Overexpression Of NIMA-related Kinase 2 Is Associated With Progression And Poor Prognosis Of Prostate Cancer

Background and objectives:The centrosome is a primary site of microtubule nucleation in cells, which plays a critical role in mitotic spindle formation and chromosome segregation. Cancer cells from a wide variety of tissue origins often exhibit multipolar spindles associating with abnormal centrosome numbers or architectures. A number of cell cycle-regulated protein kinases located at the centrosomes have been identified, which are required for mitotic progression and correct bipolar spindle formation. Among these centrosomal kinases is NIMA-related kinase 2 (NEK2) that has two splice variants, NEK2A and NEK2B. The expression of these two centrosomal kinase is regulated in a cell cycle-dependent manner.NEK2 is constitutive active serine and threonine kinase that phosphorylates multiple proteins involved in centrosome duplication and cell cycle regulation. It binds to microtubules and is enriched in the centrosome, where it contributes to centrosome splitting during the G2/M phase of the cell cycle. Aberrant NEK2 activities are oncogenic due to failure to regulate centrosome duplication, which leads to aneuploidy and neoplastic transformation. Similar to other kinases involved in spindle assembly or duplication, overexpression of NEK2 has been reported in several neoplastic diseases, such as preinvasive and invasive breast carcinomas, lung adenocarcinomas, testicular seminomas, and diffuse large B cell lymphomas. Overexpression of NEK2 in non-transformed breast epithelial cells induces centrosome over duplication; increased expression of endogenous NEK2 causes centrosome amplification in breast cancer cells that express oncogenic K-RAS.Although a large number of data demonstrate elevated expression and instability of NEK2 in cancer cells, whether NEK2 upregulation plays a role in prostate cancer (PCa) is still not clearly defined. In this study, we investigated the NEK2 expression patterns in human PCa tissue microarrays (TMA) and the effect of NEK2 depletion on cell proliferation in PCa cells. The data showed strong correlation of NEK2 expression with the prognostic outcome of PCa patients. Together, all our results indicate that overexpressed NEK2 is of prognosis value for predicting outcome of PCa recurrence.Materials and methodsPatients and tissue samplesThe study was approved by the Research Ethics Committee of Guangzhou First People’s Hospital, Guangzhou Medical University, China. Informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards.For immunohistochemistry analysis, tissue microarrays (TMA) with detail clinical information were purchased from Shanghai Outdo Biotech Co, LTD (Cat#: HPro-Ade180PG-01), which includes 99 primary PCa tissues and 81 adjacent non-cancerous prostate tissues from patients without the chemotherapy or radiotherapy before the surgery. The Taylor dataset contains microarray data for mRNAs from 149 primary PCa tissues and 29 adjacent noncancerous prostate tissues. The dataset contains patient survival time and follow-up examination between from 1 to 175 months (median,51 months) post the surgery. The date of prostatectomy was considered as day 1 for the survival analyses. The PSA recurrence was considered the biochemical recurrence-free endpoint. Death other than unexpected causes were considered the endpoint of the overall survival.Cell culture, transfection and treatmentFor cell viability assays,2x103 cells were seeded in 96-well plates and cultured for 24,48, and 72 hours. Cells were then incubated with 20 μl CCK-8 solution (Cat No:C0038, Beyotime, China) for 4 hours at 37℃. The absorbance was measured at the wavelength of 495 nm with a spectrophotometer. Data were expressed as mean±SD of three independent samples. For RNA interference, cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s instructions and harvested at 24h,48h,72h time point for protein and RNA analyses.Quantitative real-time RT-PCRTotal RNA was isolated from cells and prostate tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription was carried out using the ReverTra Ace reagents kit (TOYOBO, Osaka, Japan). In addition, real-time RT-PCR was carried out with the MiniOpticon real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using the SYBR Green master mix (TaKaRa, Otsu, Japan). The thermal cycling conditions comprised of one cycle at 94℃ for 4 minutes,40 cycles at 94℃ for 15 seconds,60℃ for 15 seconds and 72℃ for 15 seconds. All data were analyzed using the Opticon Monitor software (ver3.1, Bio-Rad) and the expression of NEK2 was calculated as relative expression level to GAPDH internal controls using the comparative cycle threshold (CT) method as described previously.Western blotting analysisProteins were extracted 48-hours post-transfection for Western blot analyses. Proteins (40 μg) were fractioned on SDS-PAGE and transferred onto Hybond nitrocellulose membranes (GE Healthcare). The membranes were blocked with 5% skim milk in PBS-Tween 20 and probed with anti-NEK2 (bs-5732R, Bioss Co Ltd., China) or anti-β-actin antibody (sc-47778, Santa Cruz, USA). The specifically bound antibodies were detected with horse radish peroxidase-conjugated human anti-Rabbit antibody and visualized with the SuperSignal West PICO Chemiluminescent Detection Kit (Pierce Biotechnology). β-actin was used as an internal loading control.Tumor growthAll nude mice with an average age of 6-8 weeks were purchased from the Guangdong Medical Laboratory Animal Center. The NEK2 silenced cells were injected on the left side flank and the control cells on the right side flank. Briefly, 2X106 cells were suspended in 0.1ml culture medium, and then mixed with the Matrigel (BD Biosciences,NO.356234) at the ratio of 1:1. The injections were carried out in a sterilized hood. We measured the tumor with caliper and calculated the tumor size at three different time points, then collected the tumor tissue to carry out