Effects of single-nucleotide polymorphisms in the mTORC1 pathway on the risk of brain metastasis in patients with non-small cell lung cancer

Primary lung cancer is among the most common malignant tumors with the highest mortality worldwide (Chen et al. 2016; Siegel et al. 2016). Non-small cell lung cancer (NSCLC) accounts for almost 80% of all lung cancer cases (Hoffman et al. 2000) and has the highest incidence of brain metastasis (BM) (Nguyen et al. 2009). Approximately, 20–65% of patients with NSCLC will have BM during the course of the disease and ultimately die from it (Chen et al. 2014; Olmez et al. 2010; Preusser et al. 2012). Despite great progress in systemic therapy including surgery, radiotherapy, chemotherapy, and targeted therapy, the prognosis of BM patients remains generally poor, with a median survival time of approximately 13.7 months (Baek et al. 2018). Without any treatments, this further decreases to approximately less than 2 months (Chen et al. 2017). BM has become an important cause of lung cancer morbidity and mortality (Nayak et al. 2012). Therefore, identifying predictive factors of BM is essential for its prevention and improving patient survival.

Some previous studies have reported higher incidence of BM in those who are younger, female, have stage IIIB/IV disease, non-smokers, and with EGFR mutation (Hsiao et al. 2013; Wei et al. 2018). Other studies have also suggested that the size of the primary tumor, cell type, and intrathoracic lymph node stage were associated with BM (Mujoomdar et al. 2007). However, these studies did not take genetic factors and molecular mechanisms into account. BM develops via a complicated process that encompasses lung cancer cell invasion, migration, arrest, extravasation, and formation of micro-metastases (Hanahan and Weinberg 2011; Paduch 2016). It has been reported that matrix metalloproteinase (MMP) overexpression promotes the transmigration of lung cancer cells to the brain parenchyma through the blood vasculature and enhance the risk of BM (Feng et al. 2011; Hu et al. 2010). In addition, the expression of chemotactic factors including CXCL12 and its receptor CXCR4 was significantly higher in BM than in primary lung cancer; CXCR4 may improve the adhesion and chemoresistance of lung cancer cells, leading to BM (Chen et al. 2011; Paratore et al. 2011).One study of mice has shown that WNT/TCF signaling plays an important role in the formation of brain and bone metastasis through LEF1 and HOXB9 (Nguyen et al. 2009). Recently, Aljohani et al. revealed that the Keap1-Nrf2-ARE pathway mutations facilitate cancer cell migration to the distant sites in patients with BM (Aljohani et al. 2018). Despite extensive studies on the key molecular mediator of BM, its incidence rate has not markedly increased, highlighting the importance of identifying novel genetic biomarkers for BM.

Single-nucleotide polymorphisms are among the most common genetic variants and play an important role in the development, invasion, and prognosis of lung cancer. Therefore, SNPs may be a valuable molecular marker for assessing the risk of BM. Some studies have demonstrated that genetic variants of the phosphoinositide 3-kinase-Akt pathway and autophagy-related gene ATG16L1 are related to an increased risk of BM in patients with NSCLC (Li et al. 2013b, 2017). In this study, we focus on the relationship between genetic variation in mTORC1 pathway and the risk of BM patients in NSCLC.

In mammalian cells, mTOR signaling pathway has two forms: mTORC1 and the mTORC2. Of these, mTORC1 is composed of mTOR, mLST8, and RPTOR (Yang and Guan 2007). It has been proven to be a central regulator of numerous cellular processes, including sensing nutrient signaling, controlling translation and protein transport of mRNA in cells, and protein degradation (Sarbassov et al. 2005). More importantly, it is closely related to the occurrence and development of cancers (Dazert and Hall 2011). Previous studies have confirmed that alterations in the mTORC1 complex are associated with the occurrence of several cancers, including prostate cancer, liver cancer, and colon cancer and are sensitive to rapamycin inhibition (Chen et al. 2015; Kaibori et al. 2015; Shuhua et al. 2015). Another study revealed that functional SNPs of the mTORC1 gene may increase the risk of esophageal squamous cell carcinoma individually or collectively (Zhu et al. 2013). In addition, two similar studies have confirmed that mTORC1 polymorphisms are associated with the prognosis of gastric cancer (GC), and the risk of death increased more than twofold when GC patients have three risk genotypes of the mTORC1 SNPs (Xue et al. 2018). Collectively, these studies suggest that mTORC1 polymorphisms may be important biomarkers of cancer development, progression, and prognosis. However, no study has evaluated the potential role of functional SNPs in the susceptibility to BM from NSCLC.

