High expression of spindle and kinetochore- associated protein 1 predicts early recurrence and progression of non-muscle invasive bladder cancer

Abstract

BACKGROUND:

Spindle and kinetochore-associated protein 1 (SKA1) is a component of SKA, which is essential for proper chromosome segregation. Recently, SKA1 was found to be over-expressed in several types of human cancers. However, reports on the relationship between SKA1 expression and the prognosis of bladder cancer, in particular, are lacking.

OBJECTIVES:

To clarify the clinical significance of SKA1 as a prognostic biomarker for early recurrence and progression of patients with non-muscle invasive bladder cancer (NMIBC).

METHODS:

The differential expression levels of SKA1 of 148 NMIBC tissues were determined by immunohistochemical staining. Quantitative real-time PCR and western blot analysis were further performed to confirm the immunohistochemistry results. Recurrence and progression free interval were assessed by Kaplan-Meier method and differences between groups calculated by log-rank statistics. The prognostic value of SKA1 for early recurrence and progression was analyzed by multivariate Cox proportional hazard regression models.

RESULTS:

SKA1 expression was significantly different in various NMIBC tissues. Kaplan-Meier analysis revealed that patients with high SKA1 expression showed high early recurrence ( $p<$ 0.001) and progression ( $p<$ 0.05) rates. Although univariate Cox regression analysis revealed that several other factors had an impact on recurrence and progression, upon multivariate analysis, high SKA1expression was the only independent predictor for early recurrence (hazards ratio [HR], 0.246; 95% confidence interval [CI], 0.131–0.461; $p=$ 0.000) and progression (HR, 0.194; 95% CI, 0.052–0.715; $p=$ 0.014).

CONCLUSIONS:

High SKA1 expression is associated with early recurrence and progression in patients with NMIBC, indicating SKA1 may serve as a promising prognostic biomarker for this disease.

1. Introduction

Bladder cancer (BC) is the most commonly occurring urinary malignant tumor with an estimated 76,960 new cases and 16,390 deaths in the United States in 2016 alone [1]. It is a heterogeneous disease with various clinical outcomes. Approximately 75% of newly diagnosed BC cases are non-muscle invasive BC (NMIBC) that show a high rate of recurrence (50–70%) and progression (10–30%) despite local therapy. The remaining BC cases present with muscle invasion and have poor outcomes despite systemic therapy [2]. The transurethral resection of bladder tumors (TURBT) and postoperative intra-vesical instillation therapy are the major recommendations for the treatment of NMIBC [3]. Including the cost of surveillance and treatment of recurrences, BC is the ninth most costly cancer in the United States [4]. Although several biomarkers and clinical parameters exist that can be used as prognostic indicators, reliable predictive indicators for the recurrence and progression of NMIBC are lacking [5]. Thus, specific molecular markers for assessing the risk of recurrence and the progression of primary NMIBC are needed urgently.

Spindle and kinetochore-associated protein 1 (SKA1) was first discovered as human mitotic spindle component in 2005 [6]. SKA1, along with SKA2 and SKA3, constitutes the SKA complex, which plays a key role in the stability of kinetochore-microtubule structure and is essential for stabilizing kinetochore-spindle microtubule attachment during mitosis [7]. Depletion of SKA1 leads to critical defects in chromosome segregation, while its over-expression results in nucleation of interphase microtubules [8]. Moreover, depletion of the SKA1 and SKA2 complex causes cells to maintain a prolonged metaphase-like state characterized by occasional loss of individual chromosomes and the persistent activation of the spindle checkpoint [9]. Depletion of SKA3 leads to premature loss of sister chromatid cohesion [10]. Extensive depletion of all three SKAs causes a chromosome congression defect [11]. Several reports have described how SKA1 was found to be over-expressed in several human cancers, including prostate cancer [8], oral adenosquamous carcinoma [12], hepatocellular carcinoma [13], gastric cancer [14], and non-small cell lung cancer [15]. Previously, we have shown that the down-regulation of SKA1 using an RNA interference lentivirus system in human BC cell lines inhibited cell proliferation and impaired the ability to form colonies [16]. However, only one report exists describing the relationship between SKA1 expression and a cancer prognosis, in this case papillary thyroid carcinoma [17]. To our knowledge, the association between SKA1 expression and the prognosis of human NMIBC has not been described. To investigate the role of SKA1 in the recurrence and progression of NMIBC, we examined the expression of SKA1 and analyzed the relationship between SKA1 expression and clinicopathological factors of NMIBC.

