High expression of KPNA2 defines poor prognosis in patients with upper tract urothelial carcinoma treated with radical nephroureterectomy

This study was approved by the Review Board of the Peking University First Hospital (Beijing, P.R. China). Informed written consent was obtained from all patients. We retrospectively analyzed the clinical and pathologic data from 607 patients who underwent RNU for UTUC from January 2002 to December 2010 at our institute.176 patients with incomplete follow-up data, Among the remaining 431 patients with complete follow-up data, 255 were excluded from the study: 81 had concomitant/previous bladder tumors, 45 had bilateral synchronous UTUC, 34 underwent other surgeries instead of radical nephroureterectomy, 39 had a follow-up period of less than 12 months, 48 patients with a tumor less than 1.0 cm (diameter) (not suitable for formation of a tissue microarray), 8 had a positive surgical margin. At last the remaining 176 patients were included in this study, 81 cases with open surgery, 95 cases with laparoscopic surgery, All UTUC patients undergoing radical nephroureterectomy, not receiving routine lymphadenectomy. Only for those high risk patients, Such as preoperative imaging examination suspected lymph node metastasis, high stage and mulifocality, the routine lymphadenectomy was performed. None of the included patients received preoperative chemotherapy, although postoperative chemotherapy or radiotherapy was administered at the time of recurrence or metastasis. The clinical stage was assigned using the American Joint Committee on Cancer TNM Staging System for Renal Pelvis and Ureter Cancer (7th edition, 2010) [19].

In total, we observed 78 male and 98 female patients with a median age of 69 years (29–86) and a median follow-up of 36 months (12–108). The patients went to follow-up appointments on a routine schedule, including regular cystoscopy, urine cytology and clinical examinations. Cystoscopy was performed every three months for the first two years, then cystoscopy intervals were extended to 1 year thereafter. Chest X-ray, serum creatine, abdominal ultrasound, and computed tomography were examined at the same time. We defined bladder recurrence as finding a subsequent bladder tumor during cystoscopy and confirmed it by pathology. The time of first bladder recurrence was used as the endpoint for the study. The follow-up of patients without tumor recurrence was censored to the date of their last visit. In case of death not related to the bladder cancer, follow-up was censored to the date of death. Metastasis or retroperitoneal recurrence was documented.

The tissue microarrays were constructed as previously described [20].IHC was performed on formalin-fixed paraffin -embedded tissue microarrays (TMAs) sections that consisted of paired normal and UTUC specimens from 176 patients. IHC was completed using an SP reagent kit (Zhongshan Biotechnology Company, Beijing, China) according to the manufacturer’s instructions. An anti-KPNA2 antibody (1:800 Abcam plc, Cambridge, UK) was used as the primary antibody. For a negative control of the staining procedure, the primary antibody was omitted. Two surgical pathologists reviewed and scored the slides independently for KPNA2 expression without knowledge of the clinical data.

To analyze the KPNA2 staining, the percentage of cancer cells with nuclear immunoreactivity was quantified. Based on an analysis of different cut-off levels and previous studies [18,21], high expression of KPNA2 was defined as strong nuclear staining in at least 10% of the carcinoma cells.

The normal urothelial cell line SV-HUC-1 and the urothelial transitional cell carcinoma cell lines 5637, RT4, T24, UM-UC-3 and J82 were obtained from the American Type Culture Collection. SV-HUC-1 was cultured in Ham’s F-12 medium. The 5637, RT4 and T24 cells were grown in RPMI 1640 medium. J82 and UM-UC-3 cells were maintained in DMEM. All culture media were supplemented with 10% fetal bovine serum (HyClone) and penicillin sodium (100 U/ml)/streptomycin sulfate (100 μg/ml) (Invitrogen). All cells were grown in a humidified atmosphere incubator with 5% CO₂ at 37°C.

Cells were transfected using the INTERFERINTM reagent (PolyplusTransfection, Strasbourg, France) according to the manufacturer’s instructions. A pool of two sequence-validated and knockdown-warranted KPNA2-siRNA was used (Homo-1111: 5′-GACUCAGGUUGUGAUUGAUTT-3′ and 5′-AUCAAUCACAACCUGAGUCTT-3′; homo-1400:5′-CCGUUGAUGAACCUCUUAATT-3′ and 5′-UUAAGAGGUUCAUCAACGGTT-3′) (GenePharma, Shanghai, China). Commercial FAM-tagged, negative control siRNAs (NC siRNA) (5′-UCCUCCGAACGUGUCACGUTT-3′,5′-ACGUGACACGUUCGGAGAATT-3′) (GenePharma) were used as an efficiency control and as a control for unspecific side effects. Cell lysates were prepared for western blotting 48 h after transfection to determine the efficiency of gene expression ablation.

