Background

Lung cancer is a clinical malignant tumor with highest incidence, with about 2 million new lung cancer cases annually, and its mortality rate is also the highest worldwide [1]. 80 to 90% of lung cancer are diagnosed as non-small cell lung cancer (NSCLC), which is mainly classified into adenocarcinoma (ADC) and squamous cell carcinoma (SCC) [2]. Lung cancer patients’ survival varies depending on lymph node metastasis (LNM), clinical stage, geographic region, and other factors. The 5-year survival of NSCLC patients in clinical stageIis about 57%, which is far more than that of patients with stage IV (that is less than 5%) [3]. Unfortunately, more than half of the NSCLC patients are diagnosed in advanced clinical stages, often accompanied by LNM and distant metastasis, which are important reasons for the poor prognosis [3, 4]. Therefore, it is an urgent need to find new biomarker for early detection of LNM of NSCLC and predict the prognosis.

Recently, the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signal pathway is over-activated in numerous tumors, including NSCLC, and associated with tumor angiogenesis, invasion, metastasis and so on [5,6,7]. Ribosomal protein S6 (S6), a part of 40S small subunit of eukaryotic ribosome, is a famous downstream effect media of the AKT/mTOR pathway [8, 9]. As the first ribosomal protein proved to undergo inducible phosphorylation, S6 is mostly induced by activated 70 kDa S6 Kinases (S6K) at five phosphorylation sites (Ser235, Ser236, Ser240, Ser244, Ser247), mainly Ser236 [9,10,11,12]. Phosphorylated S6 (p-S6) plays a crucial part in protein synthesis, cell size control and cell proliferation as increasing the affinity of the ribosome and mRNAs and improving the efficiency of protein translation [13]. In addition, p-S6 is also the key effector of mTOR in regulating cell size, whose decreased expression results in smaller cell size and reflects the growth defect [14, 15]. Overexpression of p-S6 is found in various solid tumors, such as gastric cancer, glioblastomas, and renal cell carcinomas (RCCs), and associated with poor prognosis [16,17,18]. S6 phosphorylation was even associated with malignant potential and glucose metabolism of intraductal papillary mucinous neoplasm of the pancreas [19]. In addition, the phosphorylation of S6 is considered to contribute to acquired resistance to MAPK pathway inhibitors in cancers, suggesting that p-S6 plays an essential role in the mechanism of anti-cancer drugs [20, 21].

However, the prognostic implications of p-S6 are poorly understood in NSCLC. In this present research, we have measured the level of p-S6 expression in tissue microarrays (TMAs) of non-cancerous lung tissues (Non-CLT) and NSCLC by immunohistochemistry (IHC) and decreased p-S6 expression in NSCLC cell lines with mTOR inhibitor (RAD001), to estimate not only the association between p-S6 and clinicopathological/prognostic features of NSCLC patients, but also the alteration of NSCLC cell migration and invasion ability.

Material and Methods

Patients and tissue samples

All tissue specimens which were collected by surgical resection acquired from the Thoracic Surgery Department of the Second **er S, Kopitz J, Duensing S, Pahernik S, Hohenfellner M, et al. The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker. OncoTarget. 2016;7(1):418–32. https://doi.org/10.18632/oncotarget.6225 ." href="/article/10.1186/s12885-022-09664-4#ref-CR18" id="ref-link-section-d1449378e2835">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 find 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 marker for LNM in patients with NSCLC. Ribosomal protein S6, as a famous effector of mTOR signal pathway, mostly involved in the regulation of cell size and proliferation [9]. However, there is rarely report about the effect of p-S6 on metastasis of NSCLC. 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 a major part in promoting invasion and metastasis of NSCLC. Similar to our discoveries, previous studies have reported that overexpressed p-S6 is positively related to LNM in RCCs, colorectal cancer, and epithelial ovarian cancer [18, 32, 33]. On the other hand, inhibition of p-S6 expression or S6 gene knockdown can significantly suppress the cell invasion and migration of several kinds of human cancers, such as esophageal cancer and colorectal cancer cells [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 indicated that the OS rate of NSCLC patients with highly expressed p-S6 was obviously lower than that of those with low level of p-S6. Multivariate analysis showed that high expressed p-S6 was an 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 identified 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 significantly 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 novel strategy in targeted therapy of NSCLC.

Conclusions

In summary, a significant 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 LNM of NSCLC patients. In addition, overexpressed p-S6 may be a novel poor prognostic biomarker for NSCLC patients.