2. Materials and methods

2.1 Patients and tissue samples

The study was approved by the Research Ethics Committee of the Second Hospital of Dalian Medical University, Dalian, China. Informed consent was obtained from all patients. All specimens were handled and made anonymous according to accepted ethical and legal standards.

Consecutive paraffin-embedded and fresh samples were collected from 148 patients with BC admitted to our hospital between January 2010 and December 2011. Fresh tissue specimens were harvested and placed immediately in liquid nitrogen, and stored until the isolation of RNA and protein. All cases were diagnosed as transitional cell carcinomas; other histological variants were excluded. The diagnosis and the histological grade of the BC of each case were confirmed by two pathologists. The grade and stage of the BC of each patient were defined according to the World Health Organization 1973 criteria for grade and the 2002 TNM classification system for stage. None of the patients received preoperative chemotherapy or radiotherapy. Treatment results were assessed by cystoscopy and urinary cytology. Follow-up information was obtained from the medical records of patients who fulfilled inclusion criteria including tumor stage, grade, size, tumor number, and the development of tumor recurrence. Briefly, patients were assessed postoperatively every 3 months for the first 2 years, and then every 6 months thereafter. Recurrence was defined as the relapse of a primary NMIBC having a lower or equivalent pathological stage and grade, and progression as disease having a higher TNM stage or grade when relapsed.

2.2 Immunohistochemistry analysis

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue, which was cut into 5- $\mu$ m thick sections. The sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions and distilled water. Endogenous peroxidase activity was blocked through incubation with methanol containing 0.3% hydrogen peroxide for 20 min. After rinsing in phosphate-buffered saline (PBS), the sections were heated in an autoclave at 120 ${}^{\circ }$ C for 2 min in citric acid buffer (0.01 M, pH $=$ 6.0) for antigen retrieval. The sections were incubated overnight at 4 ${}^{\circ }$ C with primary monoclonal anti-human SKA1 antibody (rabbit anti-SKA1 antibody; 1:500; Sigma Chemical Co., St. Louis, MO, USA). After washing with PBS, the slides were incubated with pre-diluted secondary antibody, followed by further incubation with diaminobenzidine. Negative controls were stained without primary antibody during the immunohistochemistry assay. The slides were evaluated by two independent pathologists using a double-blind method. SKA1 expression was quantified using a visual grading system, as reported previously [16]. Briefly, SKA1 expression was based on the extent of staining (percentage of positive tumor cells graded on a scale from 0 to 3: 0 $=$ negative, 1 $=$ 1–30%, 2 $=$ 31–60%, and 3 $>$ 60%) and the intensity of staining (graded on a scale of 0–3: 0 $=$ none, 1 $=$ weak staining, 2 $=$ moderate staining, and 3 $=$ strong staining). The combination of the extent (E) and intensity (I) of staining was obtained by the product of E $\times$ I, labeled EI, and varied from 0 to 9 for each area. The mean EI score was calculated for each BC specimen. EI scores of 0–3 were considered low expression and EI scores $>$ 3 were considered high expression.

