TRIM22 governs tumorigenesis and protects against endometrial cancer-associated cachexia by inhibiting inflammatory response and adipose thermogenic activity

Abstract

Background

Endometrial cancer (EC) is one of the most common cancers in women, with a short overall survival and poor prognosis. Besides the biologically aggressive EC properties, Cancer-associated cachexia is the main factor. However, the detailed mechanism underlying EC-related cachexia and its harmful effects on EC progression and patient prognosis remains unclear.

Methods

For clinical specimen and the vitro experiment, we detected TRIM22 expression level, EC patients’ survival time, EC cell functional change, and adipose thermogenic changes to identify the function of TRIM22 in EC progression, EC-associated cachexia, and their molecular mechanisms. Then, for the vivo experiment, we exploited the xenografts in mice to identify the function of TRIM22 again, and to screen the drug therapeutic schedule.

Results

Herein, we demonstrated that TRIM22 inhibited EC cell growth, invasion, and migration. Interleukin (IL)-6 mediated brown adipose tissue activation and white adipose tissue browning which induced EC-related cachexia. TRIM22 suppressed the EC cells’ secretion of IL-6, and IL-6 mediated EC-related cachexia. Mechanistically, TRIM22 inhibited EC progression by suppressing the nucleotide-binding oligomerization domain 2(NOD2)/nuclear factor-kappaB (NF-κB) signaling pathway, with the purpose of impeding the production of IL-6. Moreover, we revealed that TRIM22 inhibited EC-associated cachexia by suppressing the IL-6/IL-6 receptor (IL-6R) signaling pathway. Therapeutically, we demonstrated that combination treatment with a TRIM22 inducer (progesterone) and a thermogenic inhibitor (IL-6R antibody) synergistically augmented the antitumor efficacy of carbotaxol (carboplatin and paclitaxel), in vivo.

Conclusion

Our data reveals that TRIM22-EC-IL-6-cachexia cross-communication has important clinical relevance and that the use of combined therapy holds great promise for enhancing the efficacy of anti-ECs. (Fig. graphical abstract)

Graphical abstract

Background

Endometrial cancer (EC), a malignant tumor derived from endometrial epithelium, is one of the most common cancers in women, with increasing incidence and disease-associated mortality worldwide [1, 2]. The 5-year overall survival rates of EC are only 17% and 15% for advanced stages III and IV, respectively [3, 4]. Aggressiveness is the leading cause of the EC patients’ deaths. No cancer-specific drugs are available for patients with EC. Current chemical antitumor regimens have large toxic side effects and can easily develop drug resistance in long-term applications, resulting in tumor recurrence [5]. Regardless of the cancer itself, cancer-related cachexia is a major cause of patients’ death [6,7,8]. Therefore, there is an urgent need to explore comprehensive and effective therapy for both EC and associated cachexia to improve patient prognosis and overall survival.

Cachexia, a complex metabolic syndrome characterized by weight loss with atrophy of fat and skeletal muscle, is one of the main complications of cancer [9,10,11,12]. This disorder impedes the effectiveness of anticancer therapies and shortens the survival of patients with cancer [13,14,15]. Patients with many types of cancer suffering from cachexia, such as Lewis lung cancer [16], clear cell renal cell carcinoma [17], breast cancer [18], and pancreatic cancer [19], often have poor prognosis and survival. Recently, EC-related cachexia was shown to influence patient survival time and poor prognosis in a clinical cohort study [20–21]. However, the detailed mechanisms underlying EC-induced adipose tissue atrophy and its harmful effects on EC progression and patient prognosis remain unclear.

Brown adipose tissue (BAT) activation and browning of white adipose tissue (WAT, brown-like cells, or beige cells) are triggered during cancer cachexia progression, increasing thermogenic activity [17, 22–23]. In this context, adipocytes adopt a multilocular lipid droplet morphology, enhance mitochondrial biogenesis, and express uncoupling protein-1 (UCP1) and thermogenesis-related genes [17, 22,23,24,25]. The adipocytes thermogenesis is activated when exposed to tumor-secreted cytokines, including parathyroid hormone–related protein [25], zinc-alpha-2-glycoprotein-1 [26], and growth/differentiation factor 15 [27]. Interleukin-6 (IL-6), a typical inflammatory factor in cancer tumors, is a major regulator of cancer-associated cachexia [22, 28]. Nevertheless, whether BAT activation and WAT browning are associated with EC-related cachexia and the underlying mechanisms remain unclear.

TRIM22 is a RING finger E3 ubiquitin ligase [29]. It was originally identified as an interferon-induced protein [30,31,32] and regulates the nuclear factor-κB (NF-κB) signaling [33]. TRIM22 inhibits the replication of diverse viruses such as HIV-1 [30], HCV [34], and HBV [35] by inhibiting the NF-κB signaling pathway, but TRIM22 activates the NF-κB signaling pathway in autoimmune diseases [36–37]. We have previously demonstrated that TRIM22 inhibited NF-κB activity in EC [38]. However, whether the inhibition of inflammation is associated with EC-associated cachexia remains unclear.

This study demonstrated that EC was an aggressive disease, causing cachexia during tumor progression. TRIM22 inhibited EC progression and EC-associated cachexia. Mechanistically, TRIM22 interacted with NOD2, subsequently suppressing the NF-κB signaling pathway and blocking IL-6 production. TRIM22 inhibited EC-associated cachexia by attenuating BAT activation and WAT browning by inhibiting the IL-6/IL-6R signaling pathway. Thus, TRIM22 may be a promising clinical therapeutic target for patients with EC. Combination treatment with a TRIM22 inducer (progesterone) and a thermogenic inhibitor (IL-6R Ab) synergistically augments the antitumor efficacy of carbotaxol (carboplatin and paclitaxel) potentially, prolonging the overall survival of EC patients. (Fig. graphical abstract)

Methods

Patient sample analyses

Ethics approval for the study was obtained from the Fujian Medical University Union Hospital (Fuzhou, China). These experiments were performed in accordance with the Declaration of Helsinki. All EC specimens were collected from the Fujian Medical University Union Hospital (Fuzhou, China) from August 2021 to December 2022. All the atypical endometrial hyperplasia (AEH) samples were collected from the Fujian Medical University Union Hospital (Fuzhou, China) from September 2021 to September 2022. The clinical samples used in the experiments were as follows: twenty-five normal endometrial tissues, sixty-eight endometrial cancer tissues and fifty AEH endometrial tissues were applied to immunohistochemistry (IHC); Four pairs of endometrial cancer tissues, para-carcinoma tissues and normal endometrial tissues (matched for each patient) were applied to western blot analysis. The patient characteristics were shown in Table 1. All specimens were collected with the consent of the patients. All EC samples were diagnosed and evaluated according to the International Federation of Gynecology Oncology (FIGO) criteria (2009). All the EC specimens must have been pathologically diagnosed as endometrial adenocarcinoma and accepted initial surgery. All the normal endometrial tissues were collected from uterine fibroids patients who had been hysterectomy, excluding the patients who combined with other systemic diseases, such as hypertension, diabetes, disease of immune system, and used hormone, such as oestrogen, steroid hormone, before the operation. Additionally, all the AEH patients were diagnosed according to the endometrial tissue pathology through diagnostic curettage and accepted the high-dose oral progesterone therapy (megestrol acetate, 250 mg, daily or medroxyprogesterone acetate, MPA,500 mg, daily) for 3–6 months [39, 40]. Curettage was carried out again after high-dose oral progesterone therapy every 3 months. The maximum duration of treatment was 6 months. Those patients were divided into two groups, progesterone effective group (twenty-nine AEH endometrial tissues) and progesterone ineffective group (twenty-one AEH endometrial tissues), according to the recurettaged endometrial tissue pathology. Progesterone effective group referred to those patients who were sensitive to progesterone and whose recurettaged endometrial tissue pathology was normal endometrial tissue after progesterone therapy. Progesterone ineffective group referred to those patients who were insensitive to progesterone and whose recurettaged endometrial tissue pathology was the same as pre-treatment, even evolved to early EC after progesterone therapy. The patient characteristics were shown in Table 2.

