Upregulation of itchy E3 ubiquitin protein ligase contributes to endometrial cancer through promoting forkhead box P1 degradation

Patients and samples collection

Forty-seven patients with EC were recruited from Yantai Muping District Traditional Chinese Medicine Hospital between September 2023 and February 2024. All cases were confirmed as stage I/II through histopathological analysis.^[15] The patients’ clinical information was listed in Table 1. After data collection, the authors were able to access information that could identify individual participants.

Fresh EC samples and tumor-adjacent control tissue were obtained from patients who had not undergone pre-operative chemotherapy or radiation. Tumor type and grade were validated histologically. After resection, tissues were flash-frozen in liquid nitrogen immediately after collection and subsequently stored at −80°C for later analysis.

Ethical approval and informed consent

The Human Research Ethics Committee of Yantai Muping District Traditional Chinese Medicine Hospital approved this study with approval number September 05, 2023. All study participants provided written informed consent before their involvement. The research protocol adhered to the ethical principles outlined in the Declaration of Helsinki (version2024).^[16,17]

Database

The messenger RNA (mRNA) levels of ITCH and FOXP1 in 541 patients with EC and IHC results for 9 EC samples were collected through the Human Protein Atlas (HPA) (Website: https://www.proteinatlas.org/).

IHC

Fresh EC samples and tumor-adjacent control tissue were fixed in 4% paraformaldehyde for 24 h. After being dehydrated in graded ethanol and xylene, tissues were embedded in paraffin. Sections (4 μm) were baked at 60°C for 45 min in an incubator, then deparaffinized and rehydrated through a series of washes, from xylene (Cat. XX0060, Sigma-Aldrich; Darmstadt, Germany) to ethanol (Cat. 493511, Sigma-Aldrich; Darmstadt, Germany). Proteinase K (Cat. 17916, Thermo Fisher Scientific, Waltham, MA, USA) pre-treatment was performed for 10 min at room temperature. Sections were blocked by 5% bovine serum albumin for 30 min at room temperature and then incubated with the rabbit anti-ITCH polyclonal antibody (1:300 dilution, Cat. ab108515, Abcam, Cambridge, MA, USA) or the rabbit anti-FOXP1 polyclonal antibody (1:500 dilution, Cat. ab134055, Abcam, Cambridge, MA, USA) at 4°C overnight. After triple washing with phosphate-buffered saline with Tween 20 (PBST), the sections were exposed to horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (1:1000 dilution, Cat. ab6721, Abcam, Cambridge, MA, USA). Following triple PBST washes, signal detection was performed using a 3,3’-diaminobenzidine substrate kit (Cat. ab64238, Abcam, Cambridge, MA, USA) as per manufacturer’s protocol. Microscopic imaging was subsequently conducted for visualization.

Two independent pathologists evaluated the immunohistochemical staining, assessing both the staining intensity and the proportion of positively stained tumor cells. The intensity was categorized on a scale of 0–3 (0: Negative, 1: Weak, 2: Moderate, and 3: Strong), while the percentage of stained tumor cells was quantified from 0% to 100%. The pathologists then collaboratively reviewed their assessments to reach a consensus. An H-score, which can range from 0 to 300, was computed by multiplying the positivity intensity by the percentage of tumor cells exhibiting staining.^[18]

Cell culture

HEC-1A (Cat. CL-0099) and KLE (Cat. CL-0133), sourced from the Wuhan Procell Life Technology (Wuhan, China), were used as experimental models. All the cell lines included in this study were authenticated by short tandem repeat profiling. These cells were grown in Dulbecco’s Modified Eagle’s Medium (Cat. 11965092, Thermo Fisher Scientific, Waltham, MA, USA), enriched with 10% fetal bovine serum (Cat. A5670801, Thermo Fisher Scientific, Waltham, MA, USA), and supplemented with 100 IU/mL of penicillin and 10 μg/mL streptomycin (Cat. 15070063, Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained in a humidified incubator at 37°C under a 5% CO₂ atmosphere. Mycoplasma detection test using 4’,6-diamidino-2-phenylindole (DAPI) staining was employed to exclude contaminated cells.

