P-S6 was significantly overexpressed in NSCLC
Firstly, we detected the intracellular localization and the level of p-S6 expression by IHC in a total of 403 samples, of which 350 were NSCLC. The results indicated that p-S6 was mainly observed in the cell cytoplasm of lung SCC, ADC and Non-CLT (Figure 1). Quantification of p-S6 expression showed that its percentage of high expression was 30.5% (47/154) in lung SCC, 62.2% (122/196) in lung ADC and 15.1% (8/53) in Non-CLT, respectively. As shown in Supplementary Figure 1, compared with Non-CLT, the level of p-S6 protein was obviously increased both in lung SCC and ADC (p = 0.031, p < 0.001, respectively).
Association between p-S6 and clinicopathological features
As data showed in Table 1, p-S6 in female was higher than that in male (p < 0.001). Patients with lung ADC had obviously higher p-S6 expression level than patients with SCC (p < 0.001). Overexpressed p-S6 was significantly correlated with LNM (p = 0.001). Moreover, patients with high p-S6 expression level suffered a lower overall survival (OS) rate than that with low expression (p = 0.005). However, there was not significantly association between p-S6 expression and other features, such as clinical stages, pathological grade and age (all p > 0.05).
Table 1
Analysis of the association between the expression level of p-S6 and clinicopathological features of NSCLC (n=350)
Clinicopathological features (n)
|
p-S6
|
N
|
High (%)
|
Low (%)
|
P-value
|
Age(years)
|
|
|
|
|
≤50
|
95
|
48(50.5)
|
47(49.5)
|
|
༞50
|
255
|
121(47.5)
|
134(52.5)
|
0.632
|
Gender
|
|
|
|
|
Male
|
266
|
114(42.9)
|
152(57.1)
|
|
Female
|
84
|
55(65.5)
|
29(34.5)
|
0.000*
|
Clinical stage
|
|
|
|
|
Stage Ⅰ−Ⅱ
|
151
|
75(49.7)
|
76(50.3)
|
|
Stage Ⅲ
|
199
|
93(46.7)
|
106(53.3)
|
0.519
|
LN status
|
|
|
|
|
LNM
|
210
|
117(55.7)
|
93(44.3)
|
|
No LNM
|
140
|
52(37.1)
|
88(62.9)
|
0.001*
|
Histological type
|
|
|
|
|
SCC
|
154
|
47(30.5)
|
107(69.5)
|
|
ADC
|
196
|
122(62.2)
|
74(37.8)
|
0.000*
|
Pathological grade
|
|
|
|
|
Well/ Moderate
|
152
|
77(50.7)
|
75(49.3)
|
|
Poor
|
198
|
92(46.5)
|
106(53.5)
|
0.452
|
Survival status
|
|
|
|
|
Alive
|
187
|
77(41.2)
|
110(58.8)
|
|
Dead
|
163
|
92(56.4)
|
71(43.6)
|
0.005*
|
*Chi-square test, statistically significant difference (P < 0.05). Abbreviations: ADC, Adenocarcinoma, SCC, squamous cell carcinoma; LNM, lymph node metastasis. |
Impact of p-S6 expression on LNM in NSCLC
Among the 350 NSCLC patients, 210 cases had LNM and 140 cases were free from LNM. NSCLC patients with LNM had obviously higher p-S6 expression compared with those without LNM (p = 0.001, Table 1). We also evaluated the level of p-S6 in primary NSCLC tissues and its matched LNM lesions. As shown in Figure 2, regardless of lung SCC or ADC, the percentage of high p-S6 expression is significantly higher in metastatic lymph node lesions with compared with the matched primary cancer (p = 0.001, p = 0.022, respectively). To determine whether p-S6 expression was the independent predicted parameter for LNM in NSCLC, a multivariate logistic regression analysis was carried out. As mentioned in Table 2, highly expressed p-S6 (p = 0.001), clinical stages (p < 0.001), and age (p = 0.010) were notably correlated with LNM status of NSCLC patients. These results indicated that overexpressed p-S6 is an independent factor for LNM of NSCLC regardless of other parameters.
Table 2. Multivariate logistic analysis of LNM factors in NSCLC patients
Variables
|
|
|
|
95.0% CI for Exp (B)
|
B
|
S.E.
|
Wald
|
P-vaiue
|
Exp(B)
|
Lower
|
Upper
|
p-S6 expression
|
0.921
|
0.270
|
11.644
|
0.001*
|
2.512
|
1.480
|
4.262
|
Clinical stages
|
1.957
|
0.264
|
54.968
|
0.000*
|
7.068
|
4.220
|
11.880
|
Histological type
|
-0.026
|
0.276
|
0.009
|
0.925
|
0.974
|
0.567
|
1.674
|
Pathological grade
|
-0.051
|
0.256
|
0.039
|
0.843
|
0.951
|
0.575
|
1.571
|
Age
|
-0.759
|
0.293
|
6.688
|
0.010*
|
0.468
|
0.263
|
0.832
|
Gender
|
0.607
|
0.324
|
3.516
|
0.061
|
1.835
|
0.973
|
3.462
|
Abbreviations: S.E., standard error; Exp(B), odds ratio;CI, confidence interval; LNM, lymph node metastasis.*P< 0.05.