Table 1 Distribution of the participants and tissue characteristics

Table 2 Distribution of the AEH participants and tissue characteristics (pathological diagnosis)

Xenograft studies

For mouse xenograft assay, 4 ~ 5-week-old female BALB/c nude mice were randomly divided into three groups. 1 × 10⁷ Ishikawa cells (scramble control and TRIM22 OE) mixed with Matrigel (1:1 vol/vol; Corning, Inc.) 1:1 with a total volume of 100 µL, then were injected subcutaneously into each mouse of the two groups among the three groups (n = 9/group). Non-tumor-bearing control mice receive PBS only. Observation records were recorded twice a week, including mouse weight, subcutaneous transplant tumor volume, and dietary dose. The tumor volume is calculated as V = 1 / 2 × length (mm) × width² (mm). After 27 days, the three group mice were euthanized and the tumor were gathered. The mouse blood, brown fat and visceral adipose tissues were collected for further study, including immunohistochemical analysis, ELISA analysis, and hematoxylin and eosin (H&E) staining.

In addition, in order to explore new treatments for endometrial cancer, we again conducted xenograft studies. For xenograft development, all the mice were divided into two groups (tumor-bearing mice and non-tumor-bearing control mice), 1 × 10⁷ Ishikawa cells were mixed with Matrigel 1:1 in a total volume of 100 µL, then were injected subcutaneously into each nude mouse. Non-tumor-bearing control mice receive PBS only. For in vivo drug studies, 5 mice (n = 5) from the tumor-bearing group are used for each treatment group. Treatment begins after the tumor reaches about 0.8 cm in diameter. We randomly divided the mice from the tumor-bearing group into 6 groups, and the following drugs and dosing regimens were used: vehicle, carboplatin and paclitaxel (carbotaxel, carboplatin 50 µg/g body weight, paclitaxel 20 µg/g body weight dilution instillation once a week) [41], progesterone (2 mg/day diluted with water, oral gavage daily) [42], progesterone and carbotaxel, progesterone and IL-6R Ab (100 µg/g body weight intravenously once a week) [43], progesterone combined with carbotaxel and IL-6R Ab. Mouse body weight is monitored at the beginning of treatment, then weekly, and at the end of treatment. Tumor size is measured with calipers once a week. Tumor samples and brown fat and visceral adipose tissues including IngWAT and GonWAT were collected for further study.

The BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., a joint venture of Charles River Laboratories. All animal experiments were approved by the Ethics Committee and performed in accordance with institutional guidelines.

EC cell culture and treatment

As described in previous studies [44], the EC cell lines (KLE, Ishikawa, and RL-952) were cultured in DMEM/F-12 medium (HyClone), which was supplemented with 10% fetal bovine serum (FBS) (Bioindustries, Israel), 1% penicillin and streptomycin (P/S) (HyClone)) in a 5% CO2 atmosphere humidified at 37 °C. To analyze the MPA effect on Ishikawa and KLE cells, Ishikawa and KLE cells (1 × 10⁴ cells) were grown to 80% confluence in 6-well culture plate wells in DMEM/F12 with 10% FBS for 24 h. And then exposed to medium with solvent (ethyl alcohol, 0.1%(v/v)) alone or with 10, 30, 50 µM MPA for 48 h. In addition, 0.01% (v/v) ethyl alcohol was used as the solvent control. Then these cells were used for all experiments in the study.

Conditioned medium (CM) Preparation and treatment

To prepare conditioned medium (CM), Ishikawa cells and TRIM22 OE Ishikawa cells were cultured to 80% confluence with DMEM/F12 and 10% FBS in 10-cm dishes. The medium was thrown away and the cells are further cultured in serum-free DMEM/F12 for 24 h. The medium was then collected, centrifuged at 1,000 g for 10 min, and filtered through a 0.22 mm filter (Millipore, Billerica, MA). CM is used to differentiate adipocytes.

For adipocyte differentiation, mouse mesenchymal stem cell line C3H10T1/2 cells were purchased from the National Experimental Cell Resource Platform (ShanghaiTech University). Cells were treated with a brown fat-inducing cocktail containing 10% fetal bovine serum, 20 nM insulin, 1 mM dexamethasone, 0.5 mM isobutyl methylxanthine, 125 nM indomethacin, and 1 nM 3,3,5-triiodo-l-thyronine (T3). The medium is then replaced every two days with only insulin and T3 added. During brown adipogenesis, cells are treated with the CM prepared above for 6 days. On day 6, fully differentiated adipocytes were used for all experiments in this study.

Immunohistochemical analysis

Tumor tissue and adipose tissue were taken from mouse xenograft model. Both human endometrial cancer tissue and normal endometrial tissue are from the above stipulated hospital. The paraffin-embedded tissue was cut into 4 μm thick sections and fixed on a glass slide. These sections are deparaffinized in xylene, rehydrated by gradually decreasing ethanol concentration, and incubated in boiling citric acid buffer for epitope retrieval. After washing with phosphate buffered saline (PBS), tissue sections were pre-incubated with 3% bovine serum albumin and then incubated overnight at 4 °C with a suitable primary antibody (detailed in supplementary). After TBST washing, slides were incubated with secondary antibody for 20 min at room temperature, and cold TBST washed with streptavidin-biotin-peroxidase for 30 min. Finally, 3,3′-diaminobenzidine and hematoxylin were detected for staining. The brown color detected with light microscopy indicated the presence of an antigen that binds to the antibody (Olympus Corp.). The mean integrated optical density (AOD) of IHC images was measured using Image J software. AOD = Integrated Optical Density (IOD)/Area. AOD value was used to assessed IHC staining intensity.

Western blot analysis

Endometrial cancer tissue, matched adjacent normal tissues, normal endometrial tissues and cells were washed 2 times with ice-cold PBS and scraped into cold lysis buffer (1% Triton X-100, 10% 150 mM NaCl, 50 mM Tris, pH 7.4) on ice. At the same time, to detect the different distributions of NF-κB-p65, we used NE-PER™ core and cytoplasmic extraction reagents (Thermo Fisher Scientific, Inc.) to extract cytoplasmic lysates and nuclear lysates. These different lysates were boiled in Laemmli sample buffer 10 min. Equivalent amounts of protein were resolved in 10% polyacrylamide gel electrophoresis, transferred to 0.45 μm PVDF membrane (Merck Millipore), and incubated with the corresponding antibodies (detailed in supplementary). Reactive bands were visualized with ECL plus reagent (ECL; Merck Millipore). Relative expression was quantified with Image J software.

Total RNA extraction and real-time reverse transcription-PCR

Total RNA from cells and tissues was isolated with Trizol (15596-026, Invitrogen) and cDNA was prepared with Superscript III reverse transcriptase (Invitrogen). Real-time reverse transcription (RT)-PCR was performed using the SYBR Green PCR Master Mix (Toyobo, Osaka, Japan). Fluorescence detection was performed using the ABI Prism 7500 sequence (Applied Biosystems, Foster City, CA). Comparative CT(2^−△△CT) was used to analyze the relative changes of gene expression. Results indicate the number of transcripts relative to GAPDH (internal control). The PCR forward and reverse primers are shown in Table 3.