Bromodeoxyuridine/5-bromo-2’-deoxyuridine (BrdU) staining

BrdU (Cat. 2750, Sigma-Aldrich; Darmstadt, Germany) was diluted in cell culture medium to prepare a 10 μM BrdU labeling solution. The cells were incubated in the BrdU labeling solution for 8 h at 37°C in a CO₂ incubator. Following incubation, the BrdU labeling solution was removed from the cells, and the cells were washed twice with phosphate-buffered saline (PBS) for approximately 5 s per wash. Cells were fixed with 4% paraformaldehyde in pH 7.4 PBS for 10 min at ambient temperature, followed by permeabilization using 0.1% Triton X-100 in PBS for another 10 min. Treat fixed cells for 15 min in 2N HCl. After 3 times wash with PBS, the cells were incubated with mouse BrdU antibody (1:500 dilution, Cat. 2750, Sigma-Aldrich; Darmstadt, Germany) overnight at 4°C. After another 3-time washes by PBS, the cells were incubated with diluted Alexa Fluor^® 594-labeled Goat Anti-Mouse immunoglobulin G (IgG) (1:1000 dilution, ab150116, Abcam, CA, USA) for 2 h at 25°C. Cells were mounted with antifade mounting medium with DAPI (H-1200-10, Vector Laboratories, CA, United States), and the images were captured. Five random fields were captured for each group and the proliferative cells were presented by the percentage of BrdU-positive cells divided by all cells (DAPI positive).

Cell proliferation assay

The viability of cells was determined through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HEC-1A or KLE cells were seeded into a 96-well plate at a density of 2 × 10³ cells/well. Cells were then transfected with the ITCH overexpression vector or the control vector, respectively, for 48 h. Twenty microliter MTT (5 mg/mL) (Cat. 475989, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) solution was added into each well and incubated for 4 h. After added dimethyl sulfoxide (Cat. D8418, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) to each well, a 96-well plate reader (Multiskan FC Microplate Photometer, Thermo Fisher Scientific, Waltham, MA, USA) was used to record the absorbance at 570 nm.

Cell apoptosis analysis

Cells were digested by 0.25% trypsin (Cat. 25200056, Thermo Fisher Scientific, Waltham, MA, USA) and collected by centrifugation at 400 g for 10 min. The cells were washed with ice-cold PBS for 3 times and then resuspended in 100 μL Annexin V binding buffer at a final concentration of 5 × 10⁶ cells/mL. Add 1 μL fluorescein isothiocyanate-labeled annexin V (Annexin V-FITC) Conjugate (Cat. ab14085, Abcam, Cambridge, MA, USA) and 12.5 μL propidium iodide solution (Cat. ab14085, Abcam, Cambridge, MA, USA) into each sample tube and incubate for 10 min on ice in the dark. Apoptotic cells were identified using flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA). The data were processed and analyzed using FlowJo software (v10.4.1) from Tree Star, Inc., Ashland, OR, USA.