2.3 RNA extraction and quantitative real-time PCR assays

Total RNA was extracted from frozen tissue samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Total RNA (3 $\mu$ g) was reverse transcribed to cDNA using a First Strand cDNA Synthesis kit (Takara; Kusatsu, Japan). The method of PCR has been described previously [16]. In brief, the PCR cycling profile was as follows: denaturation at 94 ${}^{\circ }$ C for 2 min, followed by 35 cycles of annealing at 94 ${}^{\circ }$ C for 30 s, and extension at 72 ${}^{\circ }$ C for 1 min, followed by 10 min at 72 ${}^{\circ }$ C. The expression of mRNA was examined by quantitative real-time (qRT)-PCR with a SYBR Premix Taq and Applied Biosystems 7500 Sequence Detection system (ThermoFisher Scientific, Waltham, MA, USA). The relative expression levels of mRNA were normalized to GAPDH expression and the amplification results for qRT-PCR were calculated using the 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method. The PCR primers used were SKA1 (forward): 5-TGATGTGCCAGGAAGGTGAC-3, SKA1 (reverse): 5-CAAAGGATACAGATGAACAACAGC-3, and GAPDH (forward): 5-GTGGACATCCGCAAA GAC-3, GAPDH (reverse): 5-AAAGGGTGTAACGC AACTA-3 [15, 16, 17]. All primers were verified using Primer Express Software Version 2.0 (ThermoFisher Scientific).

2.4 Western blot analysis

Sample tissues were homogenized on ice in 10 mM Tris buffer (pH 7.4) with 1 mM EDTA and Roche complete protease inhibitor cocktail (PhosSTOP EASYpack; Sigma-Aldrich, St Louis, MI, USA), centrifuged at 10,000 $\times$ g for 30 min and then the concentration was determined by Bradford assay (BioRad, Hercules, CA, USA). In brief, protein samples were separated on 12% polyacrylamide gels and transferred to polyvinylidene difluoride membranes; 5% skim milk powder solution was added, and after a 1 h incubation at room temperature, transferred proteins were incubated with primary antibodies (1:500 dilution SKA1 and GAPDH; Sigma-Aldrich) at 4 ${}^{\circ }$ C overnight. Goat anti-rabbit IgG secondary antibody (Santa Cruz Technology, Santa Cruz, CA, USA) labeled with horseradish peroxidase and diluted at 1:1000 was added and incubated at room temperature for 1.5 h. The membrane was washed three times in Tris-buffered saline/Tween 20. The relative level of SKA1 protein was determined by the grayscale value of various protein bands and the ratio of the grayscale value of SKA1 to GAPDH protein.

2.5 Statistical analysis

The chi-square test was used to analyze the association between SKA1 expression and clinicopathological parameters of BC. The Kaplan-Meier method was used to assess time to recurrence and progression, with differences assessed using a log-rank test. The prognostic value of SKA1 for recurrence and progression was analyzed by multivariate Cox proportional hazard regression models. Statistical analysis was done with SPSS 17.0 software. All statistical tests were two-tailed, and statistical significance was assumed for $p<$ 0.05.

Table 1

Correlation between SKA1and the clinicopathological features of enrolled patients

Characteristics	Total		Low expression		High expression		${\chi }^2$	$p$ value
	N	(%)	N	(%)	N	(%)
No.	148		79	53.4	69	46.6
Gender							2.136	0.144
Male	119	80.4	60	50.4	59	49.6
Female	29	19.6	19	65.5	10	34.5
Age (years)							0.149	0.699
$<$ 65	69	46.6	38	55.9	31	44.1
$⩾$ 65	79	53.4	41	51.9	38	48.1
Stage							0.023	0.878
Ta	42	28.4	22	52.4	20	47.6
T1	106	71.6	57	53.8	49	46.2
Grade							0.064	0.800
Low	81	54.7	44	54.3	37	45.7
High	67	45.3	35	52.2	32	47.8
Tumor number							1.827	0.177
Unifocal	88	59.5	51	58.0	37	42.0
Multifocal	60	40.5	28	46.7	32	53.3
Tumor size							8.054	0.05
$<$ 3 cm	108	73.0	50	46.3	58	53.7
$⩾$ 3 cm	40	27.0	29	72.5	11	27.5
Recurrence							21.02	0
No	97	65.5	65	67.0	32	33.0
Yes	51	34.5	14	27.5	37	72.5
Progression							6.343	0.012
No	134	90.5	76	56.7	58	43.3
Yes	14	9.5	3	21.4	11	78.6

Figure 1.