Table 3 Nucleotide primer sequences used for real-time quantitative PCR

Lentivirus infection

To analyze TRIM22 function in human EC cells, we applied lentiviral vector to overexpress TRIM22. We cloned TRIM22 isoform a (NM_006074) into the BamHI and AgeI sites of the lentivirus expression vector Ubi-TRIM22-3FLAG-SV40-EGFP-IRES-puromycin (GV358). For lentiviral vector preparation, Human embryonic kidney (HEK) 293T cells were co-transfected with a TRIM22 isoform a construct, pHelper 1.0 and pHelper 2.0 using Lipofectamine3000. Supernatants were collected from 48 h post-transfection and filtered to exclude cellular debris. The resulting lentivirus was concentrated. Above process was performed by GeneChem Co., Ltd. According to the manufacturer’s instructions, Ishikawa cells were infected with TRIM22 overexpression lentiviral (TRIM22 OE Ishikawa cell) and the scramble control lentiviral (scramble con Ishikawa cell), respectively. The TRIM22 primer sequences were shown in the supplementary material.

Short hairpin RNA (shRNA) transfection

To generate TRIM22-knockdown RL-952 cells, NF-κB-p65-knockdown Ishikawa cells, NOD2- knockdown Ishikawa cells, we purchased synthesized target sequences for scrambled shRNA (scramble con) and TRIM22 shRNA, NF-κB-p65 shRNA, NOD2 shRNA from Shanghai Root Chemical Co., LTD. Transfections were performed transiently with Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions, and transfectants were used for the indicated experiments following 48 h of the recovery. The target sequences were shown in the supplementary material.

Cell proliferation assay

To detect cell proliferation, we performed Edu incorporation assays and CCK8 assays.

For the Edu incorporation assays, endometrial cells Ishikawa and KLE (1 × 10⁴) were seeded in triplicate onto 96-well plates and incubated at 37 °C in a humidified 5% CO2 atmosphere for 24 h, followed by 2 h in the medium containing 50 µM5-ethyl-2 ‘-deoxy uridines (Edu, Ribobio, China). The cells were fixed with 95% alcohol at 4˚C for 15 min. After washing with phosphate buffered saline (PBS) for three times, the cells of each well were reacted with 100 mul of 1 x Apollo reaction cocktail for 30 min. Subsequently, the DNA contents of cells in each well were stained with 100 mul of Hoechst 33,342 (5 mug/ml) for 30 min and visualized under a fluorescent microscope (Olympus Corporation). Five visual fields were randomly selected for each Edu trial, x50 magnification imaging. Anhydrous ethanol (95% alcohol) could accelerate the disappearance of GFP by increasing the permeability of the cell membrane. Therefore, GFP has the least influence on Edu. The Hoechst nuclear staining cell was recognized as Edu positive cell, and the results were represented as a percentage of the total number of cells in each region.

For the CCK8 assays, after the Ishikawa and KLE cells in the treatment with different concentration MPA were cultured in 96-well plates (2 × 10⁴ cells/well) for 48 h, then added 10mul CCK-8 solution (Beyotime Institute of Biotechnology, Shanghai, China) to each well incubating for another 1 h. The optical density (OD) value of each well of cells at 450 nm was determined by enzyme-linked immunosorbent instrument (Bio-Rad, CA, USA).

Transwell migration and invasion assay

To detect the cell migration and invasion, we used Transwell chamber (pore size, 0.8 μm; Merck Millipore). The chamber was precoated with Matrigel (BD Biosciences, United States) for invasion. Endometrial cancer cells (approximately 6 × 10⁴ for migration and 4 × 10⁴ cells for invasion assay) were placed in the upper chamber with 100 mul serum-free medium, and 600 mul culture medium containing 10% FBS was placed into the lower chamber. After incubating for 24 h, after removing the Matrigel and cells in the upper surface of the filters, the cells on the bottom of the filters were fixed with anhydrous ethanol and stained with hematoxylin at the room temperature for 15 min. Cells count was performed under an inverted microscope (Olympus Corp.) with five randomly selected fields.

Enzyme-linked immunosorbent assay (ELISA)

According to the manufacturer’s protocol (D6050, R&D Systems), the level of IL-6 protein in animal serum and CM was detected by solid-phase sandwich ELISA. The sensitivity of IL-6 is 0.7 pg/ml, and the detection range is 3.12 ~ 300 pg/ml.

Seahorse XFe96 measurements

OCR of mature adipocytes and tumor cells was measured using the Seahorse Bioscience XF(e)96 Flux Analyzer Agilent Technologies) according to the manufacturer’s protocol [45]. Briefly, C3H10T1/2 cells are differentiated for 5 days, seeded into 96-well plates at a density of 3.5 × 10³ cells/well, and allowed to adhere to the bottom overnight. It is then treated with the corresponding CM (from Ishikawa cells and TRIM22 OE Ishikawa cells) for 24 h. Mitochondrial stress testing was performed using sequential injections of oligomycin (4.5 mM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (2 mM, FCCP), and rotenone/antimycin A (1 mM). Basal, decoupled and maximum respiration parameters are automatically calculated by the WAVE software (Agilent). All parameters were normalized to the total amount of protein in a single well using the BCA protein assay (Thermo Fisher Scientific).

For ex vivo differentiation, we isolated pre-adipose cells from superficial fat (concentrated in the inguinal folds) and fat located in deeper regions (concentrated on the gonads, dorsal surface of the scapula). Freshly excised fat was collected, minced, and subsequently digested with collagenase 1 (2 mg/mL) in phosphate-buffered saline with the addition of 3.5% bovine serum albumin (Worthington Biochemical Company), then isolated the pre-adipocytes. Pre-adipocytes were cultured to 90% confluency in medium containing 5 ng/mL human basic fibroblast growth factor (Sigma-Aldrich), 10 ng/mL human epidermal growth factor, 10 ng/mL platelet-derived growth factor-BB (all from PeproTech), and 10 ng/mL mouse leukemia inhibitory factor (EMD Millipore). We seeded cells at a rate of 1.5 × 10⁴ cells per 48 wells and cultured for 2 days until confluent. Differentiation was then performed in growth factor-free medium for 10 days, and 2% FBS and a fat-inducing mixture (50µM indomethacin, 0.5µM insulin, 33µM biotin, 17µM pantothenate, 0.1µM dexamethasone, 2nM iodothyronine, 540µM isobutylmethylxanthine) were added to the medium. After 10 days, we measure OCR using mature adipocytes.

Tissue immunofluorescent staining

Histopathological sections (4 μm thick) are deparaffinized in xylene, rehydrated by gradually decreasing ethanol concentration, and incubated in boiling citric acid buffer for epitope retrieval. After washing with phosphate buffered saline (PBS), tissue sections are pre-incubated with 3% bovine serum albumin. Next, slides were hatched with primary antibodies TRIM22 and NOD2 (detailed in supplementary) overnight at 4 °C and washed 3 times with PBS for 10 min each. Sections were incubated at 37 °C in a humidity box protected from light with the corresponding secondary fluorescently conjugated antibody (1:30 00, GB21303, rabbit, Servicebio) (1:20 00, Abcam) for 1 h, and then washed 3 times with PBS for 5 min each. Then, back stain with 4’,6’-diamino-2-phenylindole (DAPI) (Servicebio, China) for 10 min at room temperature in a wet box protected from light. The slides are then examined using an inverted fluorescence microscope (Olympus Corp., Japan). TRIM22 stains were red, NOD2 stains green, and nuclei appear blue.

Co‑immunoprecipitation (Co‑IP) analysis

For Co-IP analysis, cell lysates were prepared using ice-cold native lysate, followed by 2 freeze/thaw cycles. After clarification and pre-clearing, the lysates were incubated overnight at 4 °C, and the protein-A Sepharose spheroids were pre-coated with anti-TRIM22, anti-NOD2, and anti-K63. After incubation, washed the beads with elution buffer. To detect protein expression, we used western blot.

Ubiquitination assay in vitro

An improved in vitro ubiquitination experiment was modified with some modifications as previously described [46]. Recombinant human UBE1 (100 nM), UbcH5b/UBE2D2 (1 µM), and ha-ubiquitin K63 (10 µM) were mixed in ubiquitination buffer (25 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM DTT, 2.5 mM ATP, 4 mM MgCl2) at the indicated final concentrations. Recombinant human TRIM22 and NOD2 peptides expressed in Ishikawa cells were also added to a final volume of 50 µl. The reaction mixture was incubated at 30 °C for 60 min, and then NOD2 was pulled down.

