Overexpressed P-S6 Associates with Lymph Node Metastasis and Predicts Poor Prognosis in Non-Small Cell Lung Cancer.

Background Ribosomal protein S6 (S6), a downstream effect media of the AKT/mTOR pathway, not only is a part of 40S small subunit of eukaryotic ribosome, but also involves in protein synthesis and cell proliferation during cancer development. In present study, we explore the association between phosphorylated S6 (p-S6) protein expression and clinicopathological features as well as prognostic implications in NSCLC. P-S6 was detected in tissue microarrays (TMAs) containing 350 NSCLC, 53 non-cancerous lung tissues (Non-CLT), and 88 cases of matched metastatic lymph node lesions via immunohistochemistry (IHC). Transwell assays and wound healing assay were used to assess the effects of p-S6 inhibition on NSCLC cell metastasis. 70kDa S6; Non-CLT: Non-cancerous lung

Construction of the tissue microarrays As previously described, tissue microarrays (TMAs) were constructed using para n-embedded tissue of NSCLC and Non-CLT [24]. The perforation diameter of each sample was 0.6 mm. For TMAs of NSCLC, each case included two tumor cores, of which 88 cases each also contained two matched metastatic lymph node cores. For TMAs of Non-CLT, two cores per case was included. The average score of the two cores was regarded as the nal score for each case.
Immunohistochemistry and scores IHC for p-S6 in TMAs was carried out and the conditions of antibody staining were adjusted as described previously [25]. Brie y, primary antibody for p-S6 (1:800 of Ser235/236) was applied to detect its expression level. The positive control slide and negative control slide were included in each experiment. The IgG isotype-matched antibody was applied as negative contrast to con rm the antibody speci city.
Immunoreactivity was assessed semi-quantitatively by two independent pathologists. The staining intensity score was multiplied by percentage score of positive expressed tumor cells as the total score. Speci cally, staining intensity was scored as 0 to 3 (0 for negative; 1 for mild expression; 2 for moderate expression; 3 for strong expression). The percentage score was identi ed as 0 to 4 in view of positive cytoplasmic staining: 0 = 0%; 1 = 1%-25%, 2 = 26%-50%, 3 = 51%-75%, 4 = 76%-100%. The range of total score was from 0 to 7. Cutoff level which determined according to overall survival (OS) of NSCLC patients was 2. Expression of p-S6 in tumor cells of which total score is more than 2 was considered high expression and others were regarded as low expression. Agreement between the two assessors is 96%, borderline and ambiguous cases are resolved through discussion.

Western blotting
As previously described [26], preparation of protein lysates and western blotting analysis were performed. The antibodies were used as follows: 1:1000 of Ser235/236 for anti-p-S6 and 1:5000 for anti-β-Actin.
Wound healing assay 1*10 5 SPC-A1 and A549 cells were seeded and then treated with DMSO or 5 nmol/L RAD001 at about 60% con uence and cultured until 90%. Serum free RPMI-1640 was used to replace the culture medium and wounds were scratched with the tip of 10µl pipette in each well. The size of wounds was observed using inverted microscope (Leica, Germany) and images were captured at 0h, 24h and 48h. The data presented were repeated in triplicate. Wound healing percentage (%) = (1 -scratch area at t / scratch area at 0h) * 100% (t means 24h or 48h).
Transwell assays 24-well chambers (Costar 3422; Corning, USA) were used to perform transwell migration assays. A549 and SPC-A1 cells were treated with DMSO or 5 nmol/L RAD001 for 24h at 37°C. Then 5*10 4 cells were extracted from each group and suspended in 200µl of serum-free RPMI-1640 to the upper chamber, while 10% FBS RPMI-1640 was added to the lower room. The cells could migrate through the membrane for 12h to 48h. The membrane was xed for 20 min in 4% paraformaldehyde solution and dyed with crystal violet for 20min at room temperature. The count of cells under the cell membrane was calculated. The implementation of invasion assay protocol be analogous to that of migration assay, except that Matrigel was coated on the upper cavity and the incubation time was 1 h (356234, Corning, USA).

Statistical analysis
The difference expression pattern of p-S6 between Non-CLT and NSCLC, as well as the association between p-S6 and clinicopathological features were analyzed by Chi-square (χ 2 ) test. The difference between the metastatic lymph nodes and matched primary tumors was also analyzed by Chi-square(χ 2 ) test. Multi-logistic regression method was performed to identify whether p-S6 is an independent marker for LNM of NSCLC. Survival rate curve was evaluated by Kaplan-Meier analysis, and comparisons were analyzed via log-rank test. Furthermore, cox proportional hazards model was performed for determining independent prognostic markers. The deviation between control and RAD001 groups was analyzed using one way t-test. All these statistical analyses were put into effect with SPSS Statistics software (version 24). Numerical data were presented as mean ± SEM. P < 0.05 (Two-sided) indicates that the result is statistically signi cant.

