NDRG2 was downregulated in LUAD, SOCS1, and CyclinD3 compared with normal tissues
NDRG2 protein was mainly found in the cytoplasm, and a weak expression could be found in a limited number of cell nuclei (Figure 2 A-C). The expression of NDRG2 at the protein (Figure 2 D) and mRNA (Figure 2 E) levels in LUAD was significantly lower compared with normal tissues.
To evaluate the expression of NDRG2, SOCS1, and CyclinD3 in different stages of LUAD, we divided 89 pairs of normal and LUAD samples into four groups according to the disease stage. There were 24, 15, 10, and 40 samples in stages I, II, III, and IV, respectively. The expression of NDRG2 and SOCS1 gradually decreased as the LUAD stage increases (Figure 3 B-C), while the expression of CyclinD3 gradually increased as the LUAD stage increases (Figure 3 D).
Relationship between the expression of NDRG2 and clinicopathological features of patients with LUAD
As shown in Figure 4, the expression level of NDRG2 was associated with CEA (P < 0.001).
As shown in Table 2, the expression level of NDRG2 was notably higher in LUAD tissues in stages I-II than that in stage III-IV (P<0.001). In addition, the frequencies of no vascular invasion and EGFR positivity (+) were significantly higher in patients with high expression of NDRG2 than that in patients with low expression of NDRG2 (P<0.001 and 0.001, respectively). There were no associations between expression levels of NDRG2 and other clinicopathological features, including age, sex, smoking history, and blood type (P>0.05).
Regarding the 34 patients who underwent surgery, the expression level of NDRG2 was significantly higher in stage I-II than that in stage III-IV (P=0.028). The frequencies of no vascular invasion and EGFR positivity (+) were higher in patients with high expression of NDRG2 than that in the patients with low expression of NDRG2 (0.008 and 0.030, respectively).
Prognostic implications of NDRG2 and EGFR expression
Based on the clinicopathological features of the patients with LUAD, as well as the expression levels of NDRG2, EGFR, and CEA, the survival was analyzed by the Kaplan-Meier method (Figure 5). The results showed that iodine-125 radioactive seeds brachytherapy for advanced LUAD with high expression level of NDRG2 led to significantly higher OS than in LUAD with low expression level (P = 0.0261, Figure 5 A). patients with LUAD, EGFR(+), and CEA < 2.0 ng/ml had higher OS (P < 0.0001, 0.0314, Figure 5 B-C). In addition, in operated patients with high expression of NDRG2 (Figure 5 E), EGFR(+) (Figure 5 F), and CEA < 2.0 ng/ml (Figure 5 G), higher OS was noted (P = 0.0022, < 0.0001 and 0.013, respectively).
According to the conjoined expressions of NDRG2/EGFR, the subjects were categorized into four groups: NDRG2-low/EGFR-negative(-), NDRG2-low/EGFR-positive(+), NDRG2-high/EGFR-negative(-), and NDRG2-high/EGFR-positive(+). The association between the co-expression of NDRG2/EGFR and OS was tested by the Kaplan-Meier method. In these four groups, iodine-125 radioactive seeds brachytherapy for advanced LUAD patients in the NDRG2-high/EGFR(+) group was accompanied by the best prognosis during the 5-year follow-up (P < 0.0001, Figure 5 D), and the same results were observed in operated patients (P = 0.0002, Figure 5 H).
Cox regression analysis
As shown in Table 3, NDRG2-low/EGFR(+) (hazard ratio (HR)=6.508; 95% confidence interval (CI), 2.619‑16.174; P<0.001), NDRG2-high/EGFR(+) (HR=3.519; 95% CI, 1.384‑8.949; P=0.008), and vascular invasion (HR=4.480; 95%CI, 2.291‑8.760; P<0.001) were independent prognostic factors of OS.