Low expression of NEK2 is associated with hepatocellular carcinoma progression and poor prognosis

Abstract

BACKGROUND:

NIMA-related kinase 2 (NEK2), a serine/threonine kinase, is located in the centrosome and is a member of cell cycle regulation related protein kinase (CCRK) family. Aberrant expression of NEK2 is linked with carcinogenesis and progression of various tumors.

OBJECTIVE:

To investigate the expression level of NEK2 and its relationship with clinicopathological factors in hepatocellular carcinoma (HCC).

METHODS:

Immunohistochemistry was used to measure the expression of NEK2 in 310 patients’ specimen tissues and 197 adjacent normal liver tissues of HCC cases, and the subsequent prognostic value for each sample was estimated.

RESULTS:

NEK2 expression levels in HCC were lower than in adjacent tissues (49.7% vs. 72.6%, $P<$ 0.001). First, patients with relatively low NEK2 expression had increased cancer progression and poorer prognosis than those with high expression. Second, NEK2 expression was significantly reduced in patients with large tumors ( $P=$ 0.025), with stage III Edmondson-Steiner Grading ( $P=$ 0.015). Third, patients’ tumor size positively correlated with high AFP concentration ( $P=$ 0.017). Fourth, using the Kaplan-Meier survival curve, we found a lower survival rate in patients with decreased expression of NEK2 than those with high NEK2 expression in HCC ( $P=$ 0.029, Log-rank test).

CONCLUSIONS:

Low NEK2 expression might be a useful predictor in HCC as a poor prognostic factor, and could serve as a potential therapeutic target for HCC.

1. Introduction

Hepatocellular carcinoma (HCC) is ranked the second most common cause of cancer-related mortality worldwide in men, and the sixth leading cause of cancer death in women [1]. The cause rates of HCC are the highest in East and South-East Asia and Northern and Western Africa [1]. In recent years, it has been shown that aging people have less rates of HCC in China, most likely due to reduction of HBV infection by improvement of hygiene and sanitation [2]. In China, advanced age, however, is significantly correlated with the increased incidence of HCC, and its mortality rate is as high as 54.7:10,000 cases per year [3]. Despite technological advances in early diagnosis and therapy, HCC patients still have a poor prognosis, and the average survival time since initial diagnosis is less than five years [4, 5]. Therefore, therapeutic intervention is needed for targeting new clinically applicable molecules for an early treatment of HCC.

NIMA-related kinase 2 (NEK2), which is a member of the cell cycle regulation related protein kinase (CCRK) family, is a serine/threonine kinase located in the centrosome. The role of NEK2 is reported to be multiple in cell cycle regulation. For example, the expression of NEK2 was not detectable in G1 phase, but increases in S phase and peaks in G2 phase, playing an established role in regulating centrosome separation [6, 7, 8]. NEK2 is also included in centrosome duplication [6, 9], microtubule organization, stabilization [10, 11], kinetochore attachment [12, 13], spindle assembly checkpoint [12, 13], and chromatin condensation [14]. Furthermore, NEK2 has been reported to be a novel regulator of B cell development, immunological responses, and cilia disassembly [15, 16].

In recent years, several studies found overexpression of NEK2 in different types of cancer, a role of increasing proliferation and drug resistance in cancer cells such as breast cancer [17] and myeloma [18]. In contrast, depletion of NEK2 reverses these effects [18, 19, 20]. Limited research has been carried out to elucidate the undefined association between HCC and NEK2 expression as well as the role of NEK2 in HCC development [21, 22].

In the present study, we evaluate the expression of NEK2 using immunohistochemistry, and investigate its relationship with clinicopathological characteristics such as patients’ prognosis. This study will provide insight into the function of NEK2 genes and liver cancer progression.

2. Materials and methods

2.1 Patients and samples

A total of 310 HCC tissue specimens and 197 adjacent normal liver tissue cases were obtained by surgical resection from HCC patients admitted at Zhejiang Provincial People’s Hospital from 2008 to 2015. Both kinds of tissues, verified by pathological examination, were formalin-fixed and paraffin embedded. Gender, age, tumor size, Edmondson-Steiner grades, tumor metastasis, and AFP concentration measurements were collected before surgery and saved at the hospital database. Overall survival (OS) was evaluated from the date of surgical excision of the primary tumor until the death date or the last follow-up. The Review Board of Hospital Ethics Committee approved the study, and the informed consent from each participant was obtained before data collection.