SKA1 expression in NMIBC tumor tissues from different patient groups. (A) Immunohistochemical staining of SKA1 expression. a and b Low SKA1 expression in NMIBC tissue; c and d High SKA1 exprssion in NMIBC tissue. (B) Quantitative RT-PCR analysis of SKA1 mRNA in tumor tissues from different NMIBC groups ( ${}^{*}p<$ 0.0001). (C) Western blot analysis of SKA1 protein expression in tumor tissues from different NMIBC groups.

3. Results

3.1 Patients’ characteristics

A total of 148 consecutive patients with NMIBC were enrolled in this study, including 119 male (80.4%) and 29 female patients (19.6%). The mean age was 65.8 $\pm$ 12.0 years (range from 26 to 91 years). The mean postoperative follow-up period was 68.2 $\pm$ 18.9 months (range 3 to 83 months). Of all patients 51 (34.5%) experienced recurrence and 14 (9.5%) experienced progression. The clinicopathological data for these patients are shown in Table 1.

3.2 Expression of SKA1 in NMIBC tissues

Immunohistochemical staining results revealed different SKA1 expression in NMIBC tissues. Of 148 tumor samples, 69 (46.6%) showed high SKA1 expression and 79 (53.4%) showed low SKA1 expression (Fig. 1A). To confirm these observations, we investigated mRNA expression using qRT-PCR and western blot analysis. The mRNA levels between the two groups were calculated with the comparative Ct method (2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{ct}}$ ). SKA1 mRNA levels in the high SKA1 expression group were significantly higher than those in the low SKA1 expression group ( $p<$ 0.0001; Fig. 1B). A western blot of SKA1 showed protein results consistent with mRNA results (Fig. 1C). The expression of SKA1 mRNA and protein was in accordance with immunohistochemistry staining data, indicating significant differences in SKA1 expression between the two patient groups.

Table 2

Univariate and multivariate Cox regression for disease recurrence in patients with NMIBC

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	$p$ value	HR	95% CI	$p$ value
Gender	0.701	0.330–1.491	0.356	0.748	0.349–1.605	0.456
Age	1.173	0.674–2.041	0.573	0.913	0.506–1.647	0.762
Stage	0.304	0.130–0.713	0.006	0.617	0.339–1.120	0.113
Grade	1.384	0.799–2.398	0.246	1.631	0.892–2.982	0.112
Size	0.520	0.296–0.913	0.023	0.713	0.322–1.580	0.405
Number	1.788	1.030–3.105	0.039	0.801	0.437–1.469	0.474
Expression	0.250	0.135–0.464	0.000	0.246	0.131–0.461	0.000

Table 3

Univariate and multivariate Cox regression for disease progression in patients with NMIBC

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	$p$ value	HR	95% CI	$p$ value
Gender	0.685	0.153–3.059	0.620	0.717	0.156–3.298	0.669
Age	0.858	0.301–2.446	0.774	0.487	0.162–1.464	0.200
Stage	0.030	0.000–4.102	0.162	0.338	0.110–1.043	0.059
Grade	2.692	0.902–8.032	0.076	2.915	0.873–9.732	0.082
Size	0.275	0.095–0.793	0.017	1.080	0.214–5.447	0.926
Number	3.395	1.065–10.827	0.039	1.873	0.572–6.131	0.300
Expression	0.222	0.062–0.796	0.021	0.194	0.052–0.715	0.014

Figure 2.

Kaplan-Meier curves with log-rank test results for recurrence-free and progression-free survival. (A) Recurrence-free survival plot of two patient groups with NMIBC tumors showing low and high SKA1 expression based on immunohistochemistry staining ( $p=$ 0.000). (B) Progression-free survival plot of the two groups ( $p=$ 0.02).

