Bladder cancer (BLCA) is the tenth most common malignant tumor globally, with approximately 570,000 new cases and 210,000 deaths yearly (Sung et al., 2021). Approximately 75% of patients with BLCA are diagnosed with non-muscle-invasive bladder cancer (NMIBC), whose standard therapy is transurethral resection of bladder tumor (TURBT) combined with intravesical instillation of chemotherapy or immunotherapy. However, 40% of these patients progress to muscle-invasive bladder cancer (MIBC) within 5 years (Sylvester et al., 2006; Babjuk et al., 2019). Specifically, patients with T1G3 NMIBC have a worse prognosis, with disease progression rates of 11.4% and 19.8%, and disease-specific mortality rates of 4.8% and 11.3% in 1 and 5 years, respectively (Cambier et al., 2016). In addition, patients with MIBC are often associated with lymph node metastasis or extravesical invasiveness. A large-sample study showed that the 5-year recurrence-free survival (RFS) of patients with MIBC was about 60%, and cancer-specific survival (CSS) was about 65% after radical cystectomy and pelvic lymphadenectomy (Shariat et al., 2006). However, the CSS of patients with MIBC progressing from NMIBC descended to 35%. The progression to MIBC and bladder cancer related death in high-risk NMIBC were relatively early events occurring within 48 months (van den Bosch and Alfred Witjes, 2011). The RFS of BLCA depends on the lymph node involvement and tumor stage. Studies have shown that with the progression of the tumor stage, the RFS continues to decline: the 5-year RFS of patients with pT1, pT2, pT3, and pT4 BLCA was 76%, 74%, 52%, and 36%, respectively (Stein et al., 2001). Unfortunately, this aggressive biological behavior, coupled with limited treatment options, resulted in a median survival time of only 15 months for patients with metastatic BLCA (von der Maase et al., 2005). This meant that the current treatment strategies based on patient imaging and histopathology could no longer meet the needs of the concept of precise treatment of BLCA. Therefore, exploring diagnosis and prognosis biomarkers for BLCA has great clinical significance.

In this study, we utilized the weighted gene co-expression network analysis (WGCNA) to explore gene modules exhibiting a highly correlated with tumor clinicopathological features (Zhang and Horvath, 2005; Langfelder and Horvath, 2008). This algorithm has been applied to analyze gene expression data in yeast and mice (Carlson et al., 2006; Miller et al., 2010), and has since been extended to explore the biological significance of human tumor-related genes, such as finding the molecular signature of subtypes of non-small-cell lung cancer and the prognostic biomarkers of breast cancer and BLCA (Tang et al., 2018; Niemira et al., 2019; Liu et al., 2021a).

Thus, our study aimed to identify prognostic biomarkers of bladder urothelial carcinoma using WGCNA and validate their expression characteristics, thereby providing novel targets for precision medicine in BLCA.

More than 1.6 million people live with BLCA worldwide. About 1 in 100 men and 1 in 400 women are diagnosed with BLCA sometime in life globally. BLCA has the highest lifetime treatment costs per patient among all cancer types, with an annual cost of 3.6 billion euro in the United States and almost 5 billion euro in Europe (Richters et al., 2020). Urothelial carcinomas account for 90% of bladder malignant tumors histologically (Lobo et al., 2020). While intravesical Bacillus Calmette-Guerin instillation therapy remains a crucial option for the patients with intermediate and high-risk NMIBC after TURBT, the research into the molecular biology and genetics of BLCA has revolutionized the treatment of MIBC and advanced disease, including immunotherapy and checkpoint inhibition, targeted therapy, and antibody-drug conjugates (Lenis et al., 2020). Therefore, identifying valuable diagnostic, therapeutic, and prognostic targets for BLCA to achieve personalized and precise treatment of BLCA is of clinical significance.

