Mitosis maintains the genetic properties of any multicellular organism throughout entire life cycle. Every time the mother cell divides, the chromosomes are evenly distributed among the daughter cells due to the action of the mitotic spindle [1]. The NDC80 complex which is an extremely conserved kinetochore protein from yeast to humans, is an approximately 57 nm elongated heterotetramer consisting of NDC80 (NDC80P, HEC1, nuclear division cycle 80), NUF2 (NUF2P, NUF2 component of NDC80 kinetochore complex), SPC24 (SPC24P, spindle pole body component 24), and SPC25 (SPC25P, spindle pole body component 25). At its SPC24/SPC25 globular end, the complex connects the centromere-proximal inner kinetochore layer to the microtubule-binding outer kinetochore layer at its NDC80/NUF2 globular end [2, 3]. It is crucial that spindle microtubules attach to the chromosome kinetochores through the NDC80 complex during cell division [4]. NDC80 has been considered as a major player in cancers’ initiation, development, and spread in the last few years. NDC80 was abundantly expressed in various cancers and promoted tumor growth through the reduction of apoptosis and increasing cell cycle efficiency, resulting in poor survival and prognosis of cancer patients [5, 6, 7, 8, 9]. SPC25 overexpression has been found in recent researches to enhance tumor growth and metastasis in hepatocellular carcinoma (HCC), as well as a worse prognosis for HCC patients [10, 11, 12, 13]. The same conclusion appears in the study of lung cancer [14, 15, 16], prostate cancer (PCa) [17, 18], breast cancer (BC) [19], and neurodegeneration [20]. SPC25 is also necessary for chromosomal alignment, spindle formation, and effective spindle checkpoint signaling during meiosis, according to research [21].

The number of deaths linked to endometrial cancer (EC) is rising, with an estimated 1–2% yearly increase in the disease’s incidence. But no fresh medications were authorized for the treatment of EC. There are currently only two Food and Drug Administration (FDA)-approved medications for the treatment of EC, despite the fact that numerous medications are approved for the treatment of ovarian, fallopian tube, and primary peritoneal cancers. This highlights the need for novel therapies to treat advanced, recurrent, and metastatic EC [22].

Our research goal was to determine SPC25’s differential expression, prognostic value, biological functions, and its potential in immune infiltration in uterine corpus endometrial carcinoma (UCEC) by providing a more systematic and comprehensive perspective. Thus, we looked into the associations between SPC25’s expression and various clinicopathological features, clinical outcomes in various clinical subgroups, and coexpression network analyses in UCEC. Moreover, we focused on the methylation level of SPC25 in UCEC and its corresponding predictive significance, and explored the potential mechanism of SPC25 expression and methylation status in UCEC progression.

In our results, SPC25 was shown to be significantly associated with clinical stage, histologic grade, histological type, radiation therapy, tumor invasion, weight, primary therapy outcome, molecular subtype, immune subtype, and TP53 muation status. Additionally, elevated SPC25 expression might result in poorer overall survival (OS), disease-specific survival (DSS), and progress free interval (PFI), especially in distinct clinical subgroups of UCEC. Furthermore, we also obtained the SPC25 co-expressed genes, which included 1981 positively correlated genes and 2213 negatively correlated genes. These genes primarily took part in chromosome segregation, DNA replication, cell cycle checkpoint, spindle organization, and negative regulation of cell cycle process. Co-expressed genes were enriched in Cell cycle, Spliceosome, DNA replication, RNA transport, and Oocyte meiosis. Most importantly, the majority of the CpG sites in the SPC25 DNA sequence were hypomethylated, which decreased patient survival and had a negative correlation with immunosuppressive state in the tumor microenvironment (TME). Therefore, it was hypothesized that hypomethylation of the SPC25 promoter caused the overexpression of SPC25 mRNA and protein, and this in turn decreased the expression of chemokines and their receptors, which reduced the aggregation of major immune cells to the tumor site and exerted antitumor immunity, ultimately worsening the disease and shortening the patient’s survival time.

Based on these findings, we concluded that SPC25 would modify the composition and immunological processes of TME, which in turn might alter antitumor immunity and impact the course and outcomes of disease. So we speculated that SPC25 might be a significant prognostic biomarker and that SPC25 might work as a potential molecular biomarker to predict immunotherapy response or a novel anticancer immunotherapy target to achieve promising treatment outcomes in UCEC.