Chromatin immunoprecipitation (ChIP) assay

Five million HEC-1A or KLE cells were cross-linked for 5 min at 37°C using 1% formaldehyde. The cell pellet was resuspended by sodium dodecyl sulfate (SDS) lysis buffer (1% SDS [Cat. 7910-OP, Sigma-Aldrich; Darmstadt, Germany], 10 mM ethylenediaminetetraacetic acid [EDTA] [Cat. 4005-OP, Sigma-Aldrich; Darmstadt, Germany], 50 mM Tris-HCl [pH 8.1]) and subjected to a 15-min incubation on ice. After sonication, cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C to remove the debris. The supernatant was harvested and subsequently diluted 1:10 in the appropriate dilution buffer (0.01% SDS, 1.1% Triton X-100 [Cat. T8787, Sigma-Aldrich; Darmstadt, Germany], 1.2 mM EDTA [Cat. 4005-OP, Sigma-Aldrich; Darmstadt, Germany], 167 mM NaCl [Cat. S3014, Sigma-Aldrich; Darmstadt, Germany], 16.7 mM Tris-HCl [pH 8.1]), and then subjected to a 1-h incubation at 4°C with Protein-A/G Sepharose beads (Cat. LSKMAGAG, Sigma-Aldrich; Darmstadt, Germany) with a rotating machine. Cell lysates that had been precleared were incubated at 4°C with 1 μg of FOXP1 antibody or IgG for overnight. Subsequently, protein-A/G Sepharose beads (Cat. LSKMAGAG, Sigma-Aldrich; Darmstadt, Germany) were used to capture the immune complexes. The beads underwent three washes by LiCl Buffer (0.25 M LiCl [Cat. L9650, Sigma-Aldrich; Darmstadt, Germany], 1% Triton X-100 [Cat. T8787, Sigma-Aldrich; Darmstadt, Germany], 1% deoxycholic acid [Cat. D2510, Sigma-Aldrich; Darmstadt, Germany], 1 mM EDTA [Cat. 4005-OP, Sigma-Aldrich; Darmstadt, Germany], 10 mM Tris-HCl [pH 8.1]) and two washes with TE buffer (1 mM EDTA [Cat. 4005-OP, Sigma-Aldrich; Darmstadt, Germany], 10 mM Tris-HCl [pH 8]). DNA isolation was then performed on the beads. The enrichment of the KRAS proto-oncogene GTPase (KRAS) promoter region was measured using quantitative real-time polymerase chain reaction (RT-PCR).

RT-PCR

The enrichment of KRAS promoter segments was quantified using RT-PCR with SYBR Green RT-PCR Master Mix (Cat. 4309155, Thermo Fisher Scientific, Waltham, MA, USA). Each sample within each group was measured in triplicate, and the experiment was conducted at least 3 times. The relative enrichments were determined using the ²(-ΔΔCt) method.

Primer sequences:

KRAS-a-F: 5’-CAGGGACTGCTCTGGCGT-3’

KRAS-a-R: 5’-GTAAGAAACCGTTAACG-3’

KRAS-b-F: 5’-GCTGGGCTCCGGGAAG-3’

KRAS-b-R: 5’-AAACGTCGCCCACTCAAACA-3’

KRAS-c-F: 5’-TCTGCTCCACTTTTTCCC-3’

KRAS-c-R: 5’-TCGGGAGGGGAGAGAA-3’

Immunoprecipitation (IP)

Cells were harvested and subsequently lysed in IP buffer supplemented with a protease inhibitor cocktail, 1% Triton X-100, 20 mM Tris (pH 7.5), 20 mM N-ethylmaleimide, and 150 mM NaCl. Two milligrams of the cellular supernatant were incubated at 4°C with 5 μg anti-ubiquitin antibody (Cat. ab32058, Abcam, Cambridge, MA, USA) or 5 μg anti-Flag antibody (Cat. F1804, Sigma-Aldrich; Darmstadt, Germany) coupled to protein-A/G beads (Cat. LSKMAGAG, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) overnight, to enrich for specific proteins. The protein complexes were washed 4 times with TBS buffer and eluted using sample loading buffer for immunoblotting. Total protein concentration was quantified using a Pierce bicinchoninic acid (BCA) Protein Assay Kit (Cat. 23227, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol (Cat. 23225, Thermo Fisher Scientific, Waltham, MA USA). Following measurement, the proteins were stored at −80°C for future use.

Protein extraction and immunoblotting

The initial step in protein extraction involves lysing the cells. Ice-cold RIPA (Radioimmunoprecipitation Assay) buffer (Cat. 89901, Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitors is added to the cells to disrupt the cell membrane and release the proteins. The protease inhibitors prevent protein degradation by proteases. Following cell lysis, the cells are collected using a cold plastic cell scraper and centrifuged to remove debris.