Inhibition of p-S6 reduced the cell migration and invasion of NSCLC
The alteration of cell migration and invasion ability of NSCLC were assessed after inhibition of p-S6 expression level inNSCLC cell lines (A549and SPC-A1).Firstly, we evaluated the inhibition efficiency by Western blotting after the cell lines were treated with RAD001. Figure 3 showed that p-S6 expression was significantly reduced after a 24 h treatment with RAD001 at 5 nmol/L.Secondly, to detect the effect of p-S6 on the cell migration, wound healing and transwell migration experiments were conducted. Figure 4 showed that compared with the control group, the wound healing percentage of RAD001 group was decreased by 10.19% and 20.44% at 24h and 48h in A549 cells (p = 0.018, p = 0.001, respectively), by 5.90% and 7.55% in SPC-A1 cells (p = 0.028, p = 0.048, respectively), respectively. Figure 5A revealed that the inhibition of p-S6 expression inA549 and SPC-A1 cell lines decreased the migration ability by transwell migration assay (both p < 0.001). Moreover, the cell invasion assay demonstrated that p-S6 inhibition in NSCLC cell lines reduced the number of invaded cells (both p < 0.001) (Figure 5 B). Together, these results revealed that inhibition of p-S6 expressioncould reduce the cell migration and invasion of NSCLC.
Impactof p-S6 expression on patients’prognosis
In univariate survival analysis,survival rate was estimated by Kaplan-Meier method in 350 NSCLC patients, and comparison was performed via log-rank test. NSCLC patients with highly expressed p-S6 had a poor prognosis compared to those with lowly expressed p-S6 (p= 0.013) (Figure6A & Table 3). The OS rate of NSCLC patients with advanced clinical stage (stageⅢ) was lower relative tothosewith early stages (stageⅠ-Ⅱ) (p< 0.001) (Figure6B & Table 3). Of note, patients without LNM have higherOSthan thosewith LNM (p< 0.001) (Figure6C & Table 3). Meanwhile, compared with NSCLC patients with well and moderated pathological grade, lower OS could be seen inthose with poor differentiation (p= 0.003) (Figure6 D& Table 3).
Table 3
Summary of univariate/multivariate analysis for overall survival in patients with NSCLC (n=350)
Variables
|
Univariate analysis
|
Multivariate analysis
|
Average survival time (SE)
|
95%CI
|
P-value
|
Exp (B)
|
95.0%CI
|
P-value
|
p-S6
|
|
|
|
|
|
|
High expression
|
44.561(3.050)
|
38.583-50.540
|
0.013*
|
1.549
|
1.105-2.173
|
0.011*
|
Low expression
|
67.500(5.336)
|
57.041-77.958
|
|
|
|
|
Clinical stages
|
|
|
|
|
|
|
Stage Ⅰ−Ⅱ
|
68.200(4.195)
|
59.977-76.423
|
0.000*
|
1.980
|
1.356-2.893
|
0.000*
|
Stage Ⅲ
|
47.862(3.834)
|
40.347-55.378
|
|
|
|
|
LN status
|
|
|
|
|
|
|
LNM
|
44.969(3.201)
|
38.695-51.243
|
0.000*
|
1.609
|
1.098-2.360
|
0.015*
|
No LNM
|
74.932(5.919)
|
63.330-86.533
|
|
|
|
|
Histological type
|
|
|
|
|
|
|
SCC
|
63.464(5.825)
|
52.046-74.882
|
0.240
|
1.137
|
0.806-1.603
|
0.464
|
ADC
|
50.588(3.367)
|
43.988-57.188
|
|
|
|
|
Pathological grade
|
|
|
|
|
|
|
Well/moderated
|
58.662(3.509)
|
51.784-65.541
|
0.003*
|
1.517
|
1.094-2.104
|
0.013*
|
Poor
|
54.226(4.164)
|
46.066-62.387
|
|
|
|
|
Age
|
|
|
|
|
|
|
≤50
|
45.395(3.633)
|
38.274-52.515
|
0.454
|
1.014
|
0.718-1.430
|
0.939
|
༞50
|
61.199(4.064)
|
53.234-69.165
|
|
|
|
|
Gender
|
|
|
|
|
|
|
Female
|
54.566(4.467)
|
45.810-63.321
|
0.210
|
0.718
|
0.484-1.064
|
0.099
|
Male
|
57.363(3.886)
|
49.747-64.978
|
|
|
|
|
Abbreviations: CI, confidence interval; Exp(B), odds ratio; SE, standard error; LNM, lymph node metastasis; |
* P< 0.05. |
To further investigated whether the overexpressed p-S6 was an independent prognostic index for NSCLC patients, Cox regression analysis was carried out. The results shown in Table 3 indicated that up-regulated p-S6 might be a poor prognostic marker for patients with NSCLC (p = 0.011), as are clinical stage (p < 0.001), LNM status (p = 0.015) and pathological grade (p = 0.013). Besides, age, gender, histological type had no significantly effect on the prognosis of patients with NSCLC.