SPC25 is an significant kinetochore component that plays an indispensable role in normal mitosis. The lack of SPC25 results in mitotic abnormality, followed cell death and the loss of SPC25 also causes multiple spindle aberrations, including spindle elongation, multipolarity, and fracture. In the case where there is no SPC25, MAD1 and HEC1 fail to correctly localize at the kinetochores during mitosis [38].

Up to now, some scholars have published some research results on SPC25. For instance, SPC25 could serve as a potential tumor-promoting factor, a metastasis promoter, a useful prognostic indicator, and a new therapy target for HCC. SPC25 mRNA expression was shown to be elevated in HCC, and greater levels of SPC25 expression were linked to a worse prognosis. SPC25 accelerated the cell cycle, allowing HCC cells to proliferate in vitro and tumor growth in vivo. In vitro, knocking down or silencing of SPC25 led to a considerable reduction in HCC cell proliferation and metastasis, a marked inhibition of invasion and migration, and increased protein level of p53 pathway components. SPC25 might promote proliferation and metastasis of HCC via p53 or via activating the FAK/PI3K/AKT signaling pathway through ITGB4 [10, 11, 12, 13].

SPC25 was considerably enhanced in lung adenocarcinoma (LUAD) tissue relative to normal lung tissue, increased cancer stem-like cell (CSC) properties and A549 cell invasion, and was an independent predictive factor for LUAD patients’ poor OS and recurrence free survival (RFS). Additionally, lung cancer cell proliferation requires SPC25 homologs expressed in extracellular matrix (ECM) stiffening. In idiopathic pulmonary fibrosis (IPF-LC), because CADM1 and SPC25 were implicated in transforming growth factor-beta 1 (TGF-beta 1) signaling, gene mutations in these two genes resulted in reduced CADM1 expression and increased SPC25 expression in lung cancer cells, revealing TGF-beta 1-induced epithelial-mesenchymal transition and cell proliferation [14, 15, 16].

In PCa, SPC25 was shown to be highly elevated. SPC25 knockdown decreased proliferation and accelerated apoptosis in PCa cells, resulting in a drop in the number of S phase cells and an increase in the number of G2/M phase cells. SPC25 also has several functional functions in regulating cell proliferation, apoptosis, invasion, transforming growth factor-beta signaling, and SUMOylation pathways in PCa, according to bioinformatic study. SPC25 was upregulated in advanced PCa, and PrC patients with higher SPC25 have lower OS than those with lower SPC25. SPC25+ cells developed considerably more tumorspheres in vitro culture than SPC25- cells, seemed to be more resistant to docetaxel-induced cell apoptosis, and created bigger tumors with a greater frequency after repeated adoptive transplantation than SPC25- cells [17, 18].

SPC25 level was higher in more aggressive BC cell subtypes, and BC patients had a worse prognosis when SPC25 was expressed to a greater extent. SPC25 increased BC cell proliferation, as evidenced by colony formation and CCK-8 experiment. In addition, treatment with DNA methyltransferase inhibitors and transcription factor inhibitors targeting SPC25 might improve prognosis in BC patients. The combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate significantly reduced the abundance of CSC, blocked CSC tumorigenicity, attenuated breast tumor growth, and improved OS in the MMTV-Neu-Tg mouse model, likely because growth-promoting signaling molecules such RAD51AP1 and SPC25 were prevented when chromatin modifiers were inhibited [19].

Specific depletion of SPC25 in microglia might prevent Alzheimer’s disease (AD) development by inhibiting microglia outgrowth [20]. During meiosis, SPC25 is needed for chromosomal alignment, spindle formation, and appropriate spindle checkpoint signaling [21].

However, as far as we know, there is currently no research that can fully assess the importance of SPC25 in UCEC. Therefore we made an effort to fill the research gap of SPC25 in UCEC. In our findings, SPC25 mRNA and protein expression were shown to be elevated in UCEC tissues and substantially connected with clinical stage, histologic grade, histological type, radiation therapy, tumor invasion, weight, primary therapy outcome, molecular subtype, immune subtype, and TP53 muation status in UCEC. Taken together, SPC25 was upregulated in UCEC and might act as a pivotal player in the carcinogenesis of UCEC.