In this study, we utilized bioinformatics analysis to identify ANLN and ASPM genes as prognostic biomarkers for BLCA. The upregulation of ANLN and ASPM genes was observed in BLCA, and their high expression was associated with poor OS in patients with BLCA. The results of RT-qPCR further confirmed the upregulation of ANLN and ASPM in BLCA, which was observed in both MIBC and NMIBC. By comparing the increasing multiples, we found that the increasing multiples of ANLN was higher in high-grade tumors. The proportion of upregulated ANLN and ASPM was 97.1% and 91.4%, respectively, indicating that the upregulation of both genes was a common occurrence in patients with BLCA.

ANLN is located on the short arm of human chromosome 7 and encodes a conserved multi-domain protein that interacts with various biological partners, acting as a scaffold to organize the cytoskeleton (Piekny and Glotzer, 2008). ANLN was originally isolated from Drosophila melanogaster embryonic extract and could bind to F-actin. Therefore, it was also called Anillin actin-binding protein (Field and Alberts, 1995). During cytokinesis, ANLN interacts with regulators of cytoskeletal components such as actin, septins, RhoA, and Ect2, among others, to facilitate cytoplasmic division, morphological changes, and ultimately, cell division into two subunits (Tuan and Lee, 2020). Notably, the injection of antibodies targeting ANLN has been shown to slow furrow ingression and lead to the failure of cytokinesis (Oegema et al., 2000). Similarly, the depletion of ANLN in human or Drosophila cultured cells induces lateral furrow oscillation that fails cytokinesis (Piekny and Maddox, 2010). ANLN has been identified as a upregulated gene in various tumors driving cell proliferation (Tuan and Lee, 2020). In non-small cell lung cancer, inhibition of the ANLN gene expression could suppress tumor cell growth, resulting in larger morphology, multiple nuclei, and ultimately cell death (Suzuki et al., 2005). Knockdown of the ANLN gene in mice hepatocytes blocked cytokinesis and inhibited liver tumor development (Zhang et al., 2018). A study using transcriptome sequencing found that ANLN was significantly upregulated in BLCA tissues (log2FC = 2.926) (Zeng et al., 2017). PCR and immunohistochemical experiments on BLCA and paracancerous normal mucosal tissues showed that the expression of the ANLN gene is upregulated in BLCA, especially in MIBC and high-grade cancers. Meanwhile, patients with BLCA in the high-ANLN expression group showed poor CSS, progression-free survival (PFS) and RFS, compared with the low-ANLN expression group (Zeng et al., 2017). Knockdown of the ANLN gene could inhibit BLCA cell proliferation, migration, and invasion, as well as induce cell cycle arrest and abnormal binucleation (Zeng et al., 2017; Chen et al., 2022). Patients with MIBC exhibiting high expression of the ANLN gene had shorter OS and disease-specific survival after radical cystectomy (Wu et al., 2019). Taken together, these findings suggested that the expression of ANLN gene was upregulated in BLCA and promoted the proliferation and invasion of BLCA cells. High expression of ANLN predicted poor prognosis, which was consistent with our analysis.

ASPM is located on the long arm of human chromosome 1, encoding a large protein of 3,477 amino acids (410 kDa). This protein comprises a putative amino-terminal microtubule-binding domain, two calponin homology domains that commonly involve in actin binding, 81 IQ (isoleucine-glutamine) repeat motifs that potentially bind to calmodulin, and a carboxy-terminus (C-terminal domain) of unknown function (Kouprina et al., 2005; Zhong et al., 2005; Paramasivam et al., 2007). The human ASPM gene produces two splice isoforms of 410 and 250 kDa, enriched in centrosomes and intermediates, respectively. This suggests that these isoforms may undertake the significant cellular functions of ASPM and participate in the regulation of mitosis (Paramasivam et al., 2007). The homologous proteins of human ASPM in Drosophila, asp, is involved in spindle microtubule organization and cytokinesis during mitosis and meiosis. Mutations in the human ASPM gene can lead to the defective proliferation of neural progenitor cells, resulting in autosomal recessive primary microcephaly (MCPH) (Bond et al., 2002; Roberts et al., 2002; Bond et al., 2003). Studies have shown that ASPM participates in spindle organization, spindle positioning, and cytokinesis in all dividing cells, not just nerve cells, and widely expresses in various fetal and adult tissue cells, including liver, lung, kidney (Kouprina et al., 2005; Higgins et al., 2010). The ASPM gene is upregulated in various tumors, including ovarian cancer, breast cancer, colon cancer, glioblastoma, and BLCA (Kouprina et al., 2005; Saleh et al., 2020). Patients with BLCA who have high expression levels of ASPM had shorter OS and PFS. The proliferation rate of the T24 cell line transfected with si-ASPM was significantly lower than that in the si-Control group (Xu Y. et al., 2020). Nude mice receiving ASPM knockdown T24 cells developed smaller tumors with lower tumor proliferative activity, indicating a significant role of ASPM in BLCA proliferation and tumorigenesis (Gao et al., 2020). Additionally, the single-cell transcriptome analysis identified a group of ASPM + basal-like cell types, the number of which significantly increased in acute bladder urothelium injury caused by uropathogenic Escherichia coli. This suggested that ASPM + urothelial cells were involved in the bladder urothelial regeneration after injury (Li et al., 2021). Furthermore, bioinformatics analysis revealed that high expression of the ASPM gene is associated with the poor prognosis of patients with BLCA, indicating that BLCA originating from this ASPM + subpopulation might be highly malignant and invasive (Li et al., 2021).

