Abstract
Background
Uterine sarcoma constitutes approximately 3–7% of all uterine cancers, with adenosarcoma and leiomyosarcoma being the major subtypes. This neoplasm is characterized by poor clinical outcomes, with frequent recurrence and metastasis, underscoring the urgent need for early detection strategies. Cyclin-dependent kinase regulatory subunit 2 (CKS2) is markedly overexpressed in uterine sarcoma. Preliminary data suggest that CKS2 overexpression correlates with advanced tumor staging, yet its mechanistic link to immune evasion via natural killer T (NKT)-cell regulation remains unexplored. This study aimed to explore how CKS2 regulates the immune response in uterine sarcoma.
Methods
Through the integration of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, a systematic analysis was conducted on the correlation between CKS2 expression levels, tumor prognostic staging, and immune cell infiltration. Stable CKS2-knockdown cell lines were constructed, and the expression changes of CKS2 were detected via quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot techniques. Through colony formation assays, TUNEL staining, invasion and migration assays, and Western blot analysis, the mechanism related to the regulatory effect of CKS2 on the malignant progression of uterine sarcoma cells was clarified in depth. Additionally, the specific mechanism by which CKS2 regulates NKT cell activity was verified at the tissue level via multiplex immunofluorescence.
Results
In uterine sarcoma, CKS2 expression was found to be significantly upregulated and closely associated with poor prognosis, advanced tumor stage, and a distinct negative correlation with NKT cell activity. In vitro experiments indicated that knockdown of CKS2 significantly inhibited the proliferation, migration, and invasion of sarcoma cells and promoted apoptosis. Mechanistically, CKS2 activated the PI3K/AKT signaling, reduced major histocompatibility complex (MHC) class I chain-related protein A (MICA) expression, and inhibited NKT cell activity, resulting in immune escape, which was effectively mitigated by PI3K inhibitors.
Conclusions
The findings suggest that CKS2 can serve as a valuable biomarker and an effective target for the prevention and screening of uterine sarcoma and can modify the antitumor immune response in uterine sarcoma.
Keywords: Uterine sarcoma, cyclin-dependent kinase regulatory subunit 2 (CKS2), natural killer T (NKT), major histocompatibility complex class I chain-related protein A (MICA), immune escape
Highlight box.
Key findings
• The high expression of cyclin-dependent kinase subunit 2 (CKS2) was significantly negatively correlated with the activity of natural killer T (NKT) cells, suggesting that CKS2 is involved in regulating the immunosuppressive microenvironment.
What is known and what is new?
• The expression of CKS2 is significantly upregulated in uterine sarcoma and is significantly associated with poor prognosis and tumor stage.
• CKS2 induces immune escape by activating the PI3K/AKT signaling pathway, downregulating MHC class I chain-related protein A (MICA) expression and suppressing NKT cell activity.
What is the implication, and what should change now?
• CKS2 could be regarded as a valuable biomarker and an effective screening target for uterine sarcoma. Moreover, it may provide guidance for the selection of subsequent immunotherapy agents.
Introduction
Uterine sarcoma is a rare and heterogeneous group of malignancies, accounting for approximately 3–7% of all uterine cancers and about 1% of female reproductive tract malignancies (1). It includes histological subtypes such as carcinosarcoma, leiomyosarcoma (uLMS), endometrial stromal sarcoma, and undifferentiated sarcoma. The aggressive nature and high recurrence rate of these tumors pose significant diagnostic and therapeutic challenges. Particularly, uLMS often mimics benign fibroids, making preoperative diagnosis difficult (2). Recent advances in imaging techniques, including magnetic resonance imaging (MRI) and radiomics-based tools, have improved differential diagnosis; for example, the PREoperative Sarcoma Score (PRESS) system has been proposed to aid in distinguishing uterine sarcomas from benign leiomyomas using MRI parameters and clinical features (3). However, despite these developments, consensus remains lacking on reliable biomarkers and effective treatment strategies. Therefore, it is crucial to identify molecular factors associated with poor prognosis and therapeutic resistance to enhance early diagnosis, risk stratification, and targeted therapy for uterine sarcomas.