Following that, we observed that increased SPC25 expression could lead to poorer OS, RFS, DSS, and PFI of UCEC patients. In addition, elevated SPC25 was associated with poorer OS, DSS, and PFI in a number of clinical subgroups of UCEC, yet cause a worse all of the OS, DSS, and PFI only in race (white), age $>$60, height $>$160, weight $>$80, BMI $>$30, menopause status (post), clinical stage (stage III), diabetes (No), and radiation therapy (No). It is of great importance that for the first time, we examined the relationships between SPC25 expression and various prognostic (OS, DSS, and PFI) in several clinical subgroups of UCEC.

SPC25 coexpression network analyses in UCEC pointed out that SPC25 regulated the cell cycle of tumor cells in the TME, which was in line with findings from other studies.

Tumorigenesis is significantly influenced by DNA methylation, a common epigenetic mechanism. Changes in the methylation status of several genes have been connected to the initiation, growth, and advancement of various malignancies [39, 40]. Consequently, we investigated the promoter methylation status of SPC25 in UCEC utilizing the UALCAN database. We discovered that the DNA methylation levels of SPC25 in cancer tissues were noticeably lower than those in healthy samples, indicating that SPC25’s overexpression in UCEC was caused by a low degree of promoter methylation status. Our research also showed that high-grade UCEC commonly had lower methylation levels of SPC25, which might indicate that the pattern alteration of SPC25 methylation encouraged the advancement of UCEC. Besides, we looked at the association between the SPC25 gene’s methylation levels and the prognosis of UCEC patients. UCEC patients who had hypomethylation at two CpG sites, including cg22278106 and cg15237047, had an excellent OS rate. However, UCEC patients’ poor OS was linked to the hypomethylation of two CpG sites, namely cg07224215 and cg20609092. All four of these CpG sites saw a reduction in methylation. Our research yielded two contradicting results. Our hypothesis was that hypomeylation of the final two CpG sites had a greater impact on the prognosis of UCEC patients than did the first two. This suggested that the latter two CpG sites could be future therapeutic targets for reducing SPC25 expression and improving the UCEC patients’ prognosis. In general, we discovered that the majority of methylation sites in the DNA sequences of SPC25 were hypomethylated in UCEC, and the degree of methylation was connected with patient outcomes. These findings revealed that SPC25 methylation levels functioned as an efficient predictive biomarker for UCEC, suggesting that SPC25 might play a crucial role in tumor progression.

Initial theories presupposed that an effective immune response might have an antitumor impact, but cancer cells have evolved a number of mechanisms, such as impaired antigen presentation and the recruitment of immunosuppressive cells, which encourage tumors to avoid the attack of immune cells. According to earlier research, immune infiltration can have an impact on a patient’s prognosis, and tumor-infiltrating lymphocyte grade is an independent predictor of sentinel lymph node status in patients with tumors [41]. In this study, SPC25 was shown to be substantially expressed in UCEC and linked to a poor outcome for those patients with UCEC. We also found that SPC25 expression had a clearly positive association with Th2 cells, T helper cells, and Th1 cells. Conversely, SPC25 was visibly negatively correlated with T cells, CTL, NK cells, neutrophils, iDC, and pDC.

The T helper cell-expressed TGF is a key player in the resistance mechanisms against cancer immunotherapy. An efficient tissue-level cancer defense response is elicited by targeted TGF- signaling inhibition in helper T cells, which can serve as a foundation for therapeutics focused on the cancer environment [42]. Th2 cells can turn macrophages into tumor-promoting cells and change the immune response from a cytotoxic to a supportive function when they are present in the TME [43]. Myeloma cell growth and function were markedly suppressed by CTL and Th1 cells, but Th2 cells promoted myeloma cell proliferation and cytokine secretion. In multiple myeloma, CTL and Th1 responses are advantageous and will result in tumor elimination after immunotherapy. In contrast, a Th2 response offers little protection and could even hasten the growth of tumors in vivo [44]. Effector cells of the adaptive immune system are CD8${}^{+}$ CTL cells, which precisely identify and eliminate cancer cells through apoptosis that is mediated by perforin and granzyme [45]. T cell depletion is a significant barrier to successful immunotherapy [46]. Cytotoxic lymphocytes called NK cells may obliterate stressed cells even in the absence of an antigen presentation. NK cells identify and eradicate tumor cells through “missing-self” activation (loss of healthy cell markers) or “stress-induced” activation (gain of stressed cell markers) [47]. Through triggering T cell responses against the tumor cells and directly lysing tumor cells, tumor-associated neutrophils perform a crucial anti-tumor function [48]. By cross-presenting foreign antigens, dendritic cells (DCs) play a crucial role in CD8${}^{+}$ T cell activation and producing anti-tumor CD8${}^{+}$ T cell immunity [49].