Luciferase reporter assay

Dual fluorescein reporter gene analysis was carried out following the manufacturer’s guidelines of Dual Luciferase Assay System Kit (Promega, Madison, WI, USA). Wild-type TRIM22 and mutant TRIM22 were amplified and cloned into the PGL3-Basic luciferase reporter vector. Next, Ishikawa cells were plated in 24-well plates in triplicate and treated with MPA for 48 h, then co-incorporated with the corresponding plasmids for 48 h. Luciferase activity was evaluated by the dual-luciferase reporter assay system (Promega). The data were normalized to Renilla internal control.

Chromatin Immunoprecipitation (ChIP) assay

ChIP assays were carried out following the manufacturer’s guidelines of EZ-Magna ChIP G Kit (Millipore, USA). Ishikawa cells were pre-treated with MPA for 48 h, then isolated the nuclei, then sonicated the nuclei to shear the DNA. Anti-TRIM22 antibody was used to immunoprecipitated chromatin. IgG was the control. The complexes were extracted from protein-G-Magnetic beads. Bound DNA was purified and amplified by PCR.

TCGA database analysis

The genes expression and survival information were downloaded from the Cancer Genome Atlas (TCGA) endometrial cancer dataset (http://tcga-data.nci.nih.gov/tcga/).

Statistical analysis

Unless otherwise noted, all data are represented as mean ± standard error mean (SEM). We used the Shapiro-Wilk test to assess the normality of the data, with a p-value threshold set at 0.05 to determine the appropriateness of parametric tests. Normally distributed data were performed using the unpaired two-tailed Student’s t-test or paired t-test, or one-way ANOVA or two-way ANOVA using GraphPad Prism 7.0 software. Non-normally distributed data were analyzed using non-parametric tests, Mann-Whitney U test. P < 0.05 was statistically significant. Concerning multiple comparisons, we employed the Bonferroni correction to adjust p-values for multiple comparisons, ensuring that our conclusions are conservative and robust against type I errors. Kaplan-Meier survival analysis was used to analyze the survival time of patients. Regarding the Kaplan-Meier analysis, we used the log-rank test to compare survival curves between groups, which provides a statistically rigorous method for assessing differences in survival times. Furthermore, to provide additional context for interpreting our results, we included effect sizes (e.g., Cohen’s d for parametric data and Cliff’s delta for non-parametric data) and confidence intervals (95% CI) for all key comparisons.

Results

Lack of TRIM22 worsen overall survival (OS) in patients with EC accompanied by body mass index (BMI) reduction

TRIM22 expression was lower in EC tissues than in normal endometrial tissues in TCGA(Fig. 1A). Patients with high TRIM22 expression have a longer survival time after EC (Fig. 1B). These results suggest that TRIM22 plays a functional role in the progression of EC. Western blot analysis showed that TRIM22 expression was lower in EC tissues (Fig. 1C and D). Subsequently, IHC analysis revealed that TRIM22 expression was significantly decreased in EC samples. Intracellular TRIM22 was expressed at different stages. There was a negative association between the expression of TRIM22 and the clinical stage of EC. (Fig. 1E-I) Moreover, during the menstrual cycle, TRIM22 protein levels were higher in the secretory phase than in the proliferative phase in normal endometrial tissues (Fig. 1G). Furthermore, Kaplan-Meier analysis of 68 patients showed that patients with EC and high TRIM22 expression had longer survival times (Fig. 1J). Similarly, EC patients who survived had higher TRIM22 expression than those who died (Fig. 1O-Q). These results suggest that TRIM22 may be a protective factor in patients with EC, which is associated with OS improvement.

Fig. 1

Lack of TRIM22 worsens overall survival (OS) in patients with EC accompanied by body mass index (BMI) reduction. (A) The TRIM22 expression level of EC tissue and its adjacent normal tissue in TCGA database. (B) The Kaplan-Meier analysis between TRIM22 and survival of EC patients in TCGA database. (C) Immunoblots of TRIM22 in 4 pairs of EC samples (N, normal endometrial tissue; A, adjacent normal tissue; T, tumor tissue). (D) Statistical pair analysis of TRIM22 expression derived from (C). (E) Statistical analysis of TRIM22 expression derived from (F and H). (F)Representative IHC staining of normal endometrial tissues from the proliferative and secretory during the menstrual cycle. (G) Statistical analysis of the expression of TRIM22 in EC tissues and normal endometrial tissues (proliferative and secretory) derived from (F and H). (H)Representative IHC staining of different stage EC tissues (stage I, II, III, IV). (I)Statistical analysis of TRIM22 expression in normal endometrial tissues and different stage EC tissues (stage I, II, III, IV) derived from (F and H). (J)The Kaplan-Meier analysis between TRIM22 and survival of EC patients according the TRIM22 expression derived from (H). (K)The Kaplan-Meier analysis between BMI and survival of EC patients (BMI: body mass index). (L)Statistical analysis of BMI of the death and survivor EC patients. (M) Statistical analysis of BMI of the death and survivor EC patients in the early stage (stage I and II). (N)Statistical analysis of BMI of the death and survivor EC patients in the advanced stage (stage III and IV). (O)Statistical analysis of TRIM22 expression of the death and survivor EC patients. (P)Statistical analysis of TRIM22 expression of the death and survivor EC patients in the early stage (stage I and II). (Q)Statistical analysis of TRIM22 expression of the death and survivor EC patients in the advanced stage (stage III and IV). (R) Statistical analysis of BMI between high-TRIM22 and low-TRIM22 expression groups based on mean TRIM22 expression levels. (S) Statistical analysis of TRIM22 expression between cachexia and non-cachexia group. Data were expressed as means ± SEM of three independent experiments. Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001

Cancer cachexia syndrome is a common complication of cancer that adversely affects patient survival. Its hallmarks are drastic weight loss and low BMI, accompanied by abnormal fat metabolism [47]. We divided the 68 EC patients into three groups based on the WHO BMI obesity categories, i.e., the normal weight group (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), and obese (≥ 30 kg/m2) [48]. Kaplan-Meier survival analysis revealed that patients with EC and a lower BMI had worse OS (Fig. 1K). Patients with EC who died had a lower BMI (Fig. 1L-N). These results suggested that the OS of EC patients was adversely associated with BMI. Moreover, we found that the BMI of the high-TRIM22 group increased significantly than the low-TRIM22 group, indicating that the level of TRIM22 decreased as the patient’s weight decreased. Notably, patients with cachexia are characterized by involuntary weight loss of more than 5% within 6 months and cannot be completely reversed by traditional nutritional support [49]. We divided EC patients into cachexia and non-cachexia group according to patient’s weight and demonstrated that TRIM22 expression was significantly enhanced in the non-cachexia group than in the cachexia group. Taken together, these results indicate that the lack of TRIM22 shortens the OS of patients with EC, which might accelerate BMI reduction and subsequent cachexia, thus aggravating EC progression.

TRIM22 OE suppressed EC progression and prevented body weight loss induced by cachexia

In vitro experiments showed that overexpression of TRIM22 decreased Ishikawa and KLE cell proliferation (Fig. 2A and B, S1A and B). The migratory and invasive abilities of TRIM22 OE (TRIM22 overexpression) Ishikawa and KLE cells were markedly reduced (Fig. 2C-E, S1C-E). TRIM22 KD (TRIM22 knocked down) increased RL-952 cell proliferation, migration, and invasion (Fig. 2F-J). These results indicate that TRIM22 inhibits cell proliferation, migration, and invasion.

