Background
Ovarian cancer is a common malignant tumor of the female reproductive system. The Global Cancer Observatory 2020 statistics reported that ovarian cancer causes approximately 313,959 new cases and 207,252 deaths worldwide each year [
1]. Surgery and platinum-based chemotherapy are routine therapy options for ovarian cancer [
2,
3]. Although the prognosis of early-stage ovarian cancer is favorable with treatment, nearly 75% of women present with International Federation of Gynecology and Obstetrics (FIGO) stage III–IV, which is associated to reduced 5-year survival rates [
4]. Therefore, it is important to clarify the mechanism of ovarian tumorigenesis and explore new molecular markers.
The nucleolar and spindle-associated protein 1 (NUSAP1) is a kind of microtubule- and chromatin-binding protein involved in the regulation of spindle formation and stability, chromosome segregation, and cytokinesis [
5,
6]. Its expression peaks when cells transition from the G2 phase of the cell cycle to the mitotic phase [
7]. NUSAP1 is highly expressed in multiple malignant tumors, such as breast cancer [
8], hepatocellular carcinoma [
9], esophageal squamous cell carcinoma [
10], and glioblastoma [
11]. High NUSAP1 expression promotes malignant biological behaviors, such as tumor invasion and metastasis, and is associated with poor prognosis in patients with astrocytoma [
12], breast cancer [
13], colon cancer [
14], and cervical cancer [
15]. In addition, NUSAP1 maintains resistance to the toxic effects of anti-tubulin chemotherapeutics [
16]. Therefore, NUSAP1 has extensive research prospects in cancer. However, its prognostic value and underlying specific molecular involvement in ovarian cancer have not yet been clarified.
Our study aimed to explore the clinical significance of NUSAP1 and provide a new research direction for finding biomarkers of ovarian cancer. This study is the first to report the prognostic value of NUSAP1, as well as its involvement in signaling pathways and correlation with immune cell infiltration in ovarian cancer.
Discussion
Ovarian cancer currently lacks effective early screening and diagnostic methods and is prone to recurrence, metastasis, and drug resistance, resulting in no significant improvement in the 5-year OS of affected patients. Therefore, finding valuable tumor markers and exploring their contribution for ovarian cancer development are crucial for the diagnosis, treatment, and prognosis of patients with ovarian cancer. In this study, the expression of NUSAP1 was validated though multiple databases. IHC assay confirmed that NUSAP1 is highly expressed in ovarian cancer and is an independent risk factor for patient survival prognosis. Furthermore, database analyses indicated molecular mechanisms through which NUSAP1 may be involved in ovarian cancer development.
Numerous studies have confirmed that NUSAP1 is upregulated in breast cancer [
8], hepatocellular carcinoma [
9], and other malignant tumors, and is closely related to malignant biological behaviors, such as cell cycle regulation, invasion, and metastasis. NUSAP1 silencing inhibited DNMT1 expression, leading to the inhibition of liver and colorectal cancer progression [
9]. NUSAP1 promotes the nuclear translocation of GLI family zinc finger 1 and activates the Hedgehog signaling pathway, resulting in increased aggressiveness of astrocytoma [
12]. Li et al. found that NUSAP1 promotes metastasis and induces cancer stem cell-like properties of cervical cancer cells by activating Wnt/β-catenin signals [
15]. Moreover, Xu et al. demonstrated that inhibition of NUSAP1 suppresses cell growth and metastasis by regulating the BTG2/PI3K/Akt signals in non-small-cell lung cancer [
29]. These studies indicate that NUSAP1 is closely related to the occurrence of various tumors, and its high expression may promote the malignant biological behavior of tumors. Recently, Shen et al. proposed six hub genes, including
NUSAP1, to predict the development and prognosis of ovarian cancer based on the Gene Expression Omnibus data. However, this study did not conduct experiments to verify the results of the bioinformatics analysis and evaluate the contribution of each hub gene separately [
30]. Zhang et al. showed that knocking down NUSAP1 in ovarian cancer cells promotes apoptosis and affects the cell cycle distribution [
31]. However, its specific mechanism of function in ovarian cancer has not been clarified. Herein, through the joint analysis of Oncomine, TCGA, and CCLE databases, we found that
NUSAP1 is highly expressed in multiple malignant tumors, and its protein level is correlated with FIGO stage. Results of IHC assay showed that the positive rate of NUSAP1 in ovarian malignant tumors is significantly higher than that in other groups. In addition, a significant correlation between NUSAP1 level and FIGO stage was observed. Further analysis of the expression of NUSAP1 in different histological types of ovarian cancer showed that NUSAP1 had the highest positive rate in ovarian clear cell carcinoma. However, due to the limitation of the number of clear cell carcinoma cases, it is necessary to expand the sample size to further verify the conclusion.
