ONCOLOGY LETTERS 17: 1865-1876, 2019 Overexpression of the ASPM gene is associated with aggressiveness and poor outcome in bladder cancer ZHENGLIN XU1,2, QI ZHANG3, FRANK LUH4,5, BAIYE JIN1 and XIYONG LIU4,5 1Department of Urology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006; 2Department of Urologic Surgery, Affiliated Dongyang People's Hospital of Wenzhou Medical University, Dongyang, Zhejiang 322100; 3Department of Bioinformatics and Statistics, Hangzhou Hope Biotechnology, Inc., Hangzhou, Zhejiang 310015, P.R. China; 4Sino-American Cancer Foundation and 5Tumor Biomarker Development, California Cancer Institute, Temple City, CA 91780, USA Received April 6, 2018; Accepted October 31, 2018 DOI: 10.3892/ol.2018.9762 Abstract. Abnormal spindle-like microcephaly-associated associated with grade and tumor node metastasis (TNM) (ASPM) protein is essential for mitotic spindle function stage in bladder cancer, based on pooled GEO and TCGA during cell replication. The present study aimed to evaluate datasets (P<0.05). Stratification analysis indicated that the the hypothesis that ASPM serves a critical role in cancer clinical significance of ASPM was particularly pronounced in invasiveness and may act as a prognostic biomarker in low-grade or papillary subtypes of bladder cancer. Individual bladder cancer. In total, 6 independent worldwide bladder Cox and pooled Kaplan-Meier analyses suggested that ASPM cancer microarray mRNA expression datasets (n=1,355) with expression was significantly directly correlated with poor clinical and follow-up annotations were collected from the overall (OS) and progression-free survival (PFS) in bladder Gene Expression Omnibus (GEO) and The Cancer Genome cancer. Multivariate and stratification analyses demonstrated Atlas (TCGA) databases. Reverse transcription-quantitative that the prognostic significance of ASPM was evident in polymerase chain reaction analysis revealed that ASPM mRNA low-grade or papillary bladder cancers, yet not in high-grade expression was higher in bladder cancer tissue compared with or non-papillary subgroups. Increased expression of ASPM adjacent normal bladder mucosae in 10 paired human tissue was associated with poor OS in muscle-invasive bladder samples (P=0.004). ASPM overexpression in human bladder cancer and with poor PFS in non-muscle-invasive bladder cancer samples was consistent with the mRNA expression cancer (P<0.05). Bioinformatics analysis identified the top datasets from GEO and TCGA. Bioinformatics analysis 11 ASPM-related genes on STRING-DB.org. The expression indicated that ASPM mRNA expression was significantly of the majority of these genes was associated with poor outcomes of bladder cancer with statistical significance. Gene set enrichment analysis indicated that the high expression of ASPM could enrich gene signatures involved in mitosis, Correspondence to: Dr Baiye Jin, Department of Urology, The differentiation and metastasis in bladder cancer. Further First Affiliated Hospital, School of Medicine, Zhejiang University, analysis of TCGA datasets indicated that increased ASPM 79 Qinchun Raod, Hangzhou, Zhejiang 310006, P.R. China expression was significantly associated with higher Gleason E‑mail: [email protected] score, T stage, N stage and poor clinical outcome in prostate Dr Xiyong Liu, Sino-American Cancer Foundation, California cancer. It was also significantly associated with late TNM stage Cancer Institute, 4978 Santa Anita Ave, Suite 104, Temple City, and poor PFS in renal cell carcinoma. In summary, ASPM CA 91780, USA may serve as a novel prognostic biomarker for low-grade or E-mail: [email protected] papillary bladder cancer. Abbreviations: ASPM, abnormal spindle-like microcephaly- Introduction associated protein; MIBC, muscle-invasive bladder cancer; NMIBC, non-muscle-invasive bladder cancer; GEO, Gene Expression According to the American Cancer Society, ~81,190 cases Omnibus; TCGA, The Cancer Genome Atlas; GSEA, gene set of bladder cancer (62,380 men and 18,810 women) will be enrichment analysis; OS, overall survival; PFS, progression-free survival; HR, hazard ratio; 95% CI, 95% confidence interval; RCC, diagnosed in the USA in 2018 (1). During the same year, renal cell carcinoma; PCa, prostate cancer ~17,240 bladder cancer-related deaths are expected to occur (12,520 men and 4,720 women) (1). Bladder cancer is the fourth Key words: abnormal spindle-like microcephaly-associated most common cancer in men, but it is less common in women. protein, bladder cancer, prognostic biomarker, cancer survival, gene In general, urothelial carcinoma (transitional cell carcinoma) set enrichment analysis accounts for >90% of all bladder cancers, whereas squamous cell carcinoma accounts for 3-8% and adenocarcinoma