Cyclin-dependent kinase subunit 2 (CKS2), a member of the cell cycle-dependent protein kinase subunit family, is situated on chromosome 9q22 and plays a crucial role in early embryonic development and somatic cell division (4,5). The CKS2 protein plays a crucial role in intracellular signaling and cell division and has been extensively investigated across various cancer types (6). Aberrantly elevated expression of CKS2 may function as an oncogene, contributing to the development of malignant tumors such as colon cancer, breast cancer, and cholangiocarcinoma (7-9). Moreover, CKS2 actively participates in tumor initiation and progression by maintaining the phenotypic characteristics of cancer cells while also influencing immune infiltration and regulating the immune microenvironment (10,11). Immunotherapy has been established as an important means of cancer treatment. Activating the immune system can enhance the immune surveillance function of tumors and reduce their immune evasion (12). The accumulation of natural killer T (NKT) cells in the tumor microenvironment is crucial for establishing an immunocompetent microenvironment, which can be activated by Major histocompatibility complex I (MHC I)-related proteins on tumor cell membranes, such as MHC class I chain-related protein A/B (MICA/B), and followed by the secretion of chemokines, cytokines, and granules to kill tumor cells and virus-infected cells (13,14). Although the oncogenic role and underlying mechanism of CKS2 have been elucidated in various tumor types, there is currently a dearth of studies on its immune evasion in uterine sarcoma. Consequently, the diagnostic, prognostic, and therapeutic potential of CKS2 for uterine sarcoma remains largely unexplored.
In this study, we compared the differentially expressed genes between uterine carcinosarcoma (UCS) and uLMS, as well as their corresponding normal tissues, in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Our findings revealed that CKS2 exhibited low expression levels in normal tissues; however, its expression gradually increased with disease progression. Mechanistically, we found that CKS2 could interfere with the expression of MICA through the PI3K/AKT signaling pathway, inhibit the cytotoxic activity of NKT cells, and lead to immune evasion. We validated CKS2-PI3K-AKT-MICA axis at the cell and tissue levels to identify effective molecular targets for the prevention and treatment of uterine sarcoma. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1405/rc).
Methods
Differentially expressed messenger RNA analysis
The microarray data used in this study was obtained from TCGA (https://portal.gdc.cancer.gov/repository) and GEO (https://www.ncbi.nlm.nih.gov/geo/) databases, and the original data were downloaded in fragments per kilobase of transcript per million mapped reads (FPKM) and MINiML format. The gene expression profiles of normal uterine tissues were obtained from the Genotype-Tissue Expression (GTEx) database (https://gtexportal.org/home/).
Venn diagram
The online database Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/) provides a comparative analysis of differentially expressed genes in UCS and its normal tissues via the GTEx and TCGA datasets. The criteria for defining differential messenger RNA (mRNA) expression included an adjusted P value below 0.05 and a fold change greater than 1.5 or less than −1.5. The “limma” package in R software (The R Foundation for Statistical Computing) was used for performing a differential expression analysis of GEO mRNAs, and adjusted P values were employed to control false-positive results in the GEO dataset. The criteria for defining differential mRNA expression included an adjusted P value below 0.05 and a fold change greater than 1 or less than −1 (log scale).
Differential expression analysis and KEGG enrichment analysis
The data were normalized using the scale normalization factor through the edgeR software package, and further differential gene expression analysis was conducted. During the analysis, the Benjamini-Hochberg method was used to correct the P values for multiple hypothesis testing. A corrected P value threshold of 0.05 was set, and an absolute fold change of |log2FoldChange| greater than or equal to 1.5 was used as the criterion for screening differentially expressed genes. Additionally, to explore the functional enrichment of differentially expressed genes, this study used the clusterProfiler R package to conduct statistical enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
Sangerbox 3.0 database
Sangerbox 3.0 (http://vip.sangerbox.com/) is a web-based tool platform. The data from the GEO, TCGA, International Cancer Genome Consortium (ICGC), and other databases were integrated, followed by a batch processing of the data.
