP-overexpressing Ishikawa [Figure 4G] and HEC50B [Figure 4H] cells than that in the control group.

F4
Figure 4:
Overexpression of SRARP suppressed cell migration and invasion in EC cells. Representative picture of wound healing assays in Ishikawa (A) and HEC50B cells (B). Representative result of transwell migration (C) and invasion (D) assay in Ishikawa and HEC50B cells (original magnification ×200). (E) Quantitative analysis of wound healing assay in SRARP-overexpressing EC cells. (F) The number of migrating and invaded cells in SRARP-overexpressing EC cells. (G,H) The qRT-PCR results of FLNA and FSCN1 in SRARP-overexpressed Ishikawa and HEC50B cells. Data are based on at least three independent experiments, and shown as the mean ± SD. ∗ P < 0.01; † P ≤ 0.001. 50B-NC: HEC50B infected with control lentivirus; 50B-SRARP: HEC50B infected with lenti-SRARP; EC: Endometrial cancer; ISK-NC: Ishikawa infected with control lentivirus; ISK-SRARP: Ishikawa infected with lenti-SRARP; lenti-NC: Negative control lentivirus; lenti-SRARP: Lentivirus containing SRARP cDNA; qRT-PCR: Quantitative real-time polymerase chain reaction; SD: Standard deviation; SRARP: Steroid receptor associated and regulated protein.

Overexpression of SRARP modulated epithelialmesenchymal transition (EMT) markers and the Wnt signaling pathway in EC

Increased invasion and migration are associated with the EMT, which is characterized by the loss of the epithelial marker E-cadherin and the gain of the mesenchymal marker N-cadherin. To further explore the mechanism underlying the effects of SRARP on the migration and invasion of EC cells, we detected the expression of key genes involved in the EMT and the Wnt pathways. Western blotting [Figure 5A] revealed that E-cad (encoded by CDH1) was expressed at higher level in SRARP overexpressing Ishikawa cells than that in the control, but N-cadherin (encoded by CDH2) was not found in either group. In SRARP-overexpressing HEC50B cells, the expression level of N-cadherin was significantly lower than that in the control and the expression of E-cadherin remained undetectable. Concordant results were observed at the mRNA expression level by qRT-PCR [Figure 5B]. The significant down-regulation of WNT7A was observed at mRNA and protein levels in both SRARP overexpressing cells.

F5
Figure 5:
Overexpression of SRARP modulated EMT markers and Wnt signaling pathway in EC. (A) Western blotting analysis of N-cadherin, E-cadherin, and WNT7A expression in SRARP-overexpression EC cells. (B) Analysis of the expression of CDH1, CDH2, and WNT7A in SRARP-overexpressing EC cells by qRT-PCR. Data are based on at least three independent experiments and shown as the mean ± standard deviation. ∗ P ≤ 0.001. 50B-NC: HEC50B infected with control lentivirus; 50B-SRARP: HEC50B infected with lenti-SRARP; EC: Endometrial cancer; EMT: Epithelial-mesenchymal transition; ISK-NC: Ishikawa infected with control lentivirus; ISK-SRARP: Ishikawa infected with lenti-SRARP; lenti-NC: Negative control lentivirus; lenti-SRARP: Lentivirus containing SRARP; qRT-PCR: Quantitative real-time polymerase chain reaction; SRARP: Steroid receptor associated and regulated protein.

We also investigated other pathways that promote the EMT, including the PI3K/AKT/mTOR, RAS–RAF–MEK–ERK, and TGF-β signaling pathways.[20] Key genes in these pathways, including AKT1, AKT2, ERK1/MAPK3, ERK2/MAPK1, and TGFB were detected and no significant differences in protein and phosphorylated protein levels between groups were detected (data not shown).

SRARP was co-expressed with PR in endometrial tumors

The relationship between SRARP and PR was analyzed using TCGA data from the cBioPortal website. Setting Spearman's correlation coefficient |CC| ≥ 0.5 and P < 0.0001 as thresholds, we extracted 309 PR-correlated genes (PRCGs). SRARP ranked in the top 8% of PRCGs, with a CC of 0.653 [Figure 6A]. The co-expression of SRARP with PR was also observed in CPTAC data, with a CC of 0.640 [Figure 6B]. To further confirm the correlation of PR with SRARP, we evaluated expression levels in EC samples from our hospital. SRARP displayed significantly higher expression in the PR-positive group than in the PR-negative group [Figure 6C].