2.2 Immunohistochemical staining

We constructed three tissue microarrays by BioChip (Shanghai, China), using the paraffin-embedded specimens. A standard immunohistochemical staining method was used. Briefly, 5 $\mu$ m sections were prepared from the TMAs and baked at 70 ${}^{\circ }$ C for 2 h, followed by deparaffinization in xylene, rehydrated using a gradient of ethanol concentrations and boiled in 1 mM TE buffer with a high-pressure cooker for 3 min for antigen retrieval. Subsequently, sections were blocked with 3% hydrogen peroxide for 15 min to inhibit endogenous peroxidase activity and 10% goat non-immune serum was used to incubate each sample for 20 min to reduce non-specific background staining. Next, rabbit anti-NEK2 monoclonal antibody (Abcam, Cambridge, UK) (1:2000 dilution) was applied to each section and incubated overnight at 4 ${}^{\circ }$ C. After three times washes in a buffer, the sections were incubated with the biotin-labeled secondary antibody (Invitrogen, Carlsbad, CA) for 15 min at room temperature, followed by incubation with HRP-conjugated streptavidin (Invitrogen, Carlsbad, CA) also for 15 min at room temperature. Color development was performed, using DAB Substrate Kit (Dako, Glostrup, Denmark). Hematoxylin was used to counterstain the sections, followed by dehydration, clearing, and mounting.

Figure 1.

Differential expression of NEK2 in human HCC specimens and adjacent tissues. A. HCC tissue and paired adjacent tissues. Low expression of NEK2 in HCC tissue (a1, a2); High expression of NEK2 in the paired adjacent tissues (b1, b2). B. Immunohistochemical staining of NEK2 expression in human HCC specimens and adjacent tissues. Negative ( $-$ ) (a) or weak ( $+$ ) (b) staining were defined as negative expression; moderate ( $++$ ) (c) or strong ( $+++$ ) (d) staining were defined as positive expression.

Figure 2.

Kaplan-Meier survival curves of HCC patients based on the NEK2 expression level. Patients with low NEK2 expression level had a significantly poorer survival than those with high NEK2 expression level ( $P<$ 0.05).

2.3 Evaluation of the immunohistochemical stains

The immunohistochemical positive stained cells of NEK2 were scored independently by two pathologists, based on intensity and the proportion of stained cells. Staining intensity was assessed with a four-tiered grading system: 0 $=$ negative, 1 $=$ weak, 2 $=$ moderate and 3 $=$ strong. We scored the proportion of positively stained cells as follows: 0 for no cell stained, 1 for 1–25% of cells stained, 2 for 26–50% of cells stained, 3 for 51–75% of cells stained and 4 for $>$ 75%–100% stained cells. We multiplied scores for intensity and percentage of positive cells to determine the extent of NEK2 expression: Scores $⩽$ 6 was defined to be tumors with low NEK2 expression; and scores $>$ 6, high NEK2 expression.

2.4 Statistical analysis

We performed statistical analysis using SPSS Software, version 19.0 (Chicago, IL). The ${\chi }^2$ -tests were applied to assess the associations between NEK2 expression and clinicopathological parameters, respectively. Overall survival curves were fitted using the Kaplan-Meier method and evaluated using the Log-Rank test. It was considered statistically significant if $P<$ 0.05.

Table 1

Decreased expression of NEK2 in HCC

	Cases	NEK2 expression		$P$ value
		High	Low
Hepatocellular	310	154	156	$<$ 0.001
carcinoma
Adjacent tissue	197	143	54

Table 2

Correlation between NEK2 expression and clinicopathological features of HCC patients

Clinical	Number	NEK2 expression		P value
parameters		High (154)	Low (156)
Age (years)				0.542
$<$ 55	120	57	63
$⩾$ 55	190	97	93
Gender				0.487
Male	252	128	124
Female	59	27	32
Location				0.871
Left	58	31	27
Right	175	88	87
Left $+$ Right	17	8	9
Size				0.025
$<$ 5	161	90	71
$⩾$ 5	122	61	81
Tumor number				0.727
Single	254	125	129
Multiple	56	29	27
Edmondson grade				0.015
I $+$ II	196	108	88
III	113	46	67
Metastasis				0.960
M0	281	139	142
M1	24	12	12
Vessel invasion				0.515
Absence	115	57	58
Presence	117	53	64
HBs antigen				0.473
Negative	60	32	28
Positive	245	118	127
Cirrhosis				0.495
Negaive	103	54	49
Positive	207	100	107
AFP				0.017
$<$ 50	134	82	52
$⩾$ 50	119	55	64

Table 3

Cox-regression analysis of the clinicopathological parameters in HCC patients

Parameters	Coefficient	Hazard	95.0% CI	$P$ value
		ratio	for HR
Size	0.518	1.679	0.735–3.834	0.219
AFP	0.602	1.826	0.735–4.541	0.195
Edmondson	0.564	1.757	0.727–4.246	0.210
grade
Vessel invasion	0.725	2.064	0.767–5.555	0.151
Metastasis	1.265	3.543	1.191–10.543	0.023
NEK2 expression	$-$ 0.414	0.661	0.285–1.534	0.335