Cell culture, vector construction, and transfection
MES-SA and SK-UT-1 cells purchased from Pricella (Houston, TX, USA) and authenticated via short tandem repeat (STR) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at 37 ℃ in a humidified incubator containing 5% CO2. To construct the CKS2 short hairpin RNA (shRNA) vector, we cloned the annealed shRNA oligonucleotides into the plasmid Lentiviral KnockdOwn (pLKO) vector. A pair of nontargeting shRNAs (Table S1) was used as a scramble and pLKO. To perform lentiviral transfection, we seeded MES-SA and SK-UT-1 cells at a density of approximately 40% in six-well plates and transfected the vectors with polybrene (Sigma-Aldrich, St. Louis, MO, USA). After culturing for 48 hours, we maintained the cells in DMEM medium containing 3 µg/mL puromycin to obtain stable transfected cells (see Table S1 for details).
RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and quantitative real-time PCR (qPCR) analysis
After cell lysis using TRIzol® reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA), total RNA was extracted from the aqueous phase following chloroform mixing, precipitated with isopropanol, washed with 75% ethanol, and resuspended in nuclease-free water. Subsequently, cDNA synthesis was performed utilizing a cDNA Reverse Transcription kit (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR analysis was conducted employing SYBR Green premix (Applied Biosystems/Thermo Fisher Scientific) on an ABI 7500 real-time PCR machine (Applied Biosystems/Thermo Fisher Scientific). Data acquisition was carried out using ABI SDS 2.0.1 software package and the 2−∆∆ct method was employed for data analysis. See Table S2 for details.
Western blotting
The protein concentration was determined via a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Proteins separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred onto a polyvinylidene fluoride membrane. The primary antibodies involved in CKS2 (1:1,000; ab155078; Abcam, Cambridge, UK), including β-actin (1:1,000; 66009-1-Ig; Proteintech, Rosemont, IL, USA), AKT (1:1,000; 9272; Cell Signaling Technology, Danvers, MA, USA), P-AKT (1:1,000; 4060; Cell Signaling Technology), P-PI3K (1:1,000; 17366; Cell Signaling Technology), PI3K (1:1,000; 4292; Cell Signaling Technology), and MICA (1:1,000; ab62540; Abcam), were used for Western blot detection. The expression of related proteins was detected via Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).
Colony formation assay
To detect the effect of gene interference on the cloning ability of MES-SA and SK-UT-1 cell lines, target cells were inoculated into six-well plates containing 10% FBS DMEM medium and treated with 0.1 mg/mL of poly-L-lysine for 2 h at room temperature. Each well contained 1,000 cells. After 10 days of cell culture, the cells were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for 30 min.
Cell invasion assay
In the cell invasion experiment, 200 µL of transfected cells (1×104) in serum-free DMEM were seeded into the upper Transwell chamber with an 8-µm pore size (Corning, Corning, NY, USA) coated with Matrigel, and 600 µL of medium containing 10% FBS was added to the lower chamber. After incubation at 37 ℃ for 48 h, the cells that had invaded the bottom of the membrane were fixed with 4% paraformaldehyde at room temperature for 5 min and stained with 0.3% crystal violet dye at room temperature for 5 min. Observations were made using an optical microscope.
Cell apoptosis assay
The cell slides were arranged in a 24-well plate, seeded with 5×105 cells overnight, and subsequently fixed with methanol for 30 minutes. Apoptosis in the tumor tissue was detected via a TUNEL FITC Apoptosis Detection Kit (Vazyme, Nanjing, China) according to the provided instructions.
Wound healing assay
Cells were seeded in six-well plates. Once the confluence reached 90–100%, a line was drawn on the monolayer with a 20-µL pipette tip to create scratches. The cells were then rinsed with phosphate-buffered saline (PBS) for removal of cellular debris and subsequently cultured in fresh medium. A total of five fields were selected for observation under a microscope at both 0 and 48 h postscratching. The scratch distance was measured three times for each field to calculate the average value. The cell migration rate was determined with the following formula: cell migration rate = (0-h scratch distance − 48-h scratch distance)/(0-h scratch distance) × 100%.
Patient samples
Thirty postoperative specimens of patients with uterine sarcoma confirmed by pathology at Wuxi People’s Hospital from 2010 to 2020 were collected. The patient information collection details are shown in Table 1. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Ethics Committee of The Affiliated Wuxi People’s Hospital of Nanjing Medical University (No. KY24165) and informed consent was taken from all the patients.