The extracted proteins are then separated using SDSpolyacrylamide gel electrophoresis (SDS-PAGE) (Cat. NP0322BOX, Thermo Fisher Scientific, Waltham, MA USA), a technique that employs an electric field to separate proteins based on their size. The proteins are loaded onto a gel and subjected to an electric field, causing them to migrate through the gel at a rate inversely proportional to their size. Proteins were resolved by SDS-PAGE and subsequently electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Cat. 03010040001, Mannheim, Germany), a thin, flexible membrane used to capture the proteins. The PVDF membrane is then blocked with 5% nonfat milk to prevent non-specific antibodies. Subsequently, the membrane is incubated overnight at 4°C with the primary antibody specific to the protein of interest. The following day, membranes were probed with HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Cat. 32106, Thermo Fisher Scientific, Waltham, MA USA), which produces a signal that can be visualized using a chemiluminescence imager (Cat. L00817C, GenScript, Shanghai, China).

The β-actin signal is used for normalization, as β-actin is a protein present in all cells. Normalizing the signal for the protein of interest with the β-actin signal ensures that the results are comparable between different samples.

Antibodies information:

Rabbit anti-ITCH polyclonal antibody (1:1000 dilution, Cat. ab108515, Abcam, Cambridge, MA, USA);

Rabbit anti-FOXP1 polyclonal antibody (1:1000 dilution, Cat. ab134055, Abcam, Cambridge, MA, USA);

Rabbit anti-β-actin polyclonal antibody (1:1000 dilution, Cat. ab8227, Abcam, Cambridge, MA, USA);

HRP-conjugated goat anti-rabbit secondary antibodies (1:3000 dilution, Cat. ab6721, Abcam, Cambridge, MA, USA).

RNA extraction

Trizol reagent (Cat. 15596018CN, Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from tissue and cell samples. The concentration and purity of the obtained RNA were assessed with an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Only samples with absorbance ratios of ~2.0 (260 nm/280 nm) and 1.9–2.2 (260 nm/230 nm) were included in the study.

Quantitative RT-PCR

Complementary DNA (cDNA) was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Cat. 4368814, Thermo Fisher Scientific, Waltham, MA, USA) with 1 μg RNA sample as the template. The expression levels of candidate genes were quantified using RT-quantitative PCR (RT-qPCR) with SYBR Green RT-PCR Master Mix (Cat. 4309155, Thermo Fisher Scientific, Waltham, MA, USA), using β-actin as the loading control. Each sample within each group was measured in triplicate, and the experiment was conducted at least 3 times. The relative levels of the target genes were determined using the ²(-ΔΔCt) method.

Primer sequences:

ITCH-qF: 5’-GACAGCCTCATAGCTCATGTATC-3’

ITCH-qR: 5’-CAGGGTTCTGCTGCTTATTCT-3’

FOXP1-qF: 5’-GCAACAGTGGCATCTCATAAAC-3’

FOXP1-qR: 5’-ACTCCCAAGGGCTTGAAATTA-3’

ACTB-qF: 5’-GGCATGGGTCAGAAGGATT-3’

ACTB-qR: 5’-AAGGTGTGGTGCCAGATTT-3’

Wound-healing assay

Cells were cultured in six-well plates (Cat. 140675, Thermo Fisher Scientific, Waltham, MA, USA) until reaching 80–90% confluence. A linear scratch was created in the confluent cell monolayer using a sterile pipette tip, simulating a wound. To remove detached cells, PBS (Cat. 10010023, Thermo Fisher Scientific, Waltham, MA, USA) was used for rinsing, and fresh media was added to facilitate wound closure for 24h. The healing process was observed under a microscope (DMi8, Leica, Wetzlar, Germany).