the following experiments at 43 days after the implantation.Immunohistochemistry analysisThe specimens were fixed in 10% neutral buffered formalin and subsequently embedded in paraffin. The paraffin-embedded tissues were sectioned at 4 um thickness and then deparaffinized with xylene and rehydrated for H&E or immunohistochemistry staining. Briefly, following a brief proteolytic digestion and a peroxidase blocking, the slides were incubated overnight with the primary antibody against NEK2 (bs-5732R, Bioss Co Ltd., China) at a dilution of 1:400, at 4℃. After washing, peroxidaselabeled polymer and substrate-chromogen from DAKO EnVision System (Dako Diagnostics, Switzerland) were employed to detect the specifically bound antibodies as suggested by the manufacturer. No primary antibodies were used as the negative controls. The slides were then lightly counterstained with hematoxylin. The staining was scored by two independent experienced pathologists, who were blinded to the clinicopathological data and clinical outcomes of the patients. The scores of the two pathologists were compared, and any discrepant scores were trained through re-examining the staining by both pathologists to achieve a consensus score. The number of positive-staining cells in ten representative microscopic fields was counted, and the percentage of positive cells was calculated. The staining was subjected to arbitrative categorized to 4 groups based on the percentage of positive cells for semi-quantitative analyses as following:0 (0%),1 (1-10%),2 (11-50%), and 3 (>50%). The staining intensity was visually scored and stratified as follows:0 (negative),1 (weak),2 (moderate) and 3 (strong). A final immunoreactivity scores (IRS) were obtained for each case by adding the percentage and the intensity score.Statistical analysisThe statistical analyses was carried out with the SPSS software (Version 13.0 for Windows, SPSS Inc., IL, USA) and SAS 9.1 (SAS Institute, Cary, NC, USA). Continuous variables were expressed as mean± SD. The Kaplan-Meier method was used for the survival analyses. The chiquest trend test was used for ordinal data analysis. Differences were considered statistically significant when P was smaller than 0.05.Resultsup-regulation of NEK2 in human prostate cancer cellsQuantitative real-time RT-PCR was performed to assess the expression of NEK2 at the mRNA level in 3 PCa and 1 non-tumourigenic benign human prostatic epithelial cell lines. As shown in Figure 1, expression of NEK2 was higher in all tested PCa cells than in non-tumorigenic benign human prostatic epithelial cells at the mRNA level.Depleting NEK2 expression reduces cell proliferation in PCa cellsTo determine whether high expression of NEK2 contributed to cell proliferation, siRNA was used to deplete the expression of NEK2 in LNCaP cells. Both real time RT-PCR and Western blot analyses showed that the expression of NEK2 was reduced at both mRNA and protein levels. Cell proliferation assays demonstrated that LNCaP cells with NEK2 depletion had a lower proliferation rate than the cells transfected with scramble siRNAs as the negative control. The results suggest that high expression of NEK2 promotes cell proliferation.Knockdown NEK2 expression suppresses tumorigenicity of LNCaP cells.To determine whether depleting NEK2 expression affected the tumorigenicity of LNCaP cells, the silenced and control cells were implanted to the flanks of the same mice. The tumor sizes were measured at day 14,23, and 36 days post the implantation. It was apparent that the tumors derived from the NEK2 silenced cells were smaller than those from control cells. The tumors were harvested at day 43 after the implantation for further analyses. In addition to smaller size, the tumors with NEK2 silenced cells were relatively pale than the tumors from control cells, suggesting that tumor angiogenesis was also compromised. Immunostaining revealed that expression of NEK2 protein was reduced compared with the tumors from untranfected or scramble siRNA transfected control cells. Furthermore, high magnification images showed that NEK2 protein are mainly localized at the nucleus and cytosol.Expression levels of NEK2 were associated with the clinicopathological characteristics of PCa and tumor recurrence time of PCa patientsWe then analyzed the expression pattern and localization of NEK2 in a PCa TMA comprised of 99 PCa and 81 adjacent non-cancerous prostate tissues by immunohistochemistry staining. NEK2 immunostaining was weak or invisible in adjacent noncancerous prostate tissues. In contrast, the staining of NEK2 was strong and mainly located in the cytoplasm and nucleus of the cells. A summary of NEK2 immunostaining in PCa tissues was shown in Table 2. Apparently, the PCa with a Gleason Score≥8 had significantly higher NEK2 expression than those with a Gleason Score<8. In addition, the PCa at the high pathological stage showed increased NEK2 expression compared with PCa at the low pathological stage. Moreover, statistical analyses of the Tylor dataset also showed that PCa with a Gleason Score≥8 had higher expression of NEK2 than those with a Gleason Score <8 (P=0.002) at the mRNA level.We then use the Kaplan-Meier method to analyze the association of NEK2 expression levels with the biochemical recurrence-free time and the overall survival time of PCa patients. The median of NEK2 expression in all PCa tissues of the Taylor dataset was used as the cutoff to divide all PCa tissues into high (n=80) and low (n=80) NEK2 expression groups. The biochemical recurrence-free time of PCa patients with high NEK2 expression levels was shorter than those with low NEK2 expression levels. However, the overall survival time of PCa patients was not correlated to NEK2 expression levels.Conclusions:The above findings suggest for the first time that the overexpression of NEK2 is associated with progression in patients with PCa, which also indicate that NEK2 has the potential for serving as a biomarker for PCa prognosis.