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Prognostic value of NEK2 overexpression in digestive system cancers: a meta-analysis and systematic review

Background and objective Many studies have reported that NEK2 is overexpressed in digestive system cancers (DSCs) and is also correlated with patient survival. We performed a meta-analysis to comprehensively evaluate the prognostic role of NEK2 expression in DSCs. Materials and methods A comprehensive literature search was performed using PubMed, EMBASE, and Web of Science. Synthesized hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the influence of NEK2 overexpression on the prognosis and clinicopathological features of patients with DSCs. Results A total of 13 studies involving 1,917 patients was included. Overall, patients with high NEK2 expression had poorer overall survival (HR =1.45; 95% CI: 1.15–1.83; P=0.002) and disease-free survival/recurrence-free survival (HR =2.28; 95% CI: 1.54–3.37; P<0.0001). Furthermore, subgroup analysis also suggested that elevated NEK2 expression was associated with poorer overall survival in patients with hepatocellular carcinoma (HR =1.45; 95% CI: 1.05–2.00; P=0.02) and colorectal cancer (HR =2.03; 95% CI: 1.16–3.54; P=0.01). Additionally, NEK2 overexpression was also associated with pretreatment serum AFP level (OR =1.79; 95% CI: 1.23–2.61; P<0.01) and portal vein thrombosis (OR =2.74; 95% CI: 1.22–6.17; P=0.01) in hepatocellular carcinoma. Conclusion NEK2 might act as a useful prognostic predictor and a potential therapeutic target in DSCs. However, multicenter homogeneous studies with larger sample sizes are needed to further confirm our findings owing to some limitations in our meta-analysis.

NEK2 belongs to the serine/threonine kinase family and is important for the regulation of centrosome cell cycle, and mitosis. 6,7 In mitosis, the activation of Nek2 may lead to the phosphorylation of C-Nap1, rootletin, and β-catenin, which could promote centrosome separation by dissociating these proteins from centrosomes. [8][9][10][11][12] The increase in NEK2 expression may disturb the management of the mitotic checkpoint, subsequently contributing to the formation of centrosomal abnormalities and monopolar spindles. 7,13,14 Furthermore, uncontrolled NEK2 activity was involved in rearrangements and abnormal amplification of chromosomes, which leads to chromosome instability (CIN). 7,[14][15][16][17] Overall, the biological functions of NEK2 mentioned indicate that the overexpression and aberrant activity of NEK2 might play a key role in tumorigenesis. Recently, several studies reported that NEK2 is overexpressed in various types of cancers, such as CRC breast cancer, pancreatic cancer, and lung adenocarcinoma. [18][19][20][21][22][23][24] In addition, the expression of NEK2 is closely associated with the prognosis and clinicopathological characteristics of CRC, 25,26 pancreatic ductal adenocarcinoma (PDAC), 19 and HCC. 27 However, there were inconsistencies regarding the prognostic role of NEK2 in DSCs, 19,28,29 which might be partly attributed to the small sample size of any single study exploring the prognostic significance of NEK2. Therefore, we performed a systematic review and meta-analysis to comprehensively evaluate the prognostic value of NEK2 in DSCs by combining the data of multiple relevant studies.

Materials and methods
To maximize clarity and transparency, this meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement issued in 2009. 30

literature search strategy and selection criteria
We comprehensively searched relevant studies assessing the relationship between NEK2 and the prognosis of patients with DSCs in PubMed, EMBASE, and Web of Science. The last search was performed in March 27, 2018. The search terms included: "NEK2 or NIMA related kinase 2" and "cancer or tumor or malignancy or carcinoma or neoplasm." Studies were included if they met the following eligibility criteria: 1) the associations between NEK2 expression and the prognosis or the clinicopathological characteristics of patients with DSCs were reported and 2) the studies were published in English. On the contrary, the studies would be excluded if they met any of the exclusion criteria: 1) reviews, letters, comments, case reports, animal studies, noncomparative studies, and meeting abstracts; 2) articles with insufficient clinical data; and 3) duplicated articles.

Data extraction
Data were independently extracted by 2 investigators. The senior investigator resolved incompatibilities between the reviewers in data extraction. The basic information extracted from the literature included the name of first author, publication year, country, cancer type, the number of subjects, gender, tumor stage, detection methods, cutoff scores for NEK2 overexpression, and follow-up. The endpoints of interest included overall survival (OS), disease-free survival (DFS), recurrence-free survival (RFS), and clinicopathologic characteristics.