High NUSAP1 expression predicts poor prognosis in astrocytoma [
12], breast cancer [
13], esophageal squamous cell carcinoma [
10], and colon cancer [
14]. However, opposite results were obtained in the prognostic analysis of cervical cancer [
15,
32]. In addition, studies have suggested a correlation between NUSAP1 and chemotherapeutic resistance. In oral squamous cell carcinoma, NUSAP1 downregulation enhances the anti-tumor effect of paclitaxel by activating apoptotic pathways [
33]. Moreover, Zhang et al. reported that knockdown of NUSAP1 increased susceptibility to epirubicin in invasive breast cancer [
34]. Recent investigations showed that NUSAP1 stabilizes the ATR by sumoylation via its SAP domain, thereby promoting chemotherapeutic resistance to temozolomide and doxorubicin [
35]. In this study, we followed up cancer patients for more than 5 years to explore the prognostic value of NUSAP1. The result of Cox regression analysis, which was consistent with the results of bioinformatics analysis, showed that high NUSAP1 expression conferred poor prognosis of patients with ovarian cancer. In addition, NUSAP1 showed an even more significant correlation with prognosis in patients with early-stage. Ovarian serous cancer is the most common pathological type of ovarian cancer, and HGSC is a leading cause of ovarian cancer-related death [
36]. Five years after diagnosis, advanced-stage HGSC cases had a survival rate of 32.1% [
37]. The results of bioinformatics analysis and immunohistochemistry statistics indicated that high expression of NUSAP1 was correlated with poor OS of patients with serous ovarian cancer. However, for the patients with HGSC, our clinical sample analysis results were not consistent with the database, which may be limited by the number of cases and the heterogeneity of the study cohort.
We also explored the molecular mechanism of
NUSAP1 contributing for the tumorigenesis and progression of ovarian cancer. Database analysis showed that
NUSAP1 is involved in the regulation of the DNA repair, homologous recombination, base excision repair, replication fork, cell cycle, DNA replication, and p53 signaling pathway. Studies have confirmed that these pathways are closely related to ovarian cancer. NUSAP1 has been confirmed to help generate microtubules near chromatin, leading to chromatin-induced spindle formation, which is essential for cell proliferation [
38]. In particular, Mills et al. identified NUSAP1 as a substrate for SCF-type E3 ubiquitin ligase during S/G2 phase of the cell cycle and observed a cell cycle regulatory interaction between NUSAP1 and a SUMO (small ubiquitin-like modifier) E3 ligase complex through mass spectrometry-based proteomics [
16,
39]. In addition, inhibition of NUSAP1 expression reduced cyclin A2 and cyclin B1 levels, and induced cell cycle arrest [
10,
15,
40].
