Prognostic values of four Notch receptor mRNA expression in gastric cancer

Notch ligands and receptors are frequently deregulated in several human malignancies including gastric cancer. The activation of Notch signaling has been reported to contribute to gastric carcinogenesis and progression. However, the prognostic roles of individual Notch receptors in gastric cancer patients remain elusive. In the current study, we accessed the prognostic roles of four Notch receptors, Notch 1–4, in gastric cancer patients through “The Kaplan-Meier plotter” (KM plotter) database, in which updated gene expression data and survival information include a total of 876 gastric cancer patients. All four Notch receptors’ high mRNA expression was found to be correlated to worsen overall survival (OS) for all gastric cancer patients followed for 20 years. We further accessed the prognostic roles of individual Notch receptors in different clinicopathological features using Lauren classification, pathological grades, clinical grades, HER2 status and different choices of treatments of gastric cancer patients. These results indicate that there are critical prognostic values of the four Notch receptors in gastric cancer. This information will be useful for better understanding of the heterogeneity and complexity in the molecular biology of gastric cancer and to develop tools to more accurately predict their prognosis.


Material and Methods
An online database 35 (http://kmplot.com/analysis/) was used to determine the relevance of individual Notch receptor mRNA expression to the overall survival (OS). OS is the length of time from either the date of diagnosis or the start of treatment for a cancer patient, that patients diagnosed with the cancer are still alive. In a clinical trial, measuring the OS is one of important ways to see how well a new treatment works. Currently, they established breast cancer 35 , lung cancer 19 , ovarian cancer 36 and gastric cancer data. All cancer patients in the database were identified from Cancer Biomedical Informatics Grid (caBIG, http://cabig.cancer.gov/, microarray samples are published in the caArray project), the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov) cancer datasets 19 . They collected clinical data including gender, perforation history, Lauren classification, differentiation, stage, HER2 status and treatment. The database was established using gene expression data and survival information of 876 gastric cancer patients downloaded from Gene Expression Omnibus (GEO). Briefly, four Notch sub-members (Notch1, Notch2, Notch3 and Notch4) were entered into the database (http://kmplot.com/analysis/index.php?p= service&cancer= gastric) to obtain Kaplan-Meier survival plots in which the number-at-risk is indicated below the main plot. Hazard ratio (HR) and 95% confidence intervals, as well as log rank P were calculated and displayed on the webpage. P value of < 0.05 was considered to be statistically significant. HR is the ratio of the hazard rates corresponding to the conditions described by two levels of an explanatory variable in survival analysis.