Quality assessment and statistical analysis
The quality of eligible articles was assessed using the Newcastle-Ottawa scale (NOS), 31 which consists of 3 aspects: selection, comparability, and outcome. The score of NOS ranged from 0 to 9, and articles with scores .6 were considered as high quality.
Cochrane Collaboration' s Review Manager 5.3 (Cochrane Collaboration, Oxford, UK) was used to synthesize all the data, and Stata version 12.0 (StataCorp LP, College Station, TX, USA) was employed to perform sensitive analysis, meta-regression analysis, and publication bias assessment. Synthesized hazard ratios (HRs) with 95% confidence intervals (CIs) were used to describe the prognostic value of NEK2 on survival outcomes in DSCs, and odds ratios (ORs) with 95% CIs were used to evaluate the relationship between the clinicopathologic characteristics and NEK2 expression. Heterogeneity among the published articles was assessed by Cochran's Q and Higgins I 2 statistics. The combination of P,0.05 and I 2 .50% indicates significant heterogeneity, in which situation data were pooled using a random-effects model. Otherwise, a fixed-effects model was applied to pool the data. In order to minimize the influence of confounding factors and to explore potential source of heterogeneity, subgroup analysis was performed according to country, cancer type, sample size, and detection method. Sensitivity analysis was performed by omitting each individual article step-by-step to test the stability of the meta-analysis results. Furthermore, meta-regression analysis was also used to investigate potential source of heterogeneity. Publication bias was assessed by Begg's funnel plots 32 and Egger's test. 33 Symmetrical plots and P-value .0.05 indicate that there is no significant publication bias across the included studies.  When significant publication bias exists, the trim and fill method was used to estimate the influence of publication bias on the stability of meta-analysis results. 34

search results and study characteristics
The main characteristics of the eligible, included studies are presented in Table 1. Overall, the initial search yielded 622 publications by retrieving PubMed, EMBASE, and Web of Science entries. After excluding 59 duplicate articles, 563 articles remained for screening of the title and abstract, after which process 517 articles were excluded for being reviews and comments (n=26) and irrelevant studies (n=491). Next, 46 articles were left for full-text screening and, consequently, 33 articles were excluded for being non-DSCs (n=4), conference abstracts (n=13), and lacking extractable data (n=16). Eventually, 13 articles involving 1,917 patients were included in this meta-analysis. [25][26][27][28][29][35][36][37][38][39][40][41][42] The details of the study search and selection processes are shown in Figure 1.
All of the eligible studies were published in the most recent 5 years (2014-2017). A total of 10 studies were investigated involving the Chinese population, 2 included Japanese individuals, and 1 represented a British study. A total of 9 studies analyzed the relationship between NEK2 and HCC, 3 studies reported the role of NEK2 in CRC, and 1 study referred to PDAC. The expression of NEK2 at the protein level was detected by immunohistochemistry (IHC) in 7 studies, and 2 studies utilized reverse transcription  Table 2 The nOs quality evaluation of the included articles

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Prognostic value of neK2 overexpression in digestive system cancers polymerase chain reaction (RT-qPCR) to describe the expression of NEK2 at the RNA level, and 1 study used TCGA RNASeqV2 database. In addition, the prognostic value of NEK2 in OS was investigated in 10 studies involving 1,556 patients, and associations between NEK2 expression and DFS/RFS were described in 4 studies with 357 patients. Upon quality assessment of the included studies, all of the eligible studies were found to be of high quality and were scored as 6 or more based on NOS values (  Figure 2). Additionally, subgroup analysis was conducted based on cancer type, country, sample size, and detection level. We found that high expression of NEK2 could act as an independent prognostic factor in CRC (HR =2.03; 95% CI: 1.16-3.54; P=0.01, Figure 3) and HCC (HR =1.45; 95% CI: 1.05-2.00; P=0.02, Figure 4) Table 3.

Prognostic role of neK2 in digestive system tumors in DFs/rFs
Considering the similarity between DFS and RFS, we merged these data to evaluate the prognostic value of NEK2. Due to the absence of heterogeneity among articles (I 2 =0%, P=0.54), a fixed-effects model was used to pool the HR values. The results indicated that overexpression of NEK2 was related to significantly worse DFS/RFS (HR =2.28; 95% CI: 1.54-3.37; P,0.0001) ( Figure 5).

relationship of neK2 with clinicopathological characteristics of Dscs
Owing to the differences in clinicopathological features of various cancers, the association of high NEK2 expression

sensitivity analysis and meta-regression analysis
Sensitivity analysis was performed to test the robustness of the synthesized results of the effect of NEK2 on OS in DSCs by omitting single studies, stepwise. We found that the pooled HR for OS was not altered significantly when excluding any individual study, suggesting that our synthesized results of the effect of NEK2 on OS were rigorous ( Figure 6). The robustness of the synthesized result for DFS/ RFS was not evaluated by sensitivity analysis because of limited numbers of relevant studies.
Considering the significant heterogeneity for the pooled HR data for OS, meta-regression analysis was further conducted to identify the source of heterogeneity, based on the cancer type, country, sample size, and detection level. However, similar to the results of our subgroup analysis, the outcomes of meta-regression analysis indicated that these factors might not account for the heterogeneity either (Table 5).