for 1-2% of all cases. Approximately 75% of patients are diagnosed 1866 XU et al: ASPM PROMOTES INVASIVENESS OF BLADDER CANCER with non-muscle-invasive bladder cancer (NMIBC) (2), 50-70% corresponding genes was evaluated. Based on these findings, of whom develop disease recurrence and 10-15% develop the prognostic significance of ASPM in bladder cancer was disease progression. Urinary tumor biomarkers, including evaluated. BTA®, NMP22®, UroVysion® FISH and ImmunoCyt™, have been investigated for the purposes of improving the diagnosis, Materials and methods surveillance and staging of bladder cancer (3). However, their low specificity has limited the use of these biomarkers. Analysis of human bladder tissues by reverse transcrip‑ Currently, standard guidelines recommend the use of these tion‑quantitative polymerase chain reaction (RT‑qPCR). Ten biomarkers as an adjunctive surveillance strategy, to reduce the paired bladder cancer tissue samples (6 male and 4 female, age need for invasive cystoscopy (3). Cxbladder is another urinary from 57 to 84) were collected from the Affiliated Dongyan biomarker that measures the mRNA expression profile of five People's Hospital, Wenzhou Medical University (Dongyang, genes (IGF, HOXA, MDK, CDC and IL8R), with a sensitivity China), according to a protocol approved by the Institutional of 93% and specificity of 85%. It has been used to classify high‑ Review Board. Informed consent was obtained from each and low-risk subgroups of patients presenting with hematuria, participant. Total RNA from formalin‑fixed paraffin‑embedded to assess whether invasive cystoscopy is required (4). tissue samples was extracted using an RN30-EASYspin kit Development of bladder cancer occurs via accumulation (Aidab Biotechnologies Co., Ltd., Beijing, China) in order of molecular events. Genetic alterations may result in uncon- to evaluate ASPM mRNA expression. cDNA was produced trolled cellular proliferation, inhibition of differentiation and from 1 µg total RNA using the Promega M-MLV Reverse invasiveness of tumor cells. These changes determine tumor Transcriptase (Promega Corporation, Madison, WI, USA) growth, relapse, progression and metastasis. It is necessary to and oligo dT primers according to the standard protocol. The identify reliable bladder cancer progression-associated genes ASPM primers for qPCR were as follows: Forward, 5'‑AGC in order to improve current methods of detecting and moni- ATT CCT TTT ATC CCA GAA ACA CCTG‑3' and reverse, toring cancer recurrence and metastatic invasion. 5 ' ‑ G C T TGC AGG GGA TTT GTG ATT TCT TCC‑3'. Actin was Abnormal spindle-like microcephaly-associated (ASPM) used as a loading control. The human actin primers were gene, also referred to as abnormal spindle microtubule as follows: Forward, 5'‑CCC CAA CTT GAG ATG TAT GAA assembly, is located on chromosome 1q31 and encodes the GGCT‑3' and reverse, 5'‑TCT CAA GTC AGT GTA CAG GTA ASPM protein. Expression of ASPM is essential for normal AGCC 3'. RT‑qPCR was performed using 1 µl cDNA in a mitotic spindle function in embryonic neuroblasts and 50-µl reaction volume. The thermocycling conditions were as regulation of neurogenesis (5). Defects in the ASPM gene follows: 95˚C for 10 min, followed by 40 cycles at 95˚C for are associated with autosomal recessive primary micro- 10 sec and at 60˚C for 1 min. The experiment was performed cephaly (6,7). Neuronal depletion is associated with ASPM in triplicate. mutations and predominantly affects the anterior cortex during development (8,9). An expression study revealed that Worldwide microarray gene expression datasets. A total of ASPM mRNA levels are higher in fetal tissues, but are very six independent worldwide bladder cancer microarray data- low in adult tissue (10). It has been reported that the ASPM sets were used in the present study. Four Gene Expression gene is overexpressed in glioblastoma and malignant glioma Omnibus (GEO) bladder cancer gene expression datasets were compared with normal brain tissue (11-13). Knockdown of downloaded from www.ebi.ac.uk/arrayexpress; these datasets ASPM by small interfering RNA was shown to reduce tumor included GSE13507 (21), GSE31684 (22), E-MTAB-1803 (23) cell and neural stem cell proliferation (12). ASPM expression is and E-MTAB-4321 (24). Two bladder cancer gene expres- also associated with ependymoma recurrence in children (14). sion datasets, TCGA set1 and TCGA set2, were obtained Abnormalities in ASPM expression are associated with from The Cancer Genomic Atlas (TCGA) research network: numerous cancer types. ASPM mRNA is overexpressed Cancergenome.nih.gov. All datasets contained clinical and in hepatocellular carcinoma (10,15). Upregulation is also follow-up annotations. Datasets without prognostic