3.3 Relationship between expression of SKA1 and prognosis of patients with NMIBC

The Kaplan-Meier method was used to analyze the relationship between SKA1 expression and the prognosis of patients with NMIBC. Patients showing high SKA1 expressions experienced high early recurrence ( $p<$ 0.001) and progression ( $p<$ 0.05; Fig. 2) rates. Univariate Cox regression analysis revealed that stage (hazard ratio [HR], 0.304; 95% confidence interval [CI], 0.130–0.713; $p=$ 0.006), size (HR, 0.520; 95% CI, 0.296–0.913; $p=$ 0.023), number (HR, 1.788; 95% CI, 1.030–3.105; $p=$ 0.039) and high SKA1 expression (HR, 0.250; 95% CI, 0.135–0.464; $p=$ 0.000) showed a significant impact on the early recurrence of NMIBC. But after multivariate analysis, high SKA1expression (HR, 0.246; 95% CI, 0.131–0.461; $p=$ 0.000) was the only independent predictor of early NMIBC recurrence (Table 2). Moreover, univariate analysis showed that size (HR, 0.275; 95% CI, 0.095–0.793; $p=$ 0.017), number (HR, 3.395; 95% CI, 1.065–10.827; $p=$ 0.039) and high SKA1 expression (HR, 0.222; 95% CI, 0.062–0.796; $p=$ 0.021) were risk factors for the early progression of NMIBC. However, high SKA1 expression (HR, 0.194; 95% CI, 0.052–0.715; $p=$ 0.014) was the only independent predictor of early NMIBC progression after multivariate analysis (Table 3).

4. Discussion

BC is a heterogeneous disease with diverse biological and functional characteristics [18]. BC of stages Ta, T1 and Tis are defined as NMIBC for therapeutic purposes [19]. Most newly diagnosed BCs are NMIBCs. Despite treatment with TURBT alone or with intra-vesical instillation, 31% to 78% of NMIBCs will recur and 1% to 45% of cases will progress to muscle-invasive bladder cancer within 5 years [20]. Tables exist to predict the risks of recurrence and progression depending on the clinical characteristics of NMIBC [19]. However, a specific and sensitive biomarker to estimate the prognosis of BC has not yet been found [21].

The SKA1 gene is located on chromosome 18q21.1; it codes for a protein that is approximately 30 kDa in size and contains 255 amino acids [17]. The essential roles of the SKA1 protein in carcinogenesis has only recently been revealed and is over-expressed in several types of human cancers [8, 12, 13, 14, 15, 16, 17, 18]. In addition, Shen et al. [15] found that SKA1 contributed to cisplatin resistance in NSCLC cells by protecting these cells from cisplatin-induced cell apoptosis. However, only one report exists concerning SKA1 serving as a prognostic marker in a cancer, namely papillary thyroid carcinoma [18]. No other report has elucidated its prognostic value.

To our knowledge, this is the first report aimed at determining the predictive value of SKA1 in NMIBC, and to correlate SKA1 expression with clinicopathologic features and the prognosis of patients with NMIBC. In our previous study, we employed an RNA interference lentivirus system to deplete SKA1 expression in both BT5637 and T-24 bladder cancer cells. Cell proliferation was significantly decreased in both cell lines after SKA1 knockdown. Moreover, colony formation was impaired by SKA1 silencing. Flow cytometry analysis revealed that depletion of SKA1 led to cell cycle arrest in S phase. Furthermore, knockdown of SKA1 in T-24 cells obviously down-regulated the expression of CDK4 and Cyclin D1, and alleviated the activation of ERK2 and AKT, conducive to cell growth inhibition [16]. In order to study any correlation between SKA1 expression and human NMIBC, we examined protein and mRNA expression of SKA1 in several NMIBC samples. In this study, we found that levels of SKA1 protein and mRNA were significantly different in different NMIBC patients. High expression of SKA1 correlated with early recurrence and progression of the cancer in patients, which implied that SKA1 may be involved in the tumorgenesis and the development of NMIBC. Furthermore, the results of Kaplan-Meier survival curves and Cox multivariate analysis indicated that high expression of SKA1 may be an independent predictor of early recurrence and progression of this cancer.

In summary, our study demonstrated that high SKA1 expression was associated with the early recurrence and progression of NMIBC in patients, which indicated that SKA1 may serve as a valuable prognostic marker of this disease. However, this research was conducted as a retrospective study. Since, possible underlying functions of SKA1 involved in NMIBC tumorgenesis and progression are still unclear, further investigations to elucidate the molecular mechanisms of SKA1 are needed.