Statistical analyses

Data analysis was performed using the Statistical Package for the Social Sciences (SPSS) (version 16.0, SPSS Inc., Chicago, IL, USA). A paired t-test was employed to analyze the relative ITCH and FOXP1 mRNA levels in tumor and control tissues, as well as the enrichment of the KRAS promoter region. One-way analysis of variance with Tukey’s multiple comparison test was utilized to analyze datasets comprising more than two groups. The correlation between FOXP1 and ITCH protein levels was assessed using Pearson’s correlation coefficient. Survival analyses were performed using the Kaplan–Meier method, with inter-group differences assessed by the log-rank test. Statistical significance was set at P < 0.05.

ITCH is upregulated in EC and negatively correlated with FOXP1 protein level

To explore the potential roles of ITCH and FOXP1 in the development of EC, we first checked their mRNA levels in 47 EC tissues and adjacent non-tumor tissues. ITCH mRNA was significantly increased in EC tissues compared to non-tumor controls (P = 0.022), while FOXP1 mRNA levels were similar between EC tissues and controls (P > 0.05) [Figure 1a and b]. Meanwhile, ITCH and FOXP1 protein levels in 47 paired EC and non-tumor control tissues were examined by IHC and then quantified. FOXP1 protein levels were significantly reduced in EC tissues compared to non-tumor controls [Figure 1c]. Upon analyzing the protein levels of FOXP1 and ITCH, we discovered a significant negative correlation [Figure 1d], which was observed in 74.5% of EC patients (35 out of 47) [Figure 1e].

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Figure 1:

ITCH is overexpressed in EC and negatively correlated with FOXP1. (a and b) Total RNA was isolated from 47 paired EC and adjacent non-tumor control tissues. The mRNA levels of ITCH and FOXP1 were determined by RT-qPCR (n = 47). (c) ITCH and FOXP1 protein levels were detected by immunohistochemistry and the results were quantified by ImageJ (n = 47). The scale bar in c represents 200 μm. (d) The correlation between the signals of ITCH and FOXP1 was analyzed using Pearson’s correlation coefficient analysis (n = 47). (e) Dot plot with connecting lines showing ITCH and FOXP1 level in paired samples (n = 47). Values of P < 0.05 were considered statistically significant. ITCH: itchy E3 ubiquitin protein ligase, FOXP1: Forkhead box P1.

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Patients with higher ITCH level had shortened overall survival

To validate the roles of ITCH and FOXP1 in EC using larger cohorts, we analyzed the transcriptome data from the Protein Atlas (www.proteinatlas.org/pathology).^[19] As shown in Figure 2a, Figure S1a and b, patients with higher ITCH expression had a worse prognosis. No appreciable association was observed between the mRNA expression levels of ITCH and FOXP1 [Figure 2b]. However, an inverse relationship was detected between ITCH and FOXP1 protein expression levels [Figure 2c].

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Figure 2:

Upregulated ITCH relates to poor prognosis of patients with EC and is negatively correlated with FOXP1 level. (a) The survival data, which included information on 175 patients with EC, were downloaded from the Human Protein Atlas (https://www.proteinatlas.org/). Patients were stratified into two cohorts based on the relative level of ITCH and were subjected to Kaplan–Meier analysis with the logrank test. (b) The relationship between the mRNA levels of ITCH and FOXP1 was analyzed using the Pearson’s test (n = 175). (c) Representative results of immunohistochemistry from the Human Protein Atlas (https://www.proteinatlas.org/) detecting ITCH and FOXP1 protein levels in an EC sample (n = 9), and the results of the correlation analysis. Scale bar is the strand for 100 um. A threshold of P < 0.05 indicated statistical significance. ITCH: Itchy E3 ubiquitin protein ligase, FOXP1: Forkhead box P1, EC: Endometrial cancer.