F6
Figure 6:
Co-expression of SRARP with PR. Correlation of SRARP with PR in TCGA samples (A) and CPTAC samples (B); (C) Comparison of SRARP expression between PR-positive group (n = 17) and PR-negative group (n = 8) in samples of our hospital by qRT-PCR. All data are presented as the mean ± SEM. ∗ P < 0.001. CPTAC: Clinical Proteomic Tumor Analysis Consortium; PR: Progesterone receptor; PRH: PR-positive; PRL: PR-negative; qRT-PCR: Quantitative real-time polymerase chain reaction; SEM: Standard error of the mean; SRARP: Steroid receptor associated and regulated protein; TCGA: The Cancer Genome Atlas.

SRARP could upregulate PR expression and interact with PR to modulate the expression of PR target genes in EC cells

The high-correlation between SRARP and PR promoted us to further explore the role of SRARP in PR signaling. We detected the expression of PR in SRARP over-expressing Ishikawa and HEC50B cell lines by qRT-PCR and Western blotting. A significant increase in PGR mRNA expression was demonstrated in both cells lines [Figures 7A, B]. However, the elevation of PR protein (PR-B) expression was observed only in Ishikawa cells [Figure 7A] and not in HEC50B cells [Figure 7B], and this may be explained by the lower sensitivity of Western blotting than qRT-PCR.

F7
Figure 7:
SRARP could upregulate PR expression and interact with PR to modulate the expression of PR target genes in EC cells. PR mRNA and protein expression in Ishikawa (A) and HEC50B (B) cells were detected by qRT-PCR and Western blotting. (C) Interaction between SARAP and PR detected by Co-IP. (D) Luciferase activity in cells transfected with lenti-SRARP in response to MPA treatment. (E) Expression of PR downstream target genes detected by qRT-PCR. ∗ P < 0.05; † P < 0.01; ‡ P < 0.001. 50B-NC: HEC50B infected with control lentivirus; 50B-SRARP: HEC50B infected with lenti-SRARP; IP: Immunoprecipitation; ISK-NC: Ishikawa infected with control lentivirus; ISK-SRARP: Ishikawa infected with lenti-SRARP; lenti-SRARP: Lentivirus containing SRARP cDNA; MPA: Medroxyprogesterone acetate; PR: Progesterone receptor; qRT-PCR: Quantitative real-time polymerase chain reaction; SRARP: Steroid receptor associated and regulated protein.

To test whether SRARP interacts with PR, we performed an co-IP analysis of cells over-expressing SRARP. As shown in Figure 7C, immunoblotting confirmed the successful pull-down of SRARP by IP assays. Immunoblotting using a PR antibody revealed a distinct PR band in the SRARP-IP samples but not in the control group.

Then, a luciferase reporter assay was conducted to test whether SRARP could modulate the transcriptional activity of PR target genes. Ishikawa cells stably transfected with pGM-PRE-Luc were infected with lenti-SRARP and lenti-NC. In the presence of MPA, a synthetic progestin, the overexpression of SRARP increased luciferase activity by three-fold compared with that in the control group [Figure 7D]. In addition, the expression levels of several PR target genes (including FKBP5, HAND2, MIG6, and PAEP) were examined by qRT-PCR. As shown in Figure 7E, the up-regulation of these PR target genes in the group with SRARP overexpression was demonstrated.

Discussion

SRARP is a poorly understood gene, with only a few studies in breast cancer demonstrating a favorable prognostic effect.[8-10] Our study demonstrated that higher SRARP expression was significantly associated with better DFS and OS in patients with EC. Lower SRARP expression was significantly associated with CNH, lower grade, lower stage, positive peritoneal washes, and positive lymph node metastasis. These results suggest that high SRARP expression is a favorable prognostic factor and the gene functions as a tumor suppressor in EC. The presumed tumor-suppressor effect of SRARP was verified by lentivirus-mediated overexpression in two EC cell lines, Ishikawa and HEC-50B. The overexpression of SRARP attenuated cell proliferation by inducing G1 phase cycle arrest and suppressed the migration and invasion of both EC cell lines. The decreased migration and invasion ability of SRARP-overexpressing cells was supported by the reduced expression of the actin-binding genes FLNA and FSCN1.

The molecular mechanism underlying the inhibitory effects of SRARP on migration and invasion may include the suppression of EMT and the non-canonical Wnt pathway. The switch from E-cadherin to N-cadherin is a hallmark of cell migration and invasion as well as cancer progression.[21,22] In the present study, atypical EMT was observed because N-cadherin or E-cadherin was not detected in Ishikawa and HEC50B cells, respectively, and this may be due to the difference in genetic background between these two cell lines. E-cadherin and N-cadherin are adhesion proteins that cooperate closely with the actin cytoskeleton.[23] Actin-mediated motility is regulated by actin-binding proteins, such as FLNA and FSCN1. FLNA determines the shape and movement of cells by guiding the formation of dynamic actin stress fibers to regulate cell adhesion and migration.[18] FSCN1 is involved in organizing filamentous actin into parallel bundles and is required for the formation of cell protrusions.[19] The Wnt pathway acts as a key regulator of EMT by regulating E-cadherin and N-cadherin.[24]WNT7A, a member of the non-canonical Wnt pathway,[25] is the main component of the Wnt family expressed in Ishikawa and HEC-50B cell lines according to the CCLE website. WNT7A is associated with tumor progression and worse OS in EC.[26] The simultaneous alteration of these factors contributes to the effects of SRARP in EC cells.