Publication bias
We used Egger's and Begg's tests to evaluate publication bias in the meta-analysis of the influence of NEK2 on OS in DSCs. It was found that Begg's test had a value of 0.79, Egger's test had a value of 0.93, and the funnel plot was symmetrical, indicating that there was no significant publication bias (Figure 7). Publication bias was not assessed in the

Discussion
It is believed that abnormal cell cycle regulation is important for the occurrence and development of cancers, and NEK2 might play a crucial role in regulating cell cycle via promoting the separation of centrosome in the process of cell division and orchestrating the composition and function of centrosomes. 13,43,44 Furthermore, several studies reported that NEK2 is overexpressed in various cancers and was related to unfavorable prognosis and clinicopathological characteristics, [18][19][20][21][22][23][24] indicating the involvement of NEK2 in tumorigenesis. However, the conclusions were inconsistent with respect to the prognostic role of NEK2 in DSCs. Therefore, the current meta-analysis was performed to comprehensively evaluate the influence of NEK2 overexpression on the prognosis and clinicopathological characteristics of patients with DSCs.
In the present study, we comprehensively analyzed the survival data of 1,917 patients from 13 studies and found that overexpression of NEK2 was significantly correlated with worse OS and DFS in patients with DSCs. Considering that some confounding factors might affect the reliability of our synthesized results, subgroup analysis and meta-regression were conducted according to several potential confounding factors, such as cancer type, country, sample size, and detection level. From the results, we observed that CRC and HCC patients with elevated NEK2 expression had a poorer survival prognosis. Moreover, with different sample sizes and detection levels, the overexpression of NEK2 consistently acted as an indicator of worse OS in patients with DSCs. Additionally, we also investigated the relationship between NEK2 expression and clinicopathological characteristics in patients with DSCs. Our results indicated that high expression of NEK2 was significantly associated with serum positive AFP levels and portal vein thrombosis in HCC, which might partly explain the role of NEK2 overexpression as an indicator of poorer OS in HCC.  Meta-analysis random-effects estimates (exponential form) study ommited To date, several mechanisms have been reported to be responsible for the oncogenic roles of NEK2, which might explain the inverse association between NEK2 expression and prognosis of patients with DSCs. Elevated expression of NEK2 and its homologs could lead to abnormal aggregation of centrosomes and splitting of chromatin, and finally dramatic mitotic progress, which may contribute to the aberrant proliferation of cells. 45,46 It has been reported that NEK2 overexpression could enhance tumor progression by involving the activation of the PI3K/AKT pathway. 16,46 For instance, Li et al 36 found that overexpression of NEK2 could promote the invasion and metastasis of HCC by activating AKT signaling and upregulating the expression of MMP-2. Additionally, NEK2 overexpression was also reported to abnormally activate the MAPK and Wnt/β-catenin pathways, which contributed to the proliferation, invasion, and migration of cancer cells. 26,37,40 More importantly, Lin et al 37 and Fu et al 27 found that deletion of NEK2 could significantly reduce stem-like properties, proliferation, colony formation, migration, and invasion in in vivo tumors formed by HCC cell lines, indicating that NEK2 has certain biological functions that driver genes specifically have. Therefore, we speculated that NEK2 might act as a driver gene and exhibit the potential to be a useful molecular target in HCC and in other digestive system tumors. Of course, more rigorous studies are required to determine whether NEK2 is a driver or passenger gene in digestive system tumors.
There were some limitations to our meta-analysis, which should be considered when interpreting our results. First, all the eligible studies were retrospectively designed, which inevitably had a bias risk and influenced the dependability of our synthesized results. Second, most of the studies were conducted in Asian populations, so our meta-analysis might not represent the worldwide population. Third, although our subgroup analysis and meta-regression indicated that cancer type, country, detection method, and cutoff value might not be responsible for the source of heterogeneity, the reliability of our pooled results were still challenged by significant heterogeneity. Thus, homogeneous studies are needed to further investigate the prognostic role of NEK2 in patients with DSCs. Fourth, only 4 eligible studies with small sample sizes provided data on DFS/RFS for our meta-analysis, which might be insufficient to draw a robust conclusion in this regard. Last but not least, in this meta-analysis, some survival data were manually extracted from Kaplan-Meier curves, which also caused unavoidable bias and heterogeneity.

Conclusion
In summary, this study demonstrated that NEK2 could act as a useful prognostic indicator and as a promising therapeutic target in DSCs. However, considering the limitations mentioned above, multicenter homogeneous studies with lager sample sizes are needed to further confirm our findings. Meanwhile, the relevant mechanisms underlying OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com

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Prognostic value of neK2 overexpression in digestive system cancers the prognostic roles of NEK2 in digestive system tumors, including NEK2 interaction with other genes or molecules and tumor-associated signaling pathways, should also be further explored in the future.