Table 1. The basic clinical pathological information of the patients.
| Clinical pathological parameter | No. of patients |
|---|---|
| Total | 30 |
| Gender | |
| Male | 0 |
| Female | 30 |
| Age | |
| <60 years | 22 |
| ≥60 years | 8 |
| Stage | |
| 0 | 10 |
| I–II | 14 |
| III–IV | 6 |
| Metastasis | |
| Negative | 24 |
| Pelvic cavity | 4 |
| Abdominal cavity | 2 |
| Bone | 0 |
| Others | 0 |
| Smoking | |
| Negative | 28 |
| Positive | 2 |
Immunohistochemistry
The tissue sections (4 µm) were heated at 65 ℃ for 2 h, dewaxed in xylene, and hydrated using gradient ethanol. Antigen retrieval was carried out by heating the sections in citrate buffer (pH 6.0), and subsequently, the sections were blocked in an aqueous hydrogen peroxide solution for 20 min. Finally, the tissue sections were incubated with antibodies at 4 ℃ overnight. The antibodies used in this study are listed in Table S3. A biotinylated secondary goat anti-mouse/rabbit IgG antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) labeled with streptavidin-horseradish peroxidase (HRP) and a DAB staining kit (Zhongshan Golden Bridge Biotechnology, Beijing, China) were applied according to the manufacturer’s instructions. Positive staining areas were quantified in five fields at 200× magnification.
Multispectral fluorescent immunohistochemistry
For multispectral fluorescent immunohistochemistry, first, a slide was heated to remove residual paraffin via xylene, which was followed by rehydration in graded alcohol. Antigen retrieval was performed with buffer and microwave processing. Subsequently, a blocking solution was applied for blocking purposes. The first primary antibody was then introduced and allowed to incubate. Opal polymer HRP mouse + rabbit (Aifang, Hubei, China) served as the secondary antibody. After the slides were washed, tyramine signal amplification (TSA) dye (Aifang, Hubei, China) was applied at position 1. The slides were microwaved to detach the primary and secondary antibodies before being washed again and subjected to further blocking with a blocking solution. The second primary antibody was applied at position 2 following the same procedure; DAPI staining occurred after unbound DAPI had been washed away. Finally, the slides were covered with ProLong Gold Anti-Quenching Reagent (Aifang, Hubei, China). Five fields from single-color slides were imaged at 200× magnification with StrataQuest Image Analysis software (v. 6.0.1.181) (SAS Institute, Cary, NC, USA) to generate a spectral library for unmixing.
Statistical analysis
All statistical analyses were executed via GraphPad Prism 8.0 software (Dotmatics, Boston, MA, USA). Each experiment was repeated three times. Data are presented as the mean ± standard deviation (SD) unless otherwise stipulated. Statistical intergroup comparisons were accomplished via a two-tailed Student t-test, whereas immunohistochemical scores were analyzed via the chi-squared test. Discrepancies were regarded as statistically significant at P<0.05.
Pattern diagram
Visualization of findings was realized through the drawing tools provided by FigDraw (https://www.figdraw.com/#/paint_msgs).
Results
Elevated expression of CKS2 drives the malignant progression of uterine sarcoma
The most prevalent types of uterine sarcomas are UCS and uLMS. To screen for differential genes that facilitate the malignant progression of uterine sarcomas, we initially employed the GEPIA (http://gepia.cancer-pku.cn/detail.php) database to identify the differential genes between the tumor tissues of UCS and their paired normal samples in TCGA dataset (Figure 1A). Subsequently, we used two GEO databases (GSE64763 and GSE9511) to screen for differential genes between normal uterine tissues, uterine leiomyomas, and uLMS respectively (Figure 1B). We used Venn diagram analysis on these four sets of data and identified eight genes with significant differences (Figure 1C). Among these eight differential genes, only CKS2 was conspicuously upregulated in the tumor tissues of UCS, which increased in degree from normal tissues, to uterine leiomyomas, and to uLMS, respectively, further indicating the crucial role of CKS2 in the genesis and development of uterine sarcoma (Figure 1D,1E). We discovered in TCGA that the high expression of CKS2 was significantly correlated with the malignant progression and adverse prognosis of UCS (Figure 1F). Moreover, the expression level of CKS2 slightly higher in patients at advanced stages than in those at early stages (Figure 1G). However, we concurrently discovered that a high expression of CKS2 was accompanied by a lower abundance of NKT cells, indicating that CKS2 may facilitate the tumor immune evasion in uterine sarcoma (Figure 1H).