Several types of DNA damage repair (DDR) pathways, including nucleotide excision repair, base excision repair, MMR, homologous recombination, and non-homologous end joining, can detect DNA damage and initiate the process of cell repair. Hence, alterations in DDR pathways lead to genomic instability and cancer development [
41]. As an error-free repair mechanism, HRR is a key repair method for DNA double-strand breaks. Epigenetic modifications and mutations in HRR-related genes can cause homologous recombination deficiency, thereby enhancing the sensitivity of patients to PARP inhibitors [
42]. Studies showed that NUSAP1 knockdown reduces the sumoylation level of ATR and cause DNA damage in glioblastoma multiforme cells [
35]. In addition, NUSAP1, similar to BRCA1, plays a role in DNA double-stand break repair via the homologous recombination and single-strand annealing pathways in breast cancer, and it protects BRCA1 from proteasome-mediated degradation [
8]. The combination of NUSAP1 and BRCA1 improves the prognostic power in triple-negative breast cancer compared with the calculation based on age, menstrual status, and lymph node status [
13]. We found that
NUSAP1 is correlated with DNA repair, replication fork and homologous recombination in ovarian cancer. In addition, both the CCLE and cBioPortal database showed a positive correlation between the expression of
NUSAP1 and
BRCA1 in ovarian cancer. Therefore, NUSAP1 in ovarian cancer may participate in HRR by regulating the levels of ATR and BRCA1/2; thus, low expression of NUSAP1 in ovarian cancer implies sensitivity to PARP inhibitors. Studies have shown that the acquisition of a drug resistant phenotype is related to the accumulation of epigenetic variations in tumor suppressor and DNA repair genes [
41]. DNMTs are a class of epigenetic regulatory enzymes, of which DNMT1 is involved in maintaining the methylation status after DNA synthesis and DNMT3A/B are involved in the de novo methylation of DNA [
43]. It was reported that DNMT inhibitors induce a BRCAness phenotype through the downregulation of homologous recombination and NHEJ genes, thereby sensitizing non-small cell lung cancer cells to PARP inhibitors [
44]. In addition, combination of DNMT inhibitors and PARP inhibitors promotes the accumulation of reactive oxygen species (ROS), thereby sensitizing breast and ovarian cancer cells to PARP inhibitors in a ROS-cAMP/PKA-dependent manner [
45]. Therefore, we further evaluated the correlation between
NUSAP1 and methyltransferases, and found that
NUSAP1 was positively correlated with
DNMT1,
DNMT3A and
DNMT3B. Therefore, patients with high expression of NUSAP1 can be treated with the combination of DNMT inhibitors and PARP inhibitors to enhance treatment efficacy and overall survival.
The MMR system is responsible for repairing DNA base mismatches [
46]; thus, MMR gene mutations and hypermethylation in the promoter region will result in microsatellite instability (MSI), which are mostly caused by inactivation of
MLH1,
PMS2,
MSH2, or
MSH6 [
47]. Solid tumors with deficient MMR usually have immunogenicity and extensive T-cell infiltration, which make them more responsive to the treatment with immune checkpoint inhibitors (ICIs) [
48,
49]. Although the response rate to ICIs in ovarian cancer is usually lower than in other cancer types, a proportion of ovarian cancers exhibiting MSI show an increased number of CD3 + and CD8 + tumor infiltrating lymphocytes [
50,
51]. Herein, we analyzed the correlation between
NUSAP1 and key MMR genes, which revealed that
NUSAP1 is correlated with
MSH2 and
MSH6. Furthermore, we analyzed the correlation between
NUSAP1 and immune infiltration level.
NUSAP1 showed a strongest correlation with neutrophil tumor infiltration. Neutrophils are the most abundant white blood cells and are closely related to inflammation; however, the role and characteristics of neutrophils in cancer are controversial. Studies showed that neutrophils have more anti-tumor phenotype during early stages of tumor progression, whereas become more tumorigenic during aggressive stages [
52,
53]. Neutrophils maintain tumor growth by suppressing T cell activation, accelerating gene mutation, tumor cell proliferation, angiogenesis and metastasis [
54]. It has been observed that high infiltration with tumor-associated neutrophils is associated with poor prognosis in multiple cancers [
55]. Comprehensive meta-analysis showed that high neutrophil-to-lymphocyte ratio is associated with shorter PFS and OS in ovarian cancer [
56]. In addition, the infiltration of CD8 + T cells in tumors was found to be associated with favorable prognosis in ovarian cancer [
57]. Therefore, high
NUSAP1 expression may cause increased infiltration of neutrophils and decreased infiltration of CD8 + T cells, leading to the progression and poor prognosis of ovarian cancer. Altogether, NUSAP1 may represent a new biomarker for predicting the efficacy of immunotherapy in ovarian cancer.
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