Total proteins from cell lines were extracted in lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) and quantified using a BCA protein assay (Thermo Scientific, Rockford, IL, USA). Each extracted protein sample was separated by 10% SDS-PAGE. After transferring the separated proteins to a PVDF membrane (Pall, Pensacola, FL), the membrane was incubated overnight at 4°C with antibodies against KPNA2 (Abcam Plc, Cambridge, UK, 1:1000), PARP (CST, Danvers, MA, 1:1000), PCNA (CST, Danvers, MA, 1:1000), or β-actin (Santa Cruz Biotechnology, 1:1000). After four washes with TBST, membranes were incubated with the appropriate HRP-conjugated secondary antibody at 37°C for 1 h. The protein bands were detected using Immobilon^TM Western Chemiluminescent HRP substrate (Millipore) and scanned using GeneSnap (Syngene, Cambridge, UK) acquisition software.

The cell proliferation capacity of siRNA-transfected cultures was determined using Cell Counting Kit-8 solution (Dojindo, Gaithersburg, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, cells were seeded at a concentration of 5 × 10³ cells/100 μl/well in 96-well culture plates and treated with 10 μl/well of Cell Counting Kit-8H solution during the last 2 h of culturing. The optical density of the wells was measured at 450 nm using a Multiscan microplate spectrophotometer (Thermo LabSystems, Milford, MA).

Cell migration was assessed using a Boyden chamber assay. A total of 5 × 10⁴ cells in 100 μl serum-free medium were seeded onto the upper chambers of a 24-well Boyden Chamber insert (Costar #3422) with uncoated 8-μm pores. Medium with 10% FBS was added to the lower chambers as a chemoattractant. After 24 h of incubation, cells remaining on the upper surface of the membrane were removed with a cotton swab, and cells that migrated through the membrane filter were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed under a microscope (Olympus BX40 with a DP70 digital camera). The migrating cells were manually counted per high-power field for each condition, and five fields were randomly selected per membrane.

Apoptosis was evaluated by using an Annexin-V/PI apoptosis detection kit (KeyGen Biotech, Nanjing, China) following the manufacturer’s instructions. Cells were cultured in 6-well plates at a concentration of 2-3 × 10⁵ cells/2 ml/well and transfected with siRNA. Cells were collected 48 h after transfection and then resuspended in 500 μl binding buffer, followed by the addition of 5 μl Annexin V-FITC and 5 μl PI dye. After an incubation of 10–15 min at room temperature in the dark, cells were analyzed using a BD FACStar flow cytometer (Becton Dickinson, San Jose, CA).

ΔΨm was estimated using a mitochondrial membrane sensor kit containing the cationic lipophilic fluorochrome JC-1 dye (KeyGEN Biotech, Nanjing, China) according to the manufacturer’s protocol. Briefly, 48 h after siRNA transfection, the cells were collected and resuspended in 500 μl JC-1 working solution for 20–30 min. Next, the cells were analyzed using a BD FACStar flow cytometer (Becton Dickinson, San Jose, CA).

A total of 5 × 10³ cells were seeded in 96-well cell culture plates. After a 48 h siRNA treatment, apoptosis rates were measured based on the activation of effector caspases 3 and 7 using the Caspase-Glo^TM3/7 Substrate kit (Promega, Mannheim, Germany) according to the manufacturer’s instructions. All samples were performed in triplicate.

A two-sided Fisher’s exact test or Pearson’s 2-sided χ² test was used to study significant differences between immunohistochemical and clinicopathologic data. To compare two independent samples, the nonparametric Mann–Whitney test was used. Survival curves for patients with low or high KPNA2 expression were plotted using the Kaplan-Meier method, with log-rank tests for statistical significance. Uni- and multi-variable Cox regression analyses were used to test the prognostic relevance of clinicopathologic/immunohistochemical data. Only predictive factors that were significant in the univariate analysis were used in the multivariate analysis (Cox’s proportional hazards model). SPSS version 17.0 (SPSS) was used to complete the statistical analysis. P < 0.05 was considered significant.