Fig. 2

TRIM22 OE suppressed EC progression and prevented body weight loss induced by cachexia. (A) Ratio of Edu-positive scramble control and TRIM22 OE Ishikawa cells in (A). (B) Representative images of scramble control and TRIM22 OE Ishikawa cells that cultured in trans-well plates. (C) The average number of migration Ishikawa cells in (C). (D) The average number of invasion Ishikawa cells in (C). (E) Representative images showing Edu incorporation in scrambling control and TRIM22 KD RL-952 cells. (F) Ratio of Edu-positive RL-952 cells in (F). (G) Representative images of scramble control and TRIM22 KD RL-952 cells that cultured in trans-well plates. (H) The average number of migration RL-952 cells in (H). (I) The average number of invasion RL-952 cells in (H). (J) A subcutaneous tumor growth curve from scramble control and TRIM22 OE Ishikawa cells at indicated time points (n = 9 mice per group). (K) Representative images of xenograft tumors in (K) at day 27 (n = 9 mice per group). (L) Weights of xenograft tumors in (L) at day 27 (n = 9 mice per group). (M)Representative H&E staining and IHC staining of Ki67 in xenograft tumors derived from (L). (N) Statistical analysis of Ki67 expression in xenograft tumors derived from (L) using IHC staining. (O) Statistical analysis of lipid aera in xenograft tumors derived from (L) using H&E staining. (P) A body weight curve from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (Q) Statistical analysis of body weight gain of xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (R) Statistical analysis of carcass weight of xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (S) Statistical analysis of the mass of BAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (T) Statistical analysis of the mass of IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (U) Statistical analysis of the mass of GonWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. Data were expressed as means ± SEM of three independent experiments. Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001

Next, xenograft mouse experiment showed that TRIM22 overexpression exerted a significant inhibitory effect on tumor growth. The mean tumor volume of the mice in the TRIM22 OE group was smaller. (Fig. 2K and L) Consistently, the tumor weight was lower in the TRIM22 OE group (Fig. 2M). Furthermore, hematoxylin and eosin (H&E) staining showed little lipid droplet cavitation in the TRIM22 OE group (Fig. 2M and P). IHC staining revealed that Ki67 expression was lower in the TRIM22 OE group (Fig. 2M and O). These results indicated that TRIM22 inhibited tumor growth in vivo.

Given that TRIM22 suppressed EC growth, we evaluated whether TRIM22 inhibited EC cachexia-associated body weight loss. Xenograft mouse experiment showed that TRIM22 OE attenuated the body weight/gain reduction induced by EC (Fig. 2Q and R, S1F). The carcass weight/gain of the TRIM22 OE group was markedly increased (Fig. 2S, S1G). Furthermore, the adipose tissue mass (BAT; inguinal WAT, IngWAT; epididymal WAT, GonWAT) in the TRIM22 OE group was significantly higher (Fig. 2T-V). Meanwhile, we also demonstrated that TRIM22 could overcome the muscles atrophy (Fig. S1H-J). These results indicate that TRIM22 OE inhibits EC growth and prevents EC cachexia-related body weight loss.

TRIM22 OE reversed EC-enhanced thermogenic activity in BAT

Cancer-associated cachexia manifests as lipid catabolism and mobilization, especially for pre-cachexia and the cachexia progression [50,51,52]. BAT activation was the earliest manifestation of lipid catabolism, promoting thermogenesis [52, 53]. H&E staining of BAT showed that TRIM22 OE prevented adipocyte size reduction. Moreover, IHC staining revealed that TRIM22 OE inhibited the increase of UCP1 expression of BAT tissue induced by EC. (Fig. 3A-C) TRIM22 OE blocked the upregulation of thermogenic genes (Ucp1, Pgc1α, Pgc1β, Prdm16, and Fgf21). Consistently, TRIM22 OE reversed the elevated expression of genes related to fatty acid oxidation (Ppara, Elov3, and Cpt1a), lipolysis (Hsl and Atgl), and mitochondrial biogenic transcription factor (NRF2). (Fig. 3D) These results indicate that TRIM22 OE overcome the enhanced thermogenic activity of BAT during EC progression.

Fig. 3

TRIM22 OE reversed EC-enhanced thermogenic activity in BAT. (A) Representative images of H&E and IHC-stained sections of BAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (B) Statistical analysis of lipid aera in BAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells in (A) using H&E staining. (C) Statistical analysis of UCP1 expression in BAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells in (A) using IHC staining. (D) mRNA level of genes of BAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells, including thermogenic genes, fatty acid oxidation related genes, lipolysis related genes, mitochondrial biogenic transcription factor. Data were expressed as means ± SEM of three independent experiments. Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001

TRIM22 OE blocked EC-induced Browning of WAT

Previous reports demonstrated that WAT browning, a phenotypic switch from WAT to brown-like fat (beige cells), occurs during the initial stages of cancer cachexia [17, 22, 23, 54, 55]. H&E staining showed TRIM22 OE resisted adipocyte contraction induced by EC. TRIM22 OE attenuated UCP1 protein increase. (Fig. 4A-C) Similar results were observed in GonWAT (Fig.S2A and B). TRIM22 OE weakened the browning-related genes expression increases, including fatty acid oxidation, lipolysis, and mitochondrial biogenesis (Fig. 4D). The expression of beige cell-specific markers (TMEM26, CD137, and TBX1) in the TRIM22 OE group was decreased closely to the normal level (Fig. 4E-G). Ex vivo respiration measurement with a seahorse in IngWAT showed that TRIM22 OE decreased the increase of OCR induced by EC (Fig. 4F). These results indicate that TRIM22 OE ameliorates the browning of WAT and prevents EC cachexia.

Fig. 4

TRIM22 OE blocked EC-induced browning of WAT. (A) Representative images of H&E and IHC-stained sections of IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (B) Statistical analysis of lipid aera in IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells in (A) using H&E staining. (C) Statistical analysis of UCP1 expression in IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells in (A) using IHC staining. (D) mRNA level of genes of IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells, including thermogenic genes, fatty acid oxidation related genes, lipolysis related genes, mitochondrial biogenic transcription factor. (E) mRNA level of gene of beige cell marker (TMEM26) in IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (F) mRNA level of gene of beige cell marker (CD137) in IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (G) mRNA level of gene of beige cell marker (TBX1) in IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (H) Mean OCR from Seahorse in IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. Data were expressed as means ± SEM of three independent experiments. Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001

TRIM22 inhibited the enhancement of EC cells-mediated thermogenesis activity in adipocytes through the IL-6/IL-6R signaling pathway

Inflammation plays an important role in cancer-associated cachexia pathophysiology, such as IL-6 [56,57,58,59,60,61,62]. ELISA analysis showed that TRIM22 OE restored IL-6 concentration in the serum of mice closely to normal levels (Fig. 5A). TRIM22 OE reversed the upregulation of IL-6 mRNA level (Fig.S3A). qRT-PCR analysis showed that TRIM22 OE inhibited the elevated expression of IL-6R and its downstream gene, suppressor of cytokine signaling 3 (SOCS3), in BAT. (Fig. 5B and C) Similar results were observed in the IngWAT (Fig. 5D and E). These results suggest that TRIM22 OE attenuates the stimulatory effects of the IL-6/IL-6R signaling pathway.