Figure 1.
CKS2 can be regarded as a prognostic predictor for uterine sarcoma. (A) Via the GEPIA database, differential genes between the tumor tissues of uterine sarcoma and uterine carcinosarcoma from TCGA dataset and their paired normal tissues were identified. (B) Differential genes were screened in the GEO database according to groupings. (C) The Venn diagram was used to screen the common differentially expressed genes of the four above-mentioned groups. Group A is the differential genes in TCGA as depicted in (A), Group B is the normal myometrium tissue versus uterine fibroid in GSE64763, and Group C is the leiomyosarcoma versus uterine fibroids in GSE64763, and Group D represents the differential genes in the leiomyosarcoma versus uterine fibroids within GSE9511. (D) Verification of the coexpressed genes in tumor and normal tissues in TCGA database. (E) Detection of the expression level of the CKS2 gene in the GEO database. (F,G) The correlation of CKS2 with the prognosis and stage of uterine sarcoma was examined via TCGA database. (H) The correlation between the expression of CKS2 and NKT cells. Red and blue respectively represent the distribution of the number of genes expressed at different levels. **, P<0.01; ***, P<0.001. CI, confidence interval; CKS2, cyclin-dependent kinase subunit 2; FC, fold change; GEO, Gene Expression Omnibus; GEPIA, Gene Expression Profiling Interactive Analysis; HR, hazard ratio; NKT, natural killer T; TCGA, The Cancer Genome Atlas; UCS, uterine carcinosarcoma.
CKS2 knockdown inhibited proliferation and apoptosis while aggravating invasion and migration of uterine sarcoma cells in vitro
To further examine the influence of CKS2 on cellular functions, we employed recombinant lentiviruses carrying the CKS2 shRNA sequence to infect uterine sarcoma cells (MES-SASH-CKS2 and SK-UT-1SH-CKS2). We compared the expression of CKS2 in the control lentivirus-infected and SH-CKS2 lentivirus-infected uterine sarcoma cells, and the outcomes demonstrated that the protein level of CKS2 was markedly decreased in the knockdown group (Figure 2A). Cell colony formation indicated that CKS2 depletion suppressed the clonogenic growth of MES-SA and SK-UT-1 cells (Figure 2B). The TUNEL experiment confirmed that knockdown of CKS2 could promote apoptosis in uterine sarcoma cells (Figure 2C). The Transwell invasion and wound healing assay also indicated that the invasion and migration capabilities of uterine sarcoma cells infected with SH-CKS2 were significantly decreased as compared to those of the control group (Figure 2D,2E). MICA/B plays a crucial role in activating NKT cells and are expressed on the surface of tumor cells, which are regulated by signaling pathways (15). Subsequently, we analyzed the relationship between MICA/B and CKS2 expression using RT-PCR. The results indicated that the downregulation of CKS2 expression was associated with an upregulation of MICA/B in uterine sarcomas, with a significant increase in MICA expression (Figure 2F). Cellular immunofluorescence experiments also confirmed that at the protein level, knockdown of CKS2 could promote the expression of MICA protein (Figure 2G). These results indicate that suppressing the expression of CKS2 can inhibit the proliferation, invasion, and migration of uterine sarcoma, induce apoptosis, and enhance the expression of MICA, resulting in immune activation.
Figure 2.