P-S6 was signi cantly 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). Quanti cation 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 signi cantly 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 signi cantly association between p-S6 expression and other features, such as clinical stages, pathological grade and age (all p > 0.05).

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 signi cantly 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. Abbreviations: S.E., standard error; Exp(B), odds ratio;CI, con dence 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 e ciency by Western blotting after the cell lines were treated with RAD001. Figure 3 showed that p-S6 expression was signi cantly 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.  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 upregulated 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 signi cantly effect on the prognosis of patients with NSCLC.

Discussion
In this study, we demonstratedthat the level of p-S6 protein expressionin NSCLCwas signi cantly higher than that in Non-CLT. The percentage of high p-S6 expression was obviously increased in patients with LNM than that without LNM. Inhibitionof p-S6 expression reduced the invasion andmigrationability of NSCLC cells. Our analysis indicated that NSCLC patients with increased p-S6 expression had a lower rate ofsurvival than that with low expression ones. Taking together, our results imply that p-S6 may play a signi cantpart in the progressionof NSCLC and aberrant expression of p-S6 might be a novel prognostic marker for NSCLC.Increasing evidenceshave shown that mTOR/S6K/S6 pathway plays a crucial role in p53-mediated tumor inhibition [27,28].P-S6, the key downstream effector of mTOR/S6K pathway, is involved in the occurrence and development of manycancers [9].Its expressionis ascendant in numerous tumors, such asRCCs,pancreatic cancer, and esophageal squamous cell carcinoma [18,27,29].The phosphorylation of S6 can attenuate the autophagy induced by damage-regulated autophagy modulator 1 (DRAM1) and p-S6 is a requirement for AKT-driven malignant transformation of pancreatic islet β cells [28,30].Due to the oncogenic role of p-S6, descending its expression could potentially provide a clue to nd new idea for the targeted treatment cancers. For example, the suppression of p-S6 can block the further inhibition of the therapeutic mTOR inhibitor everolimus on protein synthesis and proliferation of RCCs cells [18].Knockdown of S6K1 gene can obviously decrease the expression of cyclin D, leading to the decline of survival ability of esophageal cancer cells [29].
Activating invasion and metastasis is one of biological capabilities of cancers [31].Our study showed that NSCLC patients with LNM had higher p-S6 expression.Furthermore, the expression level of p-S6 in metastatic lymph node lesion was higher than that in matched primary lesion. Multivariate analysis indicated that the high p-S6 expression could be an independent predicted markerfor LNM in patients withNSCLC. To further clarify the metastatic ability of p-S6 in NSCLC, we inhibited p-S6 expression in A549 and SPC-A1 cell lines and found that down regulation of p-S6 weakened the migration and invasion ability of NSCLC cells. Taking together, these data suggest that p-S6 might play amajor part in promotinginvasion and metastasis of NSCLC.Similar to our discoveries, previous studies have reported that overexpressed p-S6 is positively related toLNM inRCCs, colorectal cancer, and epithelial ovarian cancer[18, 32,33].On the other hand, inhibition of p-S6 expression or S6 gene knockdown can signi cantly suppress the cell invasion andmigration of several kinds of human cancers, such as esophageal cancer and colorectal cancercells [29,34].All the mentioned above indicate p-S6 positively affects cell invasion and migration. Despite the number of studies describing the aberrant expression of p-S6 in tumors activating invasion and metastasis, the concrete biological mechanism is still unclear.One study report that the phosphorylation defect of S6 inhibits the phosphorylation of paxillin, a focal adhesion protein, leading to inhibit the formation of local adhesion [29].In this current study, we have little knowledge of how the aberrant expression of p-S6 involved in the LNM of NSCLC, further explorations for the intrinsic mechanism are required in the future.
Our results indicatedthat the OS rate of NSCLC patients with highly expressedp-S6was obviously lower than that of thosewith low level of p-S6. Multivariate analysis showed that high expressedp-S6 wasan independently prognostic indicator in NSCLC patients, which seem to favor the oncogenic role of p-S6.
Previous studies on RCCs, gastric cancer, and glioblastomas also identi ed p-S6 as a novel poor prognosis biomarker [16][17][18].Up to now, there are many inhibitors that can prevent S6 phosphorylation.
Studies have reported that inhibition of p-S6 can signi cantly reduce tumor growth, which is important for effective response to treatment of triple negative breast cancer [35,36]. In addition, mTOR inhibitor everolimus can effectively inhibit the level of p-S6, thereby reversing the resistance of HER2-mutant cancers to neratinib and exerting anti-tumor effects [37]. These further suggest that p-S6 may be a powerful biomarker in tumors and may provide novelstrategy intargeted therapy of NSCLC.

Conclusions
In summary, a signi cant overexpression of p-S6 was found in NSCLC.Inhibition of p-S6 expression could weakened the migration and invasion ability of NSCLC cells and aberrant expression level of p-S6 might be an independent predictor for LNMof NSCLC patients.In addition, overexpressed p-S6 may be a novel poor prognostic biomarker for NSCLC patients.

Supplementary Files
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