Fig. 5

TRIM22 inhibited the enhancement of EC cells-mediated thermogenesis activity in adipocytes through the IL-6/IL-6R signaling pathway. (A) ELISA of IL-6 in blood from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (B) mRNA level of IL-6R in BAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (C) mRNA level of SOCS3 in BAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (D) mRNA level of IL-6R in IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (E) mRNA level of SOCS3 in IngWAT from xenograft mice subcutaneously injected PBS, scramble control and TRIM22 OE Ishikawa cells. (F) mRNA level of TRIM22 in scramble control and TRIM22 OE Ishikawa cells. (G) ELISA of IL-6 in TRIM22 OE and scramble control Ishikawa cells condition medium (CM). (H) UCP1 mRNA in differentiated matured C3H10T1/2 cells after treated with the CM from scramble control and TRIM22 OE Ishikawa cells. (I) mRNA level of IL-6R in differentiated matured C3H10T1/2 cells after treated with the CM from scramble control and TRIM22 OE Ishikawa cells. (J) mRNA level of SOCS3 in differentiated matured C3H10T1/2 cells after treated with the CM from scramble control and TRIM22 OE Ishikawa cells. (K) OCR of differentiated matured C3H10T1/2 cells was evaluated using Seahorse after treated with CM from scramble control and TRIM22 OE Ishikawa cells. (L) Basal OCR from Seahorse in (K). (M) Uncoupled OCR from Seahorse in (K). (N) Maximal OCR from Seahorse in (K). (O) mRNA level of IL-6R in differentiated C3H10T1/2 cells when blocked the IL-6R (scramble control and IL-6R KD). (P) mRNA level of UCP1 in scramble control and IL-6R KD differentiated matured C3H10T1/2 cells after treated with the CM from Ishikawa cells. (Q)OCR of scramble control and IL-6R KD differentiated matured C3H10T1/2 cells were evaluated using Seahorse after treated with CM from Ishikawa cells. (R)Basal OCR from Seahorse in (Q). Data were expressed as means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

To directly delineate the role of IL-6 in adipocyte thermogenesis in vitro, we used a conditioned medium assay. Ishikawa cells showed lower mRNA levels of IL-6 when TRIM22 was overexpressed (Fig. 5F, S3B). IL-6 concentration in TRIM22 OE Ishikawa cell culture media (TRIM22 OE EC cell-conditioned medium) was higher (Fig. 5G). Treatment of mature adipocytes (differentiated from C3H10T1/2 cells) with TRIM22 OE EC cell-conditioned medium inhibited Ucp1 expression (Fig. 5H). After treatment with TRIM22 OE EC-conditioned medium, TRIM22 OE drastically decreased IL-6R and SOCS3 expression in mature adipocytes (Fig. 5I and J). TRIM22 OE inhibited the basal, uncoupled, and maximal OCR in mature white adipose cells (Fig. 5K-N). To confirm the inhibitory effects of TRIM22 on the IL-6/IL-6R signaling pathway-mediated enhancement of thermogenic activity in differentiated adipocytes, we blocked IL-6R expression in adipocytes (Fig. 5O), followed by treatment with the EC cell-conditioned media (scramble control and TRIM22 OE Ishikawa cell). We found no significant changes in Ucp1 expression in IL-6R knockdown adipocytes (Fig. 5P). Similar results were observed for the basal oxygen OCR (Fig. 5Q and R). These results suggest that TRIM22 inhibits the EC-induced thermogenesis activity through the IL-6/IL-6R signaling pathway.

TRIM22 and NOD2 interaction restricted EC progression by inhibiting the NF‑κB pathway

NOD2 is closely associated with human cancer development by affecting the inflammatory microenvironment [63]. Interactions between TRIM22 and NOD2 have been reported to play an important role in the regulation of inflammation [36]. NOD2 expression decreased significantly starting at stage III in TCGA (Fig. 6A and B). Kaplan-Meier analysis of TCGA showed that patients with EC and low NOD2 expression had relatively short survival times (Fig. 6C). Immunofluorescence showed that TRIM22 and NOD2 were robustly co-localized in normal endometrial and EC tissues, and the expression of TRIM22 and NOD2 was decreased in EC tissues (Fig. 6D-F). These findings suggest a potential relationship between TRIM22 and NOD2 expression in EC tissues.

Fig. 6

TRIM22 and NOD2 interaction restricted EC progression by inhibiting the NF‑κB pathway. (A) NOD2 expression level in the normal endometrial tissue and EC tissues in TCGA database. (B) NOD2 expression level in EC tissues from the different stage (I, II, III, IV). (C) The Kaplan-Meier analysis between NOD2 and survival of EC patients in TCGA database. (D) Immunofluorescent detection of TRIM22 and NOD2 in EC and normal endometrial tissue. Scale bar, 20 μm. (E) Statistical analysis of TRIM22 in (D). (F)Statistical analysis of NOD2 in (D). (G) Co-IP detection of TRIM22, NOD2 and HA-Ub in TRIM22 OE and scramble control Ishikawa cells. (H) Reciprocal Co-IP detection of TRIM22, NOD2 and HA-Ub in TRIM22 OE and scramble control Ishikawa cells. (I) Immunoblots of TRIM22, NOD2, NF‑κB-p65, IκB-α, p-NF‑κB-p65, p-IκB-α in TRIM22 OE and scramble control Ishikawa cells. (J) Immunoblots of nuclear NF‑κB-p65 and cytoplasmic NF‑κB-p65 in TRIM22 OE and scramble control Ishikawa cells. (K) Representative images showing Edu incorporation in the scramble control and NOD2 KD Ishikawa cells. (L) Ratio of Edu-positive scramble control and NOD2 KD Ishikawa cells in (K). (M) Representative images of scramble control and NOD2 KD Ishikawa cells that cultured in trans-well plates. (N)The average number of migration Ishikawa cells in (M). (O) The average number of invasion Ishikawa cells in (M). (P) Representative images showing Edu incorporation in the scramble control, NOD2 KD and NOD2 KD + TRIM22 OE Ishikawa cells. (Q) Ratio of Edu-positive scramble control, NOD2 KD and NOD2 KD + TRIM22 OE Ishikawa cells. (R) Representative images of scramble control, NOD2 KD and NOD2 KD + TRIM22 OE Ishikawa cells. that cultured in trans-well plates. (S) The average number of migration Ishikawa cells in (R). (T) The average number of invasion Ishikawa cells in (R). Data were expressed as means ± SEM of three independent experiments. Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001

Co-immunoprecipitation (Co-IP) assay showed that NOD2 was copurified with TRIM22 in Ishikawa cells, and this association increased after TRIM22 overexpression (Fig. 6G). Moreover, reciprocal Co-IP demonstrated that TRIM22 was copurified with endogenous NOD2, and the interaction was enhanced by TRIM22 overexpression (Fig. 6H). These results indicate a specific interaction between NOD2 and TRIM22.

We next determined whether TRIM22 mediated ubiquitination of NOD2. Reciprocal Co-IP assay showed that overexpression of TRIM22 enhanced NOD2 K63 specific-ubiquitination (Fig. 6G and H), indicating that TRIM22 specifically mediates NOD2 K63-linked polyubiquitination. Since NOD2-mediated NF‑κB pathway activation is linked to human inflammatory disorders [56,57,58,59,60,61], we examined whether TRIM22 and NOD2 interaction could influence the NF‑κB-p65 activity in EC. Total NF‑κB-p65 and IκB-α expression in Ishikawa cells were increased after TRIM22 overexpression (Fig. 6I). Moreover, TRIM22 OE inhibited the IκB-α phosphorylation and subsequently suppressed the NF‑κB-p65 activation in Ishikawa cells (Fig. 6I). Furthermore, TRIM22 OE decreased the nuclear NF‑κB-p65 protein level (Fig. 6J), indicating that TRIM22 inhibits nuclear translocation of NF‑κB-p65. Furthermore, the proliferative, migratory, and invasive abilities of Ishikawa cells significantly increased after NOD2 KD (NOD2 knocked down) (Fig. 6K-O). Consistently, NF‑κB-p65 knockdown significantly decreased the proliferation, migratory, and invasive abilities of Ishikawa cells, the inhibitory effect of TRIM22 on EC cells was attenuated in the reversal test (Fig. 6P-T).