Thus, this study aimed to investigate the association between eight potential functional polymorphisms of three genes (i.e., mTOR, mLST8, and RPTOR) in the mTORC1 pathways and the risk of BM in patients with NSCLC in the Chinese population. Moreover, given that BM of NSCLC is a polygenic disease, we also aimed to investigate the effect of combination of multiple SNPs on the prognosis of BM and the relationship between risk genotypes and various tumor-related indicators including serum tumor makers, Ki-67 proliferation index, and gene mutation frequency of BM from NSCLC.

BM causes serious neurological symptoms and is the leading cause of mortality in NSCLC. Despite this significant impact, the mechanisms by which BM develops remain unclear, and predictive biomarkers for BM are yet to be identified to date. In this study, we attempted to identify the molecular markers of BM by analyzing the association between eight potential functional polymorphisms of three genes in the mTORC1 pathways and the risk of BM. We found that patients with mLST8:rs26865 and mLST8:rs3160 variant genotypes had an evidently increased risk of BM. To our best knowledge, this is the first study to report that SNPs of mTORC1 pathway-related genes are associated with BM in NSCLC.

In this study, two SNPs associated with BM risk were located in mLST8, which mapped to chromosome 16p13.3 and was composed of 11 exons. mLST8 binds to the catalytic domain of mTOR and is among the crucial component proteins of the mTORC1 pathway (Yang et al. 2013). Furthermore, mLST8 not only enhances the stability of the interaction between RPTOR and mTOR, but also plays a vital role in stimulating mTOR kinase activity (Kakumoto et al. 2015). Therefore, mLST8 is crucial for the regulation of the mTOR pathway. Previous studies have revealed that mLST8 was upregulated in human colon and prostate cancer cells (Sapam et al. 2018) and contributed to tumor progression by activating the mTORC1 complex and its effector protein including translation initiation factor 4E-binding protein 1 (4E-BP1) (Yang and Guan. 2007). mLST8 upregulation could promote phosphorylation of 4E-BP1, which is known to play a role in tumor progression in various cancers including breast, ovarian, and prostate cancers (Armengol et al. 2007). Thus, we hypothesized that mLST8 is essential for tumor progression. A recent study focused on the relationship between SNPs of mLST8 and gastric cancer showed that although mLST8:rs26865 was not statistically related to gastric cancer, its hazard ratio of > 1 suggests a tendency to promote gastric cancer (Xue et al. 2018). These results suggest that our finding of increased BM risk in patients with polymorphisms of mLST8:rs26865 and mLST8:rs3160, at least in part, may be biologically reasonable.

He et al. evaluated the association between SNPs within mTORC1 genes and risk of GC in 1125 patients and 1196 healthy controls in a Chinese population, and the results showed that RPTOR rs1062935 and rs3751934 polymorphisms were not associated with the risk of GC (He et al. 2013). However, in our study, univariate Cox proportional hazard analysis showed that patients with TC/CC genotype of RPTOR:rs1062935 had significant lower risk of BM. These two inconsistent results may be explained by the difference in cancer types, sample size, and genetic susceptibility. In addition, it suggests that larger studies are needed to validate our findings.

Single SNP are well known to have relatively weak predictive power in cellular signaling pathways, while this is greatly enhanced when multiple SNPS are combined (Li et al. 2013a). When we combined two risk SNPs of mLST8:rs26865 and mLST8:rs3160, the cumulative effect of the risk genotype was “dose dependent,” that is, as the number of observed risk genotypes increased, the risk of brain metastases also increased significantly. These results suggest that multiple polymorphisms within the mTORC1 pathway have a cumulative effect and may further enhance the detective power. Nevertheless, evidence for real interactions of the risk SNPs is weak, and the mechanisms behind these high interactions are yet to be elucidated.

Approximately, 45% of patients with NSCLC have lymph node-positive disease, and higher lymph node stage was associated with the risk of BM (Waqar et al. 2018). Moreover, some studies reported that each component of the TNM staging system is correlated with the risk of BM (Mujoomdar et al. 2007). In our study, we analyzed the correlation between clinical status of BM patients and unfavorable genotypes. The results confirmed that BM patients with lymph node metastasis (N2/3) tended to have polymorphisms of rs26865 and rs3160 in mLST8. These observations further indicate that SNPs of mLST8 may be biologically feasible as a marker of BM.