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ITCH induces FOXP1 degradation through ubiquitination

ITCH is an E3 ubiquitin ligase that transfers the ubiquitin to target proteins and induces their proteasome-dependent degradation. To understand whether ITCH induces FOXP1 degradation, we constructed a vector expressing the ITCH WW domain fused with three tandem FLAG epitopes at the N terminus. Meanwhile, 3 × Flag-tagged importin β was used as a negative control for IP. As shown in Figure 3a, only the ITCH WW domain could recruit FOXP1 from the cell lysate. Furthermore, ITCH and FOXP1 were colocalized in the HEC-1A cell nucleus [Figure 3b]. These results indicated that ITCH directly interacts with FOXP1 in EC cells. Subsequently, we tested whether FOXP1 is a direct ubiquitination substrate for ITCH. As shown in Figure 3c, overexpression of ITCH promoted the ubiquitination of FOXP1 and downregulated FOXP1 levels in a dose-dependent manner, indicating that ITCH negatively regulates FOXP1 expression at the post-transcriptional level.

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Figure 3:

ITCH interacts with FOXP1 and induces its degradation. (a) HEC-1A cells were transfected with Flag-tagged ITCH WW domain or Importin-β for 48 h. The cells were then subjected to immunoprecipitation using anti-Flag antibody magnetic beads. The recruited proteins were subjected to immunoblotting using an anti-FOXP1 antibody (n = 3). (b) The subcellular localization of ITCH and FOXP1 was detected by immunofluorescence in HEC-1A cells, with DAPI labeling the cell nucleus. The images were captured by a confocal microscope at ×40 magnification, and the scale bar is 5 μm (n = 3). (c) HEC-1A cells were transfected with 1 μg FOXP1 expression vector and increasing amounts of the ITCH expressing vector (0, 0.5, 1, 2 μg). ITCH and FOXP1 expression levels were assessed by immunoblotting using 20% of the total cell lysate. FOXP1 ubiquitination was evaluated by immunoprecipitation with an anti-ubiquitin antibody using the remaining cell lysate (n = 3). ITCH: Itchy E3 ubiquitin protein ligase, FOXP1: Forkhead box P1, DAPI: 4’,6-diamidino-2-phenylindole.

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FOXP1 inhibits KRAS expression

FOXP1 is a transcription factor that exhibits tumor-suppressive properties across diverse solid tumors and regulates the expression of multiple downstream genes, including KRAS.^[20] To understand whether FOXP1 controls KRAS expression in EC, FOXP1 was overexpressed in HEC-1A and KLE cells. Both the protein and mRNA levels of KRAS were repressed by FOXP1 in a dose-dependent manner [Figure 4a-c]. To identify the FOXP1 binding site in the KRAS promoter region, ChIP assays were performed using anti-FOXP1 antibodies. Three pairs of primers were designed to confirm the enrichment of the KRAS promoter region [Figure 4d]. As shown in Figure 4e, FOXP1 was successfully enriched by the anti-FOXP1 antibody. In addition, the KRAS promoter region was also enriched by the anti-FOXP1 antibody in both HEC-1A and KLE cells [Figure 4f]. These results indicate that FOXP1 represses KRAS expression through binding to the promoter sequence and inhibiting transcription.

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Figure 4:

FOXP1 inhibits KRAS expression through binding to the promoter region. (a) HEC-1A and KLE cells were transiently transfected with increasing amounts of the FOXP1 expressing vector (0, 0.5, 1 μg) for 48 h. The samples were then assessed through immunoblotting techniques to examine the protein levels of FOXP1 and KRAS (n = 3). (b) The band density was quantified using Image J (n = 3). (c) RT-qPCR was used to examine the mRNA level of KRAS. (d) The schematic diagram of the Kras promoter region and primer design (n = 3). (e) FOXP1 in HEC-1A and KLE cells was immunoprecipitated by an anti-FOXP1-specific antibody (n = 3). (f) The immunoprecipitated DNA was then used to quantify three segments of the KRAS promoter region by qPCR (n = 3). Data were analyzed using Student’s t-test. Significance thresholds were set at P < 0.05 (^✶), P < 0.01 (^✶✶), and P < 0.001 (^✶✶✶). FOXP1: Forkhead box P1, RT-qPCR: Real-time quantitative polymerase chain reaction, KRAS: KRAS proto-oncogene GTPase.