NF-κB activation induces the production of inflammatory factors, such as IL-6 [64,65,66]. Given that tumor and serum IL-6 levels were drastically increased in EC mice (Fig.S3 and Fig. 5), we searched TCGA data and found that IL-6 expression was significantly increased in the advanced stage (Fig.S4A and B). Kaplan-Meier analysis of TCGA data showed that patients with EC and IL-6-high expression exhibited significantly shorter survival times (Fig.S4C). Moreover, we knockdown NF‑κB-p65 Ishikawa cells, then detected the IL6 in the culture medium (CM). We found that NF‑κB-p65 knockdown significantly decreased the production of IL6 in the Ishikawa cell CM(Fig.S4D). These results suggest that tumor-derived IL-6 is closely associated with EC progression and contributes to a shorter lifespan.

Together with IL-6-mediated enhancement of thermogenesis activity, as mentioned in Fig.S3 and Fig. 5, these results suggest that the TRIM22 and NOD2 interaction inhibits EC progression by inhibiting the NF‑κB signaling pathway and subsequently suppresses IL-6 production, improving the overall survival time of patients with EC.

Progesterone enhanced the Carbotaxol therapeutic effect on EC by inducing TRIM22 expression in vivo

As TRIM22 inhibited EC progression and concomitant cachexia, we attempted to identify a promising drug target to enhance anticancer effect. By analyzing the TRANSFAC data, we found a progesterone response element (PRE) in the promotor region of TRIM22 (Fig. 7A). MPA, a synthetic progesterone analog, is a well-known drug for endometrial protection, and we next examined whether MPA could enhance the PRE activity of TRIM22. Luciferase reporter showed that the transcriptional activity of the TRIM22-Luc reporter group was drastically increased in the presence of MPA (Fig. 7B). ChIP analysis showed that Ishikawa cells stably expressing PR treated with MPA significantly increased TRIM22 PRE abundance (Fig. 7C and D). MPA treatment significantly increased TRIM22 mRNA expression, whereas the MPA antagonist, RU486, reversed MPA effect on TRIM22 expression dose-dependently (Fig. 7E). These results suggest that MPA regulates TRIM22 expression by binding to its promotor region of TRIM22.

Fig. 7

Progesterone enhanced the carbotaxol therapeutic effect on EC by inducing TRIM22 expression in vivo. (A) The TRIM22 putative progesterone responsive element (PRE) and the mutant site in the TRANSFAC database. (B) The Luciferase activity of TRIM22 and progesterone in Ishikawa cells. (C) ChIP detection of the association of PR with TRIM22 PRE in Ishikawa cells. (D) Statistical analysis of the association of PR with TRIM22 PRE in (C). (E) mRNA level of TRIM22 in Ishikawa cells treated with vehicle, MPA and MPA inhibitor RU486. (F) Immunoblots of TRIM22 in Ishikawa and KLE cells treated with vehicle and different concentration MPA. (G) Statistical analysis of TRIM22 in (F). (H) The CCK8 of Ishikawa and KLE cells treated with vehicle and different concentration MPA. (I) Representative images of pre-treatment and post-treatment IHC-stained (with an anti-TRIM22 antibody) sections of endometrial tissues from atypical endometrial hyperplasia (AEH) patients who accepted the high dose progesterone treatment for 3-6months. (J) Statistical analysis of TRIM22 in (I). (K) Statistical analysis of endometrial epithelium thickness in (I). (L) Statistical analysis of the ratio of endometrial glands to stroma in (I). (M) A subcutaneous tumor growth curve of xenograft mice treated with vehicle, carbotaxol, progesterone, combination progesterone and carbotaxol, combination progesterone, carbotaxol and IL-6R Ab at indicated time points (n = 5 mice per group). (N) Statistical analysis of last tumor volume in (M). (O) Statistical analysis of the last tumor weight of xenograft mice treated with vehicle, carbotaxol, progesterone, combination progesterone and carbotaxol, combination progesterone, carbotaxol and IL-6R Ab. (P) Representative H&E staining and IHC staining of Ki67 in xenograft tumors derived from (M). (Q) Statistical analysis of Ki67 expression in xenograft tumors derived from (M). (R)Statistical analysis of TRIM22 expression in xenograft tumors derived from (M). Data were expressed as means ± SEM of three independent experiments. Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001

Furthermore, MPA increased TRIM22 levels in PR-positive Ishikawa cells dose-dependently (Fig. 7F and G) and thus inhibited cell proliferation (Fig. 7H). However, the anticancer effect of MPA was not observed in the PR-negative KLE cells (Fig. 7H). Moreover, we evaluated the anticancer effect of progesterone in 50 patients with atypical endometrial hyperplasia. TRIM22 expression was significantly increased after high-dose oral progesterone treatment in the progesterone-sensitive (effective) group (Fig. 7I and J). Statistical analysis showed that the endometrial epithelium thickness decreased in the progesterone-sensitive group, indicating a good therapeutic effect (Fig. 7K). In contrast, the ratio of endometrial glands to stroma drastically increased after high-dose oral progesterone treatment in the progesterone-sensitive group (Fig. 7L). These results demonstrate that progesterone protects the endometrium by inducing TRIM22 expression, inhibiting EC progression.

Carboplatin and paclitaxel treatment is the major adjunctive treatment for patients with EC but has serious toxic side effects and a high recurrence rate, causing a poor prognosis [67,68,69]. We attempted to identify an effective therapy to improve the therapeutic effects of carbotaxol. In Vivo subcutaneous xenograft model showed that greatest tumor growth inhibition was observed in mice treated with carbotaxol in combination with progesterone and IL-6R Ab (Fig. 7M-O). The combination of progesterone, carbotaxol, and IL-6R Ab showed the highest staining intensity of TRIM22 expression by H&E staining and IHC analysis (Fig. 7P and R). Treatment with the combination of progesterone, carbotaxol, and IL-6R Ab showed the lowest Ki67 expression (Fig. 7P and Q).

These results demonstrate that TRIM22 is a promising clinical therapeutic target for EC, enhancing its activity by progesterone or its combination with conventional treatments (e.g., carbotaxol), synergistically augmenting antitumor efficacy.

Progesterone-carbotaxol-IL-6R antibody combination therapy synergistically overcame the EC-associated cachexia in vivo

As IL-6 was an important inflammatory factor that caused the EC cachexia mentioned above, and a suboptimal response to progesterone treatment existed in some EC patients with “progesterone resistance,” it is necessary to comprehensively resist cachexia occurrence in EC progression for anticancer therapy. Next, we further explored therapies to overcome cancer-related cachexia in vivo. Treatment with a combination of progesterone, carbotaxol and IL-6R Ab significantly reversed body weight reduction, closest to that of the PBS group (Fig. 8A). Moreover, treatment with the combination of progesterone, carbotaxol, and IL-6R Ab showed the maximum body weight/gain (Fig. 8B, S5A). Similar results were obtained for the carcass weight/gain (Fig. 8C, S5B). Consistently, a greater reduction in UCP1 expression of BAT and IngWAT was observed in the combination treatment with progesterone and carbotaxol, progesterone and IL-6R Ab, or progesterone, carbotaxol, and IL-6R Ab (Fig. 8D–F). Ex vivo measurement showed that OCR of BAT and IngWAT was decreased dramatically after combination treatment (Fig. 8G and H). These results indicate that the new combination therapy (progesterone, carbotaxol, and IL-6R Ab) is a more effective anticancer program that inhibits EC progression and its associated cachexia.

