ZNF488 is an independent prognostic indicator in nasopharyngeal carcinoma and promotes cell adhesion and proliferation via collagen IV/FAK/AKT/Cyclin D1 pathway

Figures & data

Table 1 Clinical characteristics of NPC patients according to high and low ZNF488 expression

Characteristics	ZNF488 expression		P-value
Low or none, n=101(%)	High, n=57(%)
Age			1.000
<50	49 (48.5)	27 (47.4)
≥50	52 (51.5)	30 (52.6)
Gender			0.704
Male	74 (73.3)	44 (77.2)
Female	27 (26.7)	13 (22.8)
TNM stage			1.000
I–II	3 (3.0)	1 (1.80)
III– IV	98 (97.0)	56 (98.2)
T stage			0.175
T1– T2	44 (43.6)	18 (31.6)
T3– T4	57 (56.4)	39 (68.4)
N stage			0.833
N0–N1	18 (17.8)	11 (19.3)
N2–N3	83 (82.2)	46 (80.7)
M stage M0 M1	100 (99.1) 1 (0.01)	53 (99.3) 4 (0.07)	0.057
Locoregional failure Yes No	16 (15.8) 18 (31.6)	85 (84.2) 39 (68.4)	0.018
Distant metastasis			0.001
Yes	11 (10.9)	16 (28.1)
No	90 (89.1)	31 (71.9)

Notes: Two-sided P-values were calculated using Pearson’s chi-square test or continuity correction to evaluate the significance of the correlations. Bold values indicate statistical significance (P<0.05).

Figure 1 High expression of ZNF488 is correlated with poor clinical outcomes. (A) Negative ZNF488 staining in nasopharyngeal epithelium tissue, (B) negative ZNF488 staining in NPC tissue with normal rabbit IgG, (C) negative staining of ZNF488, (D) weak staining, (E) moderate staining, (F) strong staining (magnification 400×). (G) Overall survival, (H) locoregional recurrence-free survival. (I) Distant-metastasis-free survival. (J) Progression-free survival.

Table 2 Univariate analysis and multivariable cox regression analyses of ZNF488 expression levels and overall survival

Variable	Univariate analysis			Multivariate analysis
	HR	95%CI	P-value	HR	95%CI	P-value
ZNF488 expression (high vs low)	4.602	2.124–9.969	<0.001	3.314	1.489–7.386	0.003
Age (<50 vs ≥50 years)	0.915	0.447–1.874	0.484
Gender (female vs. male)	0.644	0.380–1.818	0.831
T stage (3–4 vs1-2)	3.320	1.365–8.079	0.008	2.886	1.155–7.210	0.023
N stage (2–3 vs 0–1)	1.191	0.660–2.155	0.110
Radiation dose (<70 Gy vs ≥70Gy)	2.565	1.095–6.008	0.030	4.197	1.746–10.085	0.001
Chemotherapy (no vs yes)	1.082	0.440–2.661	0.557
Distant metastasis (yes vs no)	8.077	4.008–16.278	<0.001	6.962	3.316–14.618	<0.001
Locoregional failure (yes vs no)	2.873	1.428–5.778	0.003	2.806	1.345–5.853	0.006

Note: Bold values indicate statistical significance (P<0.05).

Figure 2 Effects of ZNF488 on adhesion ability and FAK signaling pathway. (A) Immunofluoresce for ZNF488 detection. (B) Western blot to detect ZNF488 protein level in both HNE1 and CNE1 ZNF488 overexpression stable cell lines and vector. (C) Adhesion assay in HNE1 and CNE1. (D) FAK activation assay to detect the activity of pFAK (Y397). (E) Western blot to detect collagen IV, integrin α5, FAK and p-FAK(Y397). (F) Western blot to detect ERK 1/2, p-ERK1/2, Akt and p-Akt. *** P<0.001.

Figure 3 Effects of ZNF488 on cell proliferation. (A) The growth curves of MTT assays in both HNE1 and CNE1. (B) Colony formation assays were conducted. * P<0.05; *** P<0.001.

Figure 4 Effect of ZNF488 on cell cycle distribution and Cyclin D1. (A) Cell cycle analysis with flow cytometry were performed in HNE1 and CNE1. (B) Results of cell cycle analysis with histogram. (C) Proliferation index of each group. (D) S-phase fraction of each group. (E) Western blot to detect the protein level of cyclin D1, Cyclin D2, Cyclin E (C-19), and cleaved caspase 9 p10. NS, no significance. * P<0.05, ** P<0.01, *** P<0.001.