3. Results

3.1 Decreased expression of NEK2 in HCC tissues

Using immunohistochemistry we examined 310 HCC specimens and 194 normal tissues, 167 of which were paired samples. The NEK2 expression level in tumor tissues was significantly lower than that in adjacent normal tissues of HCC. Positive staining of NEK2 could be observed in 154 of 310 (49.7%) cases of HCCs, whereas 143 of 197 (72.6%) cases of adjacent tissue samples were positively stained ( $P<$ 0.001) (Table 1 and Fig. 1).

3.2 Correlation between NEK2 expression and clinicopathological characteristics

Based on the result of immunochemistry, we further investigated the association of NEK2 expression and clinicopathological features in 310 HCC samples (Table 2). NEK2 expression was reduced significantly in patients with large tumors ( $P=$ 0.025) who were also found having high AFP concentration ( $P=$ 0.017). Significantly decreased NEK2 expression was observed more frequently in tumors with stage III Edmondson-Steiner Grading ( $P=$ 0.015). However, there was no correlation with age, gender, tumor location, tumor number, metastasis, vessel invasion, HBs antigen and cirrhosis with NEK2 expression ( $P>$ 0.05).

3.3 Association between NEK2 expression and prognosis

To enable statistical evaluation of the significance of prognostic of NEK2 expression, Kaplan-Meier curve for overall survival were analyzed. The 3-year overall survival rate was 50% ( $n=$ 72) for the patients with low NEK2 expression, and 69.4% ( $n=$ 78) for the patients with high NEK2 expression, and their difference was statistically significant ( $P=$ 0.029, Log-rank test, Fig. 2). Thus, we concluded that the average survival time of patients with negative NEK2 expression was shorter than those with positive expression. Additionally, factors with possible prognostic effects in HCC, including size, AFP, Edmondson Grade, Vessel invasion, Metastasis and NEK2 expression, were analyzed by Cox regression analysis. Our results indicated that metastasis was an independent prognostic in patients with HCC ( $P=$ 0.023), while NEK2 expression was dependent ( $P=$ 0.335; Table 3).

4. Discussion

To the best of our knowledge, this study represents the first observation of expression of NEK2 and its prognostic role in more than 300 patients with liver cancer. Although many studies had reported that NEK2 was highly expressed in a variety of human cancers and usually presented poor prognosis, we obtained decreased expression of NEK2 in HCC tissues, compared with normal adjacent tissues (49.7% vs. 72.6%). Besides, the decreased NEK2 expression was negatively correlated with large tumor size, high APF concentration, high clinical stages, poor prognosis, and overall survival rates. As NEK2 regulates formation and separation of the spindle through the substrate phosphorylation, thereby regulating chromatin distribution [23], it is likely that the low expression of NEK2 might impair the cell cycle.

Given the pivotal roles of NEK2 in the regulation of cell division, abnormal expression of NEK2 may perturb its normal biological functions, which might ultimately induce cancer progression. Recent researches have suggested an abnormal regulation of the MAPK signaling pathway, which was associated with aggressive cancer progression and poor prognosis of HCC [24, 25]. Previous studies indicating high expression of NEK2 in HepG2 suggested it might activate p38 MAPK pathway [22]. The involvement of P38 MAPK signal pathway core molecules might play a crucial role in the regulation of cell proliferation, apoptosis, tumorigenesis, and tumor progression. Earlier research found that p38 MAPK pathway could downregulate cyclin D1 expression, resulting in G1 phase arrest [26]. Furthermore, p38 MAPK can induce apoptosis via various pathways, including elevation of c-myc expression, P53 phosphorylation, c-jun and c-fos activation, Bax translocation or increasing TNF- $\alpha$ expression [27, 28, 29, 30, 31]. Lyoda et al. found that the activity of p38 MAPK in hepatocellular carcinoma tissue was significantly lower than adjacent non-tumor tissue, and with a large size tumor group ( $>$ 20 mm), the activity of p38 MAPK was considerably lower than that in the smaller size tumor group [32]. According to their study and our results, we can hypothesize that NEK2 may promote apoptosis or tumor suppression factors via activating p38 MAPK pathway in HCC cells. Thus the lower expression of NEK2 promotes tumorigenesis and progression in HCC. Further studies are required to prove our hypothesis.

In conclusion, our observations have provided evidence for the roles of NEK2 in HCC; low expression of NEK2 is related to unfavorable prognosis in HCC. NEK2 may be regarded a promising therapeutic target in control HCC.