Discussion
Among four Notch receptors and ligands, Notch1 is relatively the most studied member of Notch signaling in gastric carcinogenesis 7,37-39 . With the active form of Notch 1, the Notch 1 intracellular domain (NICD) was frequently expressed in gastric cancer cell lines, and the depletion of Notch 1 by Notch 1 siRNA led to growth inhibition of gastric cancer cells 17,40 . Down-regulation of Notch1 expression by gamma-secretase inhibition (N-[N-(3,5-diflu orophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, DAPT) was also able to substantially inhibit migration, invasion, and proliferation, as well as epithelial-mesenchymal transition in gastric cancer cell lines 41 . Changes in the expression of the Notch1 intracellular domain (NICD) differentially expressed in gastric cancer, and the aberrant expression of Notch1 NICD is associated with an advanced tumor stage, tumor metastasis and overall patient survival 42 . Du et al. 43 performed a meta-analysis and showed that the expression of Notch1 protein was significantly higher in tumor tissues of gastric cancer compared to normal tissues. Specifically, stratified analyses showed that significantly increased expression of Notch1 was associated with non-cardia location, > 5 cm size, diffuse type, positive lymphovascular invasion and distal metastasis, indicating that Notch1 protein may be an oncogene. Recently, Bauer et al. 44 reported that primarily resected patients with Notch1 protein-negative tumors demonstrated worse survival and high Notch1protein expression was associated with early-stage tumors and associated with significantly increased survival in this subgroup. Their results supported that Notch1 expression indicated good prognosis in gastric cancer patients. However, whether or not Notch1 mRNA has a prognostic role in gastric cancer patients remains elusive. In this report, Notch1′ s high mRNA expression was found to be correlated to worsen OS for all gastric cancer patients followed for 20 years. Notch1′ s high mRNA expression was also found to be correlated to worsen OS in intestinal type cancer patients, but not in diffuse type cancer patients.   46 . Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of Notch2 staining, indicating a prominent role for Notch2 in chemotherapy resistance of gastric cancer 47 . Above results indicate that Notch2 seems to be a tumor oncogene in gastric carcinogenesis. Du et al. 43 reported that the expression of Notch2 protein significantly was higher in tumor tissues of gastric cancer compared to normal tissues. However, Bauer et al. 44 reported that higher Notch2 protein expression was associated with early-stage and intestinal-type tumors and with associated better survival in the subgroup of intestinal-type tumors. Their results support that Notch2 expression with early tumor stages suggest that Notch2 may act as a tumor suppressor in gastric cancer. However, there is no report about the prognostic significance of Notch2 mRNA expression in gastric cancer. In this report, Notch2′ s high mRNA expression was found to be significantly correlated to worsen OS for all gastric cancer patients, as well as in intestinal type cancer patients and in diffuse type cancer patients.
The study about Notch3 in gastric cancer is limited 43 . The expression of Notch3 protein was observed to be associated with the intestinal/glandular differentiation of gastric carcinoma cells, suggesting a role as a possible favorable prognostic indicator 48 . In this report, our results show that Notch3′ s high mRNA expression was significantly correlated to worsen OS for all gastric cancer patients, as well as in intestinal type cancer patients and diffuse type cancer patients.  Same as Notch3, the study about Notch4 in gastric cancer is also limited. In a recent report 49 , Notch4 activation was observed to promote gastric cancer growth in vitro and in vivo, while Notch4 inhibition using Notch4 siRNA had opposite effects. In addition, Notch4 activation induced expression and activation of Wnt1, β -catenin and downstream target genes, c-Myc and cyclin D1, in gastric cancer cells, while Notch4 inhibition had opposite effects. Wnt1 is one of WNT members that regulates various processes including tumor initiation, tumor growth,  cell senescence, cell death, differentiation and metastasis 50,51 . β -catenin is the primary transducer of canonical WNT signals to the nucleus and is involved in the development and progression of a significant proportion of gastric cancer cases 52,53 . Activation of the cyclin D1 oncogene, often by amplification or rearrangement, is a central mediator in the transition from G1 to S phase and a major driver of multiple types of human tumors including gastric cancer [54][55][56] . These results indicate that Notch4 seems to be a tumor oncogene in gastric carcinogenesis.
In this report, we observed that Notch4′ s high mRNA expression was also found to be significantly correlated to worsen OS for all gastric cancer patients, intestinal type cancer patients, and diffuse type of cancer patients. The HER2/neu proto-oncogene (also known as c-erbB-2) encodes a 185 kDa transmembrane glycoprotein receptor known as HER2/neu or p185, HER2 partial homology with EGFR. HER2/neu shares a receptor with intrinsic tyrosine kinase activity and has been implicated in cancer with special emphasis in breast cancer 57,58 . HER2 overexpression was detected in 6% to 35% of GC patients and led to the advent of targeted therapy with anti HER2 antibody like Trastusumab which has improved the overall survival 59,60 . In this study, we found that all the individual Notch receptors except Notch 3 are significantly associated with worsen OS in either HER2 negative or HER2 positive gastric cancer patients. Notch 3′ s high mRNA expression is only significantly associated with worsen OS in HER2 negative gastric cancer patients.
The connection between Notch signaling and carcinogenesis, as well as its crosstalk with many oncogenic signaling pathways suggest that Notch signaling, especially some Notch receptors may be candidate for drug target of gastric cancer. A number of γ -secretase inhibitors were demonstrated to inhibit Notch activation and cell growth in gastric cancer cells 41,45,61,62 . The treatment combination of γ -secretase inhibitor and 5-FU was shown to be better than that with the single treatment in the inhibition of gastric cancer cell proliferation 61 . Therefore, Notch expression status could also impact the treatment efficiency of 5-FU based adjuvant therapy and/or prognosis of gastric cancer patients. In this study, we observed that Notch 1 mRNA high expression is associated with worsen OS in surgery alone gastric cancer patients and 5-FU based adjuvant gastric cancer patients. Notch 3 and Notch 4′ s high mRNA expression is only associated with worsen OS in surgery alone for gastric cancer patients. Interestingly, Notch 2′ s high mRNA expression is only associated with better OS in 5-FU based adjuvant gastric cancer patients.
In summary, by using the KM plotter database, we accessed the prognostic roles of four Notch receptors in gastric cancer patients through KM plotter, in which updated gene expression data and survival information included data from a total of 876 gastric cancer patients. All four Notch receptors' high mRNA expression was found to be correlated to worsen overall survival (OS) for all gastric cancer patients followed for 20 years. We further accessed the prognostic roles of individual Notch receptors in different clinicopathological features including Lauren classification, pathological grades, clinical grades, HER2 status and different choices of treatments of gastric cancer patients. These results indicate that there are critical prognostic values of Notch 1-4 receptors in gastric cancer. This information will be useful for the better understanding of the heterogeneity and complexity in the molecular biology of gastric cancer and to develop tools to more accurately predict their prognosis.  Table 5. Correlation of Notch receptor mRNA high expression with different treatment of gastric cancer patients.