Most findings state that the immunogenicity of the malignancies influences the effectiveness of immune checkpoint blockade [50]. Low immunogenicity cancers tend to respond poorly to immune checkpoint-blocking treatment because T cell infiltration by malignancies is relatively small [51]. In order to preserve self-tolerance and avoid autoimmune disease, immune checkpoint molecules normally downregulate activation signals from costimulatory molecules. This method, however, might be used by tumor cells to prevent T cells from becoming activated and functioning normally, which results in T-cell depletion, tumor immune escape, and aggressive tumor growth. In our finding, we confirmed that the SPC25 expression levels were evidently negatively correlated with immune stimulators, such as CD27, CD40LG, CD276, HHLA2, TMIGD2, TNFRSF4, TNFRSF14, TNFRSF18, TNFRSF25, and TNFSF14. On the contrary, SPC25 was distinctly positively associated with immune inhibitors, including LAG3 and PDCD1LG2.

When it comes to the directed migration of immune cells, chemokines and their receptors are crucial [52, 53, 54]. In this study, we foud that SPC25 was evidently negatively correlated with chemokines and their receptors: CCL14, CX3CL1, CCR3, CCR4, CCR5, CCR6, and CX3CR1, suggesting that High SPC25 expression might prevent immune cells from migrating into the TME. Studies show that CCL14’s active form binds to CCR1, CCR3, and CCR5 to encourage the chemotaxis of monocytes, eosinophils, and T lymphocytes [55, 56]. The inflammatory receptor CCR4 stimulates the respiratory burst and phagocytosis of macrophages [57] and regulates the differentiation of M1/M2 macrophages [58]. B cell integrin-mediated adhesion is regulated by CCR6 [59, 60]. White blood cell survival and NK cell activation are both regulated by CX3CR1 [61], which might explain how SPC25 regulates immune infiltration in UCEC.

These findings suggested the possibility of tumor immune escape and antitumor immunity being implicated in the SPC25-mediated carcinogenic mechanisms of UCEC.

Tumor immunogenicity and immune cells inside the TME will be impacted by dysregulated epigenome DNA methylation [62]. We examined the connection between SPC25’s methylation status and immune infiltration in order to decipher the mechanism by which SPC25 hypomethylation promotes the progression of UCEC. Our data showed that SPC25’s methylation status was positively correlated with immune cells, immunostimulatory factors, chemokine and chemokine receptors while negatively connected with immunoinhibitors. This suggested that SPC25’s hypomethylation might contribute to immunosuppression and promote tumor progression in UCEC, which could assist to explain the SPC25 low methylation status in high-grade UCEC tumors and poor prognosis of corresponding UCEC patients. Thus, SPC25 methylation might serve as an indicator of cancer immune infiltration and potential predictor of UCEC patient response to immunotherapeutic drugs.

In conclusion, the mRNA and protein of SPC25 were overexpressed in UCEC tumor tissues and associated with a variety of clinicopathological features. The expression and methylation of the SPC25 gene were related to the prognosis of UCEC patients. SPC25 expression levels and methylation status not only correlated with the extent to which various immune cell types were infiltrating the tumor, but also had a relationship with immunomodulators and chemokines, which might have an impact on how well immunotherapy works in UCEC patients. By modifying the expression of genes involved in cell cycle and immune response, SPC25 controlled the course of UCEC. Therefore, SPC25 was a valuable prognostic biomarker for UCEC, as well as a possible therapeutic target. However, further research is required to substantiate our findings.

