Prognostic roles of mRNA expression of notch receptors in non-small cell lung cancer

Notch signalling is aberrantly activated in human non-small cell lung cancer (NSCLC). Nevertheless, the prognostic roles of mRNA expression of four Notch receptors in NSCLC patients remain elusive. In this report, we reported the prognostic roles of Notch receptors in a total of 1,926 NSCLC patients through “The Kaplan-Meier plotter” (KM plotter) database which is capable to assess the effect of 22,277 genes on survival of NSCLC patients. We found that mRNA high expression level of Notch1 was associated with better overall survival (OS) for all NSCLC patients, hazard ratio (HR) 0.78 (0.69-0.89), p=0.00019, better OS in adenocarcinoma (Ade) patients, HR 0.59 (0.46-0.75), p=1.5e-05, as well as in squamous cell carcinoma (SCC) patients, HR 0.78 (0.62-0.99), p=0.044. mRNA high expression levels of Notch2 and Notch3 were associated with worsen OS for all NSCLC patients, as well as in Ade, but not in SCC patients. mRNA high expression level of Notch4 was not found to be associated with to OS for all NSCLC patients. In addition, mRNA high expression levels of Notch2, Notch3, but Notch4 are significantly associated with the NSCLC patients who have different smoking status. Our results indicate that mRNA expression of Notch receptors may have distinct prognostic values in NSCLC patients. These results will benefit for developing tools to accurately predict the prognosis of NSCLC patients.


INTRODUCTION
Lung carcinoma is the leading cause of cancerrelated death worldwide, and among this carcinoma, non-small cell lung carcinoma (NSCLC) comprises the majority of cases [1][2]. NSCLC includes: adenocarcinoma (Ade), squamous cell carcinoma (SCC), two major histological types. In spite of the progresses in early diagnosis, radical cure treatment, and molecular targeted therapies, the majority of NSCLC patients present with advanced stage disease and recur in about one third of individuals [3][4]. Therefore, it is still needed to further investigate the molecular mechanism of initiation, development and to identify potential prognostic markers and potential drug targets of NSCLC.
The Notch signaling pathway, an evolutionarily conserved and complex signaling system is known to regulate epithelial cell and stem cell homeostasis [5][6]. Notch signalling is aberrantly activated in several human tumors including breast, colon, cervical, pancreatic, head and neck, prostate cancer, renal carcinoma, Large-cell and Hodgkin lymphomas, as well as NSCLC [7][8][9][10]. Previous reports demonstrate that Notch signaling is activated and plays a critical role in NSCLC initiation, progression, and metastasis of NSCLC [10][11][12][13]. However, the prognostic values of mRNA expression of Notch receptors in NSCLC patients have not been determined. In this report, we determined the prognostic roles of mRNA expression of four Notch receptors in NSCLC patients.