Knockdown of CKS2 expression could significantly suppress the malignant progression of uterine sarcoma cells. (A) The knockdown efficiency of CKS2 was assessed at the protein level in two UCS cell lines (MES-SA and SK-UT-1) following infection with SH-CKS2 virus and its control virus. (B) Colony-formation assay was used to detect cell proliferation. The crystal violet staining used in the images. (C) TUNEL assay applied to detect cell apoptosis. The results were observed under an inverted fluorescence microscope at 100× magnification. Scale bar: 100 µm. (D,E) Representative images of the Transwell for invasion and wound healing assays for migration. The crystal violet staining used in the Transwell assay. The results were observed under an inverted microscope at 100× magnification. Scale bar:100 µm. (F) Detection of the mRNA expression of MICA/B in CKS2 cells via RT-PCR. (G) Cellular immunofluorescence assay revealed that the decreased expression of CKS2 in cells resulted in the upregulated expression of MICA protein. The results were observed under an inverted fluorescence microscope at 200× magnification. Scale bar: 20 µm. *, P<0.05; **, P<0.01; ***, P<0.001. CKS2, cyclin-dependent kinase subunit 2; MICA, Major histocompatibility complex class I chain-related protein A; RT-PCR, reverse transcription polymerase chain reaction; UCS, uterine carcinosarcoma.
CKS2 regulated MICA protein expression through the PI3K/AKT signaling pathway
We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the differentially expressed genes for the four above-mentioned groups of data and discovered that focal adhesion and the PI3K/AKT pathway were the common signaling pathways (Figure 3A). In pediatric retinoblastoma, it has been found that CKS2 promotes the malignant phenotype of the tumor by regulating the PI3K/AKT pathway associated with the cancer (16). We observed at the protein level that the phosphorylation levels of P-PI3K and P-AKT were markedly decreased after knockdown of CKS2 protein, suggesting that CKS2 regulates the PI3K/AKT signaling pathway (Figure 3B). It has also been reported that the activation of the PI3K/AKT signaling pathway suppresses the expression of MICA protein and subsequently inhibiting the cytotoxic response of NKT cells (17). To verify whether CKS2 regulates MICA expression through the PI3K/AKT signaling pathway, we established cell lines with overexpression of CKS2 and subsequently added the PI3K inhibitor BKM120 to observe the expression of MICA protein (Figure 3C). Subsequently, we examined the impact of PI3K inhibitors on the functionality of CKS2-overexpressing cells. The experimental findings demonstrated that PI3K inhibitors significantly suppressed the augmentation of cell proliferation, invasion, and migration induced by the overexpression of CKS2, and their efficacy was marginally less potent than that resulting from knockdown of CKS2 (Figure 3D-3F). These experimental results indicate that CKS2 promotes cancer via the PI3K/AKT signaling pathway and regulates the expression of MICA protein.
Figure 3.
CKS2 regulated tumor progression and MICA protein expression through the PI3K/AKT pathway. (A) KEGG pathway enrichment was performed for four groups of differential genes. (B) Western blot analysis was used to detect the expression of P-PI3K, total-PI3K (T-PI3K), P-AKT, and total-AKT (T-AKT) proteins. (C) Protein expression was detected after the addition of PI3K inhibitor BKM120 following the overexpression of CKS2 virus. (D) Cell proliferation results. (E,F) Representative images of the Transwell for invasion and wound healing assays for migration. The crystal violet staining used in the Transwell assay. The results were observed under an inverted microscope at 100× magnification. Scale bar: 100 µm. *, P<0.05; **, P<0.01; ***, P<0.001. CKS2, cyclin-dependent kinase subunit 2; KEGG, Kyoto Encyclopedia of Genes and Genomes; OD, optical density.