However, even with our detailed and systematic research of SPC25 and cross-verification using many databases, we must admit that our current research has a number of potential limitations.

On the one hand, we explored SPC25 gene only using the public databases such as TCGA and GEO yet lacking actual clinical data. And there were variations in microarray and sequencing data from various databases, which could lead to systematic bias. Thus, further in vivo/in vitro verification experiments and follow-up multicenter, large-sample, and prospective studies should be performed to provide precise verification and high-quality evidence in order to investigate if there’s a link between SPC25 and patient survival, and to find more effective antitumor immunotherapy techniques.

On the other hand, although concluding that SPC25 expression was significantly correlated with immune cell infiltration and cancer patients’ survival, we lacked direct evidence of SPC25 influencing prognosis despite its involvement in the regulation of immune cell infiltration. In other words, the processes by which SPC25 regulates the immune system as well as its precise route remain unknown and need further study. We also need sufficient and accurate clinical trial data to to determine the advantages of anti-SPC25 medicines in inhibiting tumor growth or improve patient prognosis.

In conclusion, the discovery of SPC25 investigation in UCEC might provide a integrative understanding of its crucial function in tumor promotion and suppression, and add a comprehensive analytical basis for in-depth validation of molecular biology experiments, and even for future clinical applications of cancer therapy.

NDC80, nuclear division cycle 80; NUF2, NUF2 component of NDC80 kinetochore complex; SPC24, spindle pole body component 24; SPC25, spindle pole body component 25; HCC, hepatocellular carcinoma; PCa, prostate cancer; BC, breast cancer; EC, endometrial cancer; UCEC, uterine corpus endometrial carcinoma; OS, overall survival; DSS, disease-specific survival; PFI, progress free interval; TME, tumor microenvironment; GEO, gene expression omnibus; UALCAN, the university of alabama at birmingham cancer data analysis portal; TCGA, the cancer genome atlas; HPA, the human protein atlas; AUC, area under the curve; KM, kaplan-meier; GSEA, gene set enrichment analysis; IHC, immunohistochemistry; FPKM, fragments per kilobase per million; TPM, transcripts per million; ROC, receiver operating characteristic curve; FDR, false discovery rate; TPR, True Positive Rate; RFS, recurrence free survival; GSEA, gene set enrichment analysis; GO_BP, gene ontology biological process; KEGG, kyoto encyclopedia of genes and genomes; STRING, search tool for the retrieval of interacting genes/proteins; Th1, T helper 1 cell; Th2, T helper 2 cell; Th17, T helper 17 cell; Treg, regulatory T cells; Tgd, gamma delta ($\gamma$$\delta$) T cells; Tcm, central memory T cells; Tfh, T follicular helper cells; CTL, cytotoxic T cell; NK cells, natural killer cells; pDC, plasmacytoid dendritic cell; iDC, immature dendritic cell; LUAD, lung adenocarcinoma; CSC, cancer stem-like cell; RFS, recurrence-free survival; ECM, extracellular matrix; IPF-LC, idiopathic pulmonary fibrosis; TGF, transforming growth factor; AD, Alzheimer’s disease; DCs, dendritic cells.

The datasets generated and/or analysed during the current study are available in the TCGA database (https://portal.gdc.cancer.gov/) and GEO database (https://www.ncbi.nlm.nih.gov/geo).

YZG and LXL were responsible for the design of the study protocol. XX and SZ contributed to the writing of the study protocol and assisted in completing statistical analysis and picture drawing. LXL collected surgical specimens and performed Western blotting experiments. MZ downloaded the data, designed the experimental scheme, drew the pictures and performed statistical analyses. LXL performed the statistical analysis, drew the pictures and wrote the first draft of the manuscript. MZ also assisted LXL in writing the first draft. All authors contributed to manuscript revision, read, and approved the submitted version. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Approval was granted by the Ethics Committee of the Second Hospital of Lanzhou University (2023A-453). This study was performed in line with the principles of the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. TCGA and GEO databases are publicly available and written informed consent was obtained from the patients prior to data collection.

Not applicable.

This study was funded by Distant Cause Development Fund (grant number 071100136).

The authors declare no conflict of interest.