In cultured ER/AR/PR-positive breast cancer cells, SRARP interacts with ER or AR as a coregulator to modulate PR expression. The knockdown of SRARP by RNA interference decreases ER and PR expression.[8]SRARP also negatively regulates the AR-mediated downregulation of ER and PR expression in breast cancer cells.[8,9] Nevertheless, previous studies have not investigated whether SRARP could interact with PR. In the present study, we demonstrated the role of SRARP in PR signaling, as evidenced by its high correlation with PR levels in EC tissues and analyses of the effects of SRARP overexpression in EC cells. SRARP increased the expression of PR and interacted with PR. This interaction was further demonstrated by a luciferase reporter assay showing that SRARP activated PR, and SRARP overexpression in EC cells upregulated PR target genes. These results improve our understanding of SRARP, indicating it might function as a coregulator to regulate the transcriptional activity of many steroid hormone receptors. Unfortunately, we were unable to evaluate the relationship between SRARP and ER or AR owing to the unavailability of EC cell lines with high expression levels of ER, AR, and PR.

Although we demonstrated that SRARP could upregulate PR expression, the PR expression level in SRARP-overexpressing EC cells was still lower than that in the normal uterine endometrium and most EC tissues. It is possible that SRARP is a coregulator of ER and does not function as a transcription factor. ER is a well-defined transcription factor that directly regulates the expression of PR in the uterus.[27] However, the expression level of ER is very low in EC tissues and cells with low PR expression. Alternatively, this result may be explained by the lack of certain PRCGs that function as coregulators of PR expression, as we found that many PRCGs were expressed at very low levels in EC cell lines according to CCLE data. Hence, the overexpression of SRARP alone cannot fully recover the normal PR pathway. However, owing to its low expression in EC cell lines, we did not investigate the effect of SRARP knockdown.

Like other nuclear transcription factors, PR recruits many coregulators to form large multi-protein complexes to regulate downstream target genes.[28] The components of the PR transcriptional complex have not been fully defined and the contribution of PR coregulators to sensitivity to progestin therapy in PR-positive EC is unclear. We provide the first evidence that SRARP is a component of the PR transcriptional complex and that the interaction between SRARP and PR is necessary for MPA responsiveness. Further exploration of other main components of the PR transcriptional complex will help clarify the molecular mechanism underlying progestin resistance in EC.

Both PRA and PRB are expressed in the normal uterine endometrium.[28] Our results showed that only PRB was upregulated in SRARP-overexpressing cells, suggesting that SRARP interacts with transcription factors that only bind to the PRB promoter in EC cell lines. PRA and PRB are transcribed from the same gene by two distinct promoters and exhibit distinct functions in EC. PRB has been reported to play a key role in the pathogenesis of EC.[29] Ishikawa cells transfected with PRB have a growth inhibitory response to progesterone, whereas cells transfected with PRA do not.[30] PR was upregulated more highly by SRARP overexpression in the Ishikawa cell line than in the HEC50B cell line. The Ishikawa cell line was established from a patient with well-differentiated endometrioid EC expressing ER and PR in the early passages but not in cells over the 50th generation.[31] The HEC50B cell line was derived from poorly differentiated endometrioid EC tissue that did not initially express ER and PR.[32] The difference in genetic background and transcriptome between Ishikawa and HEC50B cells could account for the dissimilarity between these two cell lines in the effects of SRARP on PR expression.

In summary, SRARP, as a coregulator, may have pleiotropic effects and coordinate the functions of multiple nuclear transcription factors. The present study elucidated the tumor-suppressive effect of SRARP in EC and revealed that this effect is mediated by the inhibition of the EMT via the negative regulation of the non-canonical Wnt signaling pathway. We also demonstrated that SRARP is required for the transcriptional activity of PR on its downstream target genes in addition to its role in modulating PR expression. Our study provides a deeper understanding of coregulators that function in PR signaling and may provide a therapeutic strategy for targeting the coregulator of steroid receptor signaling in hormone-unresponsive EC.

Funding

This work was supported by grants from the National Key Technology R&D Program of China (Nos. 2019YFC1005200 and 2019YFC1005201).

Conflicts of interest

None.

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Keywords:

SRARP; Progesterone receptor; Epithelial-mesenchymal transition; Coregulator; Tumor suppressor; Endometrial cancer

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