CKS2 suppressed NKT cell immune surveillance by promoting MICA shedding via the PI3K/AKT pathway
We conducted CKS2 immunohistochemical staining on the 30 collected tissue specimens (10 cases of normal uterine smooth muscle tissues and 20 cases of uterine sarcoma tissues). According to the immunohistochemical scoring outcomes, CKS2 exhibited low expression in the normal uterine myometrial tissues; however, in the uterine sarcoma tissues, 6 cases had low CKS2 expression and 14 cases had high expression (Figure 4A). To determine whether CKS2 regulates MICA expression via the PI3K/AKT pathway, we used multicolor immunofluorescence technology to detect the protein expression in tissues. MICA can be expressed in endothelial cells, dendritic cells, fibroblasts, epithelial cells, and many tumors, but it has low expression in normal tissues (18). We observed that in normal myometrial tissue, CKS2, P-PI3K, and MICA all showed low expression, and the number of NKT cells in the immune microenvironment was reduced. However, in uterine sarcoma tissues with a low expression of CKS2, P-PI3K expression was decreased, MICA expression was increased, and the number of NKT cells was increased; conversely, in tissue with a high expression of CKS2, P-PI3K expression was increased, MICA expression was decreased, and the number of NKT cells was decreased (Figure 4B). NKT cells possess the capability of generating a potent antitumor response (19). Besides directly eliminating tumor cells, they can also secrete proinflammatory cytokines, such as IFN-γ and TNF-α, to inhibit cell proliferation and induce tumor cell apoptosis (20). The levels of IFN-γ and TNF-α were detected in fresh tissues, and combined with the expression of CKS2 as confirmed by immunohistochemistry, the results showed that the higher the expression of CKS2 was, the lower the levels of IFN-γ and TNF-α in its microenvironment (Figure 4C). These findings suggest that CKS2 modulates MICA expression through the PI3K/AKT pathway, suppresses the aggregation of NKT cells, and thereby disrupts the secretion of IFN-γ and TNF-α, resulting in the immune evasion of tumors.
Figure 4.
CKS2 regulated MICA expression and thus interfered with NKT cell infiltration. (A) IHC was applied to detect the expression of CKS2 protein in tissues. The results were observed under an inverted microscope at 100× magnification (scale bar: 50 µm) and 200× magnification (scale bar: 25 µm). (B) Multicolor immunofluorescence was applied to detect the expression of CKS2, P-PI3K, and MICA proteins in tissues, as well as the infiltration of NKT cells. The results were observed under an inverted fluorescence microscope at 200× magnification. Scale bar: 20 µm. (C) Flow cytometry was used to detect the levels of cytokines IFN-γ and TNF-α in fresh tissue samples. NS, not statistically significant; ***, P<0.001. CKS2, cyclin-dependent kinase subunit 2; IFN-γ, interferon-γ; IHC, immunohistochemistry; NKT, natural killer T; TNF-α, Tumor necrosis factor-α.
Discussion
Uterine sarcoma constitutes a rare and heterogeneous group of malignant neoplasms, encompassing various histological subtypes (21). Currently, surgical intervention remains the preferred treatment modality for early-stage tumors; however, the high recurrence rate and unfavorable prognosis continue to pose significant clinical challenges (22). In this study, at the tissue level, the upregulation of CKS2 expression increased in degree across normal uterine tissue, leiomyomas, and leiomyosarcomas, respectively, and was negatively correlated with the number of NKT cells. By constructing a CKS2 knockdown stable transfection virus in uterine sarcoma cells, we found that the reduction of CKS2 could inhibit the proliferation, invasion, and migration of uterine sarcoma cells; induce tumor cell apoptosis; and negatively regulate the protein expression of MICA ligand binding by NKT cells. Next, we found that CKS2 regulates MICA expression through the PI3K/AKT signaling pathway, which in turn affects NKT cell aggregation and leads to immune escape.
Giannini et al. (23) conducted a meta-analysis that compared laparoscopic and abdominal myomectomy, highlighting the perioperative benefits of minimally invasive surgery. However, in the context of uterine sarcoma, these advantages must be weighed against the risk of misdiagnosis and potential tumor dissemination. Given the diagnostic challenges in differentiating sarcoma from benign leiomyoma, molecular markers such as CKS2 may aid in preoperative risk stratification and guide appropriate surgical decision-making to ensure oncologic safety. While no CKS2-specific inhibitors are currently available for clinical use, the development of small-molecule inhibitors or RNA-based strategies targeting CKS2 could provide a novel immunomodulatory approach for patients with uterine sarcoma. Further preclinical studies, including in vivo validation and drug screening, will be essential to evaluate the safety, efficacy, and translational potential of CKS2-targeted therapies.