Spearman correlation analysis revealed a correlation between the mRNA expression of ANLN and ASPM in BLCA and paracancerous tissues. The correlation was higher in paracancerous tissues, indicating that the interaction between ANLN and ASPM also exists in normal tissues. Previous studies have confirmed ANLN and ASPM as risk factors for the prognosis of patients with BLCA through silico data analysis (Chen et al., 2019; Wang et al., 2019; Xu et al., 2019; Li et al., 2020; Liu et al., 2021b). Our study aimed to further discuss the impact of these genes on BLCA. ANLN is a highly conserved multi-domain protein that interacts with multiple cytoskeletal components during cell division (Tuan and Lee, 2020). ASPM participates in regulating microtubules, which are the cytoskeletal fibers involved in various physiological functions of cells (Akhmanova and Steinmetz, 2019). Therefore, both ANLN and ASPM are cytoskeleton-associated proteins involved in cytokinesis (Schiewek et al., 2018). The cytoskeleton plays a crucial role in maintaining the shape and internal organization of cells, as well as in cell division and migration. In particular, cell migration is initiated by an actin-dependent protrusion of the cell’s leading edge, which comprises structures known as lamellipodia and filopodia (Imai-Sumida et al., 2017). Regulating the formation of cytoskeleton and pseudopodia promotes the invasion and metastasis of BLCA (Xu N. et al., 2020). Remodeling of the intracellular cytoskeleton is one of the critical mechanisms of tumorigenesis, as the cytoskeleton directly regulates two major characteristics of cancer cells: proliferation and motility (Jiang et al., 2009; Sagona and Stenmark, 2010; Akhshi et al., 2014; Fife et al., 2014). Human cells contain four major cytoskeletal components: actin, microtubules, intermediate filaments, and septin polymers (Spiliotis and Nelson, 2006; Hohmann and Dehghani, 2019). These components have complex interactions, control cellular organization through signaling crosstalk, and play an essential role in tumor development (Jiang et al., 2009; Sagona and Stenmark, 2010; Akhshi et al., 2014). Studies have shown that down-regulating the RAS-driven actin cytoskeleton and phosphatidylinositide 3-kinase/Akt pathway could induce apoptosis and inhibits cell growth, migration and invasion in human BLCA cells (Imai-Sumida et al., 2017).

In summary, our study identified two related prognostic biomarkers for BLCA: ANLN and ASPM. High expression of ANLN and ASPM was associated with poor OS in BLCA. Furthermore, our results showed that ANLN and ASPM were upregulated in BLCA tissues. We also observed a correlation between the mRNA expression of ANLN and ASPM in both BLCA and paracancerous tissues. Notably, the increasing multiples of ANLN was higher in high-grade BLCA. These results suggest that ANLN and ASPM genes may be potentially promising targets for BLCA. We continue to explore the interaction mechanism between both ANLN and ASPM genes in vitro and in vivo experiments.