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ITCH overexpression promotes proliferation and inhibits apoptosis of EC cells

To explore the function of ITCH during the carcinogenesis of EC, ITCH overexpression HEC-1A and KLE cells were constructed. In ITCH overexpression cells, FOXP1 protein level was reduced, and KRAS protein level was increased [Figure 5a]. Meanwhile, KRAS mRNA level was increased in ITCH overexpression cells but FOXP1 mRNA level was not significantly altered [Figure 5b]. BrdU staining, MTT assay, and flow cytometry analysis were conducted on the cells to assess proliferative cells percentage, cell viability, and apoptosis. In Figure 5c-e, ITCH overexpression upregulated the number of proliferative cells, the cell viability, and repressed apoptosis of HEC-1A and KLE cells. Meanwhile, ITCH overexpression promoted the migration of HEC-1A and KLE cells [Figure 5f].

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Figure 5:

ITCH promotes EC cell proliferation and inhibits apoptosis through repressing FOXP1. (a) ITCH overexpression in HEC-1A and KLE cells was constructed. ITCH, FOXP1, and KRAS protein expression were quantified through immunoblotting, using β-actin as a loading control (n = 3). (b) The mRNA levels of FOXP1 and KRAS were detected by RT-qPCR (n = 3). (c) The proliferative cells were quantified by BrdU staining (n = 3). Scale bar = 50 μm. (d) Cell viability was assessed using the MTT assay (n = 3). (e) Flow cytometry assessment was employed to detect apoptotic cells (n = 3). (f) Wound healing assay was used to examine cell migration capacity (n = 3). Scale bar = 200 μm. Data were analyzed using Student’s t-test. Significance thresholds were set at P < 0.05 (^✶) and P < 0.01 (^✶✶). ITCH: Itchy E3 ubiquitin protein ligase, FOXP1: Forkhead box P1, EC: Endometrial cancer, RT-qPCR: Real-time quantitative polymerase chain reaction, KRAS: KRAS protooncogene GTPase, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, mRNA: Messenger RNA.

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Since KLE cells expressed very low levels of endogenous ITCH, we only knocked down ITCH in HEC-1A cells. As shown in Figure S2a, in ITCH knockdown cells, FOXP1 protein level was increased and KRAS protein level was reduced. Meanwhile, ITCH knockdown cells had reduced viability [Figure S2b] and increased apoptosis [Figure S2c]. These results indicated that ITCH contributes to the carcinogenesis of EC through inducing FOXP1 degradation to promote KRAS overexpression [Figure 6].

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Figure 6:

The schematic diagram shows ITCH upregulating KRAS expression through inducing FOXP1 degradation. (PowerPoint, version 2504, Microsoft, Redmond, WA, USA). ITCH: Itchy E3 ubiquitin protein ligase, FOXP1: Forkhead box P1, KRAS: KRAS proto-oncogene GTPase.

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Finally, we analyzed gene expression data from patients included in the TCGA project using cBioPortal (https://www. cbioportal.org/). We found that 2821 genes were positively correlated with ITCH level in EC, and these genes were functionally enriched in DNA repair, cell division, chromatin remodeling, and protein polyubiquitination and located in both cytosol and nucleus [Figure S3a]. Meanwhile, 1740 genes were negatively correlated with ITCH and these genes were functionally enriched in translation, ATP synthesis, aerobic respiration, and rRNA processing and mainly located in the cytosol, mitochondria, and nucleoplasm [Figure S3b]. Ten genes that positively correlated with ITCH were enriched in the EC term. These genes are key factors for WNT, MAPK/PI3K, and DNA repair signaling pathways [Figure S4a]. Meanwhile, 15 genes that negatively correlated with ITCH were enriched in oxidative phosphorylation. These data suggest that the upregulation of ITCH may promote oncogene expression and modulate the switch of cancer cell metabolic pattern from oxidative phosphorylation to glycolysis [Figure S4b].