CKS2 has been found to be highly expressed in various tumor tissues and to participate in regulating the activation of signaling pathways and modulating the malignant progression of tumors (24-26). We discovered, for the first time, that the high expression of CKS2 in uterine sarcoma correlates with a poor prognosis and is involved in regulating immune escape. In our study, we discovered that CKS2 could activate the PI3K/AKT pathway to facilitate the proliferation, invasion, and migration of uterine sarcoma and inhibit apoptosis. The PI3K/AKT pathway is in an activated state in uterine sarcoma and can be involved in regulating tumor invasion (27). Through a public database, we found a negative correlation between CKS2 and the number of NKT cells, which is consistent with research confirming that the PI3K/AKT pathway can inhibit the expression of MICA protein (17), thereby interfering with the activation of NKT cells. At the tissue and cellular levels, we further verified the low expression of CKS2, the decreased phosphorylation levels of P-PI3K and P-AKT, the upregulated expression of MICA protein, and the aggregation of NKT cells. Moreover, after the addition of PI3K inhibitors to the CKS2-overexpressing cell lines, an upregulation of MICA was observed, further confirming that CKS2 can regulate the expression of MICA through the PI3K/AKT pathway and thereby influence the activation of NKT cells. Buparlisib (BKM120), a PI3K inhibitor, has been tested in multiple cancers including UCS, and it has been reported that the PTEN status of the tumor might be a predictive factor for the response to the combination of PI3K inhibitors and chemotherapy (28).
Immune evasion remains a crucial element in the progression of solid tumors (29). NKT cells, as a distinct subset of T cells, possess the capacity to generate potent antitumor responses and can be recognized and activated by MICA/B; they thus have become a key point of interest in the development of effective cancer immunotherapies (30). The intratumoral accumulation of NKT cells is a requisite for the establishment of an immunocompetent tumor microenvironment. Mechanistically, intratumoral NKT cells facilitate the generation of cytokines such as IFN-γ and TNF-α to initiate antitumor adaptive immune responses and orchestrate the intratumoral infiltration of T cells, dendritic cells, natural killer cells, and myeloid-derived suppressor cells (14). The combination of NKT cell infiltration level and PD-1/PD-L1 interaction score has good performance in predicting the immunotherapeutic response of patients with non–small cell lung cancer, which helps to accurately identify patients who may benefit from immunotherapy (31). In this study, we also found that in tumor tissues with a low expression of CKS2, MICA protein was highly expressed, the infiltration of NKT cells in the tumor microenvironment was elevated, and the levels of cytokines IFN-γ and TNF-α were increased. The results showed that CKS2 could regulate NKT cell infiltration through MICA protein, thereby inducing immune escape and aggravating tumor progression.
Although progress has been made in elucidating immune escape mechanisms in uterine sarcoma, key challenges persist. The complexity of the tumor immune microenvironment and the detailed roles of immune regulators like CKS2 are not fully understood. While CKS2 is linked to tumor progression and immune modulation in other cancers, its function in uterine sarcoma immune evasion is largely unexplored. This study addresses this gap by investigating CKS2’s role, highlighting its novelty and potential clinical impact. However, several limitations exist. Our analysis based on TCGA and GEO datasets, supported by in vitro and tissue-level validation, is limited by relatively small sample sizes. The reliance on cell assays and immunofluorescence lacks in vivo and longitudinal clinical data to confirm the physiological relevance of CKS2-mediated NKT cell regulation. Additionally, although the PI3K/AKT pathway was identified as a mechanism for CKS2-driven MICA downregulation, other immune pathways are yet to be explored. Future research with larger cohorts and in vivo models is needed to validate and extend these findings.
Conclusions
In summary, our study suggests that CKS2 may contribute to the malignant progression and immune evasion of uterine sarcoma by downregulating MICA expression through the PI3K/AKT pathway, thereby suppressing NKT cell activity. These findings provide preliminary evidence supporting the potential of CKS2 as a biomarker and a possible therapeutic target in uterine sarcoma. However, further validation in larger cohorts and in vivo models is needed before clinical application.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Ethics Committee of The Affiliated Wuxi People’s Hospital of Nanjing Medical University (No. KY24165) and informed consent was taken from all the patients.
Footnotes
Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1405/rc
Funding: The work was supported by Maternal and Child Health Research Project of Jiangsu Province (No. F202302).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1405/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1405/dss
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