Serum tumor markers remain a simple and economical tool for diagnosing and predicting the prognosis of lung cancer patients. Many serum components such as CEA, CYFRA 21-1, SCC, and NSE have been investigated and considered to be markers of this malignancy (Mauro et al. 2019). With respect to the relationship of serum tumor markers and BM, previous studies have demonstrated that patients with high CEA level had a higher risk of BM and poor prognosis (Lee et al. 2012). In the present study, we confirmed that AG/GG genotype of mLST8:rs26865 was associated with high CEA level and that mLST8 SNPs rs26865 and rs3160 polymorphic variants were associated to high SCC antigen level. Meanwhile, we did not find any association between NSE and the risk of BM, and this may be because NSE is a glycolytic enzyme that is commonly expressed in neuroendocrine tumors, particularly SCLC, rather than NSCLC. Nevertheless, these findings suggested that rs26865 and rs3160 of mLST8 SNPs may play a role in the pathogenesis of BM.

To our best knowledge, Ki-67 is expressed at multiple stages of the cell cycle except in the G0 phase, and the Ki-67 proliferation index is among the most common and widely applied biomarkers of cell proliferation. A previous study has shown that Ki-67 expression is higher in human lung adenocarcinoma tissues than normal lung tissue, and a higher Ki-67 proliferation index was related to the degree of differentiation, TNM stage, lymph node metastasis, and poor OS (Li et al. 2018). Moreover, high Ki-67 proliferation index in BM was associated with unfavorable survival (Berghoff et al. 2014). Our study found that BM patients with AG/GG genotype of mLST8:rs26865 and TC/CC genotype of mLST8:rs3160 expressed a significantly higher Ki-67 index than those with AA genotype and TT genotype. This suggests that patients with mLST8 SNPs rs26865 or rs3160 polymorphic variants may have shorter OS. Coincidentally, by analyzing the effect of risk genotypes of SNPs on BM patient outcomes, we found that patients with AG/GG genotype of mLST8:rs26865 had significantly poorer OS. However, the OS was not significantly different between those with TC/CC genotype and those with TT genotype of mLST8:rs3160. These results indicated that the role of rs26865 and rs3160 polymorphic variants of mLST8 in the development of BM should be further studied.

Recently, one study profiled the characteristics of brain metastases from NSCLC and showed that the EGFR mutation rate in BM was 36.3% (Ferguson et al. 2018). Furthermore, while NSCLC patients with EGFR mutations had a higher risk of brain metastases, they had better prognosis than those with EGFR wild-type mutations (Hsu et al. 2016). In our study, the frequency of EGFR mutation was 36.9% in 130 BM patients, which is consistent with previous finding (Ferguson et al. 2018). Moreover, the frequency of EGFR mutation was significantly higher in patients with AA genotype of mLST8:rs26865 than those with AG/GG. This may indicate that patients with wild-type genotype of mLST8:rs26865 SNP, at least in part, may have better survival. Nevertheless, we did not detect other gene mutations such as TP53 and K-ras that have been previously confirmed to have a high frequency of mutations in brain metastases (Ma et al. 2018). This is due to the limitations of patient genetic testing data and underline that we should collect more complete information to better investigate the association between polymorphisms in mTORC1 pathway and BM of NSCLC.

This study has some limitations that should be considered when determining the generalizability of the results. One limitation is the relatively small sample size, and thus and this may have influence the finding of weak effect of polymorphisms in BM. Furthermore, we selected potential functional genetic variants instead of tagging SNPs of mTORC1 pathway in the analysis, and thus some other important genetic variants may have not been included. Finally, the functional mechanisms by which potential genetic variations affected BM have not yet been elucidated. Therefore, additional larger, well-designed, and in-depth studies should be performed to overcome these limitations.

In conclusion, polymorphisms of mTORC1 pathway were associated with the risk of BM. We found that rs26865 and rs3160 polymorphic variants of mLST8 were significantly associated with the risk of BM, particularly among those with lymph node metastasis (N2/3), high CEA and SCC antigen level, and high Ki-67 proliferation index and gene mutation frequency. These findings show that SNPs, combined with other predictive indicators, may be valuable biomarkers for identifying NSCLC patients with a high risk of brain metastasis.