RESULTS
It is well known that Notch signaling have four receptors, Notch1~4. All Notch receptors can be found in the database (www.kmplot.com).
For further assess the association of Notch receptors with other clinicopathological profiles, we determined the correlation with the patients' smoking status (Table  1), clinical different stages ( Table 2) and different chemotherapeutic treatment (  Table 3, only Notch3 is significantly correlated with the patients without chemotherapy.
Notch1, among four Notch receptors, is the best studied in NSCLC carcinogenesis [41][42][43][44]. Increased frequency of Notch1 activation can induce lung adenoma formation and enable progression to Ade and metastases [45]. Notch1 activation can also enhance epithelialmesenchymal transition (EMT) in lung cancer cells [46][47]. Cisplatin was also observed to activate Notch1 first, then further induce the enrichment of CD133 positive cells in lung Ade [48]. Notch1 function was found to be required for tumor initiation through suppression of p53mediated apoptosis in NSCLC [49]. But recently, Wael et al observed that Notch1 expression has a tumor inhibitory effect on Ade cells, but not SCC cells in NSCLC [50]. A meta-analysis showed that Notch1 was correlated with lymph node metastasis, TNM stages and significantly poor OS in NSCLC patients [51]. Notch1 mRNA was expressed on both mesenchymal and epithelial structures of the embryonic lung, indicating that Notch1 has a role in the vascular morphogenesis and lung development [52]. In our report, mRNA high expression of Notch1 was correlated to better OS for all NSCLC patients, also better OS in Ade and SCC patients.
Notch2 is a tumor suppressor in lung carcinogenesis [53]. But, the prognostic significance of either Notch2 protein or mRNA in NSCLC patients is not known. In our report, mRNA high expression of Notch2 was significantly correlated to worsen OS for all NSCLC patients, HR 1.29 (1.13-1.46), p=9.1e-05. Notch2 mRNA high expression was also correlated to worsen OS in Ade patients, HR 2.2 (1.72-2.81), p=9.2e-11, but not in SCC patients, HR 1 (0.79-1.26), p=0.99.
Notch3 potentially contributes to the multistep evolution of lung cancer in airway epithelium by using a transgenic mouse model [54]. High Notch3 expression may contribute the resistance to chemotherapy in lung cancer patients [55]. Notch3 was found to upregulate ZEB-1, which leads to TGF-β-induced the EMT and bone metastasis in NSCLC [56]. A report showed that Notch3 was an independent prognostic factor for patients with NSCLC [57]. In consistent with previous report, our results showed that mRNA high expression of Notch3 was found to be significantly correlated to worsen OS for all NSCLC patients, HR 1. 19  Notch4 is an oncogene in mammary tumor [58][59]. Notch4 was able to expended the tumorigenicity in mammary epithelial cells [60][61]. Although Notch4 was detected in both tumor and stromal compartments    of NSCLC [62], the significance of Notch4 in NSCLC needs to be further determined. In this report, mRNA high expression of Notch4 was not significantly correlated to OS for all NSCLC patients, Ade patients, as well as SCC patients. Nicotine can deregulate essential biological activities such as the cell apoptosis, proliferation, migration, invasion, inflammation, cell-mediated immunity and angiogenesis [63]. Nicotine might be promoting NSCLC growth and metastasis by inducing the secretion of stem cell factor [64]. There is no report regarding whether there is a direct correlation between nicotine and Notch activation in NSCLC. However, Hirata et al [65] reported that nicotine can increase ALDH-positive cells through a PKC-Notch pathway in MCF-7 cells. In addition, nicotine may impact specific Notch members in endothelial cells [66] or dendritic cells [67], thus indirectly impact NSCLC. Nicotine was also reported recently to enhance the expression of synaptic and Notch1 proteins in stressed animals [68]. In this report, we observed that high mRNA expression of Notch2, Notch3, but Notch4 are correlated with smoking status of NSCLC patients. High mRNA expression of Notch 1 is only significantly associated with smoked NSCLC patients.
Previous results suggest that Notch signaling, especially Notch receptors may be essential drug target for NSCLC patients. γ-secretase inhibitor, DAPT was demonstrated to inhibit Notch activation and cell growth in NSCLC cells [69]. However, γ-secretase inhibitors are not able to distinguish individual Notch receptors and may cause intestinal toxicity [70] by inhibiting other signaling pathways [71]. Recently, phage display technology may produce highly specialized antibodies which can recognize each Notch receptor paralogue in human patients and rodent models [72]. Based on our study that mRNA high expression of Notch2 and Notch3 was correlated to worsen OS for all NSCLC patients, thus Notch2 and Notch3 might be potential drug targets for NSCLC patients.
In summary, we demonstrated that Notch1 mRNA high expression was correlated to better OS for all NSCLC patients. Notch2 and Notch3 mRNA high expression were correlated to worsen OS for all NSCLC patients. Notch4 mRNA high expression were not correlated to OS for all NSCLC patients. These results will be useful for better understand the complexity and heterogeneity in the molecular biology of NSCLC and to develop tools to more accurately predict their prognosis. Based on our study, Notch2 and Notch3 might be potential drug targets for NSCLC patients.

MATERIALS AND METHODS
An online database [36] was used to determine the relevance and significance of Notch receptors' mRNA expression to overall survival (OS). NSCLC patients in the KM plotter database were come from the Gene Expression Omnibus (GEO, http://www.ncbi. nlm.nih.gov/geo/), Cancer Biomedical Informatics Grid (caBIG, https://biospecimens.cancer.gov/ relatedinitiatives/overview/caBig.asp), and The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov) lung cancer datasets [73]. The database was established using gene expression data and survival information of 1,928 NSCLC patients downloaded from above three datasets. The NSCLC patients were followed up 20 years. By using KM plotter, four Notch receptors (Notch1, Notch2, Notch3 and Notch4) were entered into the database (http://kmplot.com/analysis/index. php?p=service&cancer=breast) to obtain Kaplan-Meier survival plots in which the number-at-risk is indicated below the main plot. The certain gene mRNA expression above or below the median separates the cases into high expression and low expression. Hazard ratio (and 95% confidence intervals) and log rank P were calculated and displayed on the webpage.

CONFLICTS OF INTEREST
The authors have no financial interest in this project.

Author contributions
JX, XZ, DY participated in the design of the study and drafted the manuscript. JX, XZ, XC, YW, DL, YH, JJX reviewed and extracted data. JX, XZ and DY participated in the search the study and performed the statistical analysis. JX and DY wrote the manuscript. All authors reviewed the manuscript. www.impactjournals.com/oncotarget