Fig. 8

Progesterone-carbotaxol-IL-6R antibody combination therapy synergistically overcame the EC-associated cachexia in vivo. (A) A body weight curve from non-tumor bearing mice and tumor bearing mice treated with vehicle, carbotaxol, progesterone, combination progesterone and carbotaxol, combination progesterone, carbotaxol and IL-6R Ab at indicated time points (n = 5 mice per group). (B) Statistical analysis of body weight gain of non-tumor bearing mice and tumor bearing mice treated with vehicle, carbotaxol, progesterone, combination progesterone and carbotaxol, combination progesterone, carbotaxol and IL-6R Ab.(C) Statistical analysis of carcass weight of non-tumor bearing mice and tumor bearing mice treated with vehicle, carbotaxol, progesterone, combination progesterone and carbotaxol, combination progesterone, carbotaxol and IL-6R Ab.(D) Representative IHC staining of UCP1 in IngWAT and BAT from non-tumor bearing mice and tumor bearing mice treated with vehicle, carbotaxol, progesterone, combination progesterone and carbotaxol, combination progesterone, carbotaxol and IL-6R Ab.(E)Statistical analysis of UCP1 expression of BAT in (D).(F) Statistical analysis of UCP1 expression of IngWAT in (D). (G) OCR of BAT from non-tumor bearing mice and tumor bearing mice treated with vehicle, carbotaxol, progesterone, combination progesterone and carbotaxol, combination progesterone, carbotaxol and IL-6R Ab. (H) OCR of IngWAT from non-tumor bearing mice and tumor bearing mice treated with vehicle, carbotaxol, progesterone, combination progesterone and carbotaxol, combination progesterone, carbotaxol and IL-6R Ab. Data were expressed as means ± SEM of three independent experiments. Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001

Discussion

Cancer-associated cachexia, a systemic wasting condition and a multifactorial syndrome, is a late consequence of distinct cancers and contributes to significant mortality [23]. Its’ harm has received widespread attention in clinic [5], whereas the detailed mechanism between EC and its associated cachexia remains elusive. Furthermore, an effective therapy for the comprehensive administration of EC-associated cachexia is unknown. We found that TRIM22 expression was negative in EC and in patients’ OS in the TGCA EC cohort and in our cohort study. TRIM22 overcame the body weight loss in patients with EC in a previous cohort study. TRIM22 consistently suppressed body weight loss induced by cachexia in xenograft mice. Moreover, TRIM22 inhibited EC growth. Mechanistically, TRIM22 inhibited EC progression by suppressing the NOD2-dependent NF-κB signaling pathway, reducing IL-6 levels. TRIM22 overcame EC-associated cachexia by attenuating the enhancement of BAT and WAT thermogenic activities via inhibition of the IL-6/IL-6R signaling pathway. Therefore, TRIM22 improved the prognosis and OS of patients with EC. We demonstrated that progesterone could induce TRIM22 expression and that IL-6R Ab could inhibit thermogenic activity. Combination treatment with progesterone, IL-6R Ab, and carbotaxol holds great promise for enhancing the efficacy of EC, which exhibits a potential advantage in clinical applications for supplementing the shortage of conventional therapy. Recent studies have shown that many patients with cancer-associated cachexia, such as gastric cancer [70], renal cell carcinoma [17], and pancreatic cancer [71], also experienced WAT browning, causing serious weight loss, adipose atrophy, poor prognosis, and poor OS. We observed obvious WAT browning in EC-bearing mice. In contrast, characteristics related to cachexia were reversed by TRIM22 OE. In addition, we found that the BAT thermogenic activity was significantly increased in EC-bearing mice during cachexia progression. Therefore, enhanced BAT activity and WAT browning contribute to EC-associated cachexia. Meanwhile, we also detected that TRIM22 could overcome the muscles atrophy. However, the mechanism of muscles atrophy in EC-associated cachexia needs to be further study.

IL-6 superfamily members, such as IL-6, are among the most frequently reported cachexia-inducing factors. Many studies have reported that BAT activity correlates with blood IL-6 levels in mice [72,73,74]. IL-6 promotes WAT browning during cancer-associated cachexia [74,75,76]. We found that serum IL-6 levels significantly increased in EC-bearing mice. Moreover, the IL-6/IL-6R signaling pathway was activated in the BAT and WAT of EC-bearing mice. Consistently, in a conditioned medium assay, we found that differentiated mature adipocytes showed higher thermogenic activity after treatment with Ishikawa cell-conditioned medium. There were no significant changes in Ucp1 expression and OCR when IL-6R was knocked down in differentiated mature adipocytes. These results indicate that the activation of the IL-6/IL-6R signaling pathway promotes BAT activity and WAT browning during EC-related cachexia.

NF-κB is a key regulator for IL-6 production during inflammation, promoting cancer development. TRIM22 inhibited EC progression by suppressing the NOD2-dependent NF-κB signaling pathway, reducing IL-6 levels. Thus, TRIM22 further restrained BAT and WAT thermogenic activity to overcome the cachexia effect in patients with EC and prolonged their OS. NOD2 plays an anti-inflammatory role by regulating the downstream inflammatory signaling pathway, such as NF-κB [77,78,79]. NOD2 expression was drastically decreased in the advanced EC stage (III and IV) in TCGA databases. Patients with EC who had high NOD2 expression had longer OS. NOD2 has been consistently reported to be a protective factor that improves the OS [54]. IL-6 has also been reported to be an aggravating factor that accelerates patients’ death [80]. IL-6 expression was significantly increased in the advanced EC stages (III and IV). Patients with EC who had lower IL-6 expression had shorter OS. These results indicate that NOD2 is an EC protective factor that regulates anti-inflammatory functions. Other studies have also reported the paradoxical role of NOD2 in triggering pro-inflammatory cytokine production [81,82,83]. NOD2 has also been reported to promote hepatocarcinogenesis via induction of the inflammatory response [84]. The reasons for this difference, particularly the regulation of NOD2-dependent NF-κB signaling, deserve further investigation.

Current EC therapies have numerous limitations; thus, there is an urgent need to develop novel and more effective combination treatments. In this study, we validated that combination therapy involving a TRIM22 inducer, thermogenesis inhibitors, and carbotaxol substantially improved the long-term efficacy of carbotaxol in inhibiting EC progression and EC-related cachexia. This combination therapy would still be the best choice, especially for progesterone-insensitive patients with EC, who might experience “progesterone resistance”. This strategy offers a novel combination therapy for inhibiting EC progression and EC-related cachexia, bringing new hope to patients with EC.

However, there were also several limitations in the present study that should be noted. Firstly, we still need to expand the sample size in future research. Secondly, progesterone may enhance the efficacy of EC by reducing toxic side effects [85, 86], and further exploration and research in this area are still warranted. Thirdly, to further validate the consistency of our experimental results, we plan to incorporate additional models in the future, including patient-derived xenograft models, which will allow us to mimic the clinical setting as closely as possible. Fourthly, in our study, we demonstrated that TRIM22 played its role interiorly in the tumor cell, as with many reports [87,88,89]. Nonetheless, whether TRIM22 itself exerts its biological effects in the secreted style remains uncertain. TRIM22 may exert its biological effects through secretory pathways or exosome-mediated mechanisms, which will be further elucidated in future studies. Finally, many findings strongly support the notion that TRIM22 could serve as a viable therapeutic target for cancer treatment, while also highlighting its potential as a diagnostic and prognostic biomarker, including osteosarcoma [90], gastric cancer [91], ovarian cancer [92], and hepatocellular carcinoma [93]. In line with these observations, our study demonstrated that TRIM22 not only inhibits the progression of endometrial cancer but also alleviates EC-related cachexia, further suggesting its potential as a therapeutic target in this context. However, future research is warranted to explore the clinical implications of TRIM22 and its translational potential in cancer therapy.

Conclusion

In conclusion, our study found that TRIM22 inhibited EC progression by suppressing the NOD2/NF-κB signaling axis and subsequently reducing IL-6 production. Moreover, TRIM22 inhibits the thermogenic activity of BAT and WAT by suppressing the IL-6/IL-6R signaling pathway. Furthermore, our data revealed that progesterone could directly induce TRIM22 expression. Importantly, our study further verified that treatment with a combination of carbotaxol, a TRIM22 inducer (progesterone), and an inhibitor of thermogenesis (IL-6R Ab) synergistically enhanced the anticancer efficacy in EC, expanding the arsenal of therapeutic agents for combating EC. (Fig. graphical abstract)