Endothelial Nitric Oxide Synthase Traffic Inducer (NOSTRIN) is a Negative Regulator of Disease Aggressiveness in Pancreatic Cancer

Abstract

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is refractory to available treatments. Delineating critical pathways, responsible for disease aggressiveness and therapeutic resistance, may identify effective therapeutic targets. We aimed to identify key pathways contributing to disease aggressiveness by comparing gene expression profiles of tumors from early-stage PDAC cases with extremely poor survival (≤7 months) and those surviving 2 years or more following surgical resection.

Experimental design: Gene expression profiling was performed in tumors in a test cohort of PDAC (N = 50), which included short (≤7 months, N = 11) and long surviving (≥2 years, N = 14) patients, using affymetrix GeneChip Human 1.0 ST array. Key genes associated with disease aggressiveness were identified, using Cox regression, Kaplan-Meier, and pathway analyses with validations in independent cohorts for mechanistic and functional analyses.

Results: Gene expression profiling identified 1,820 differentially expressed genes between short and long survival groups with inflammatory gene network ranking first. Lower expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was associated with worst survival indicating its potential inhibitory role in disease progression. NOSTRIN overexpression suppressed migration and invasion of pancreatic cancer cells and enhanced sensitivity to chemotherapeutic drug gemcitabine. NOSTRIN inhibited production of nitric oxide (NO) by suppressing the activation of endothelial nitric oxide synthase (eNOS). Furthermore, miR-221, bound to the 3'UTR of NOSTRIN and suppressed its expression, and an increased miR-221 expression associated with poor survival in PDAC.

Conclusions: Our findings showed that NOSTRIN is a potential negative regulator of disease aggressiveness, which may be targeted for designing improved treatment strategy in PDAC. Clin Cancer Res; 22(24); 5992-6001. ©2016 AACR.

Figure 1

Schematic representation of the strategy to distinguish genes associated with disease aggressiveness in long vs. short survival groups of resected PDAC patients.

Figure 2. Identification and validation of candidate genes with prognostic significance in PDAC

a) Kaplan-Meier analysis showing that an increased expression of AQP9, IGF2BP3 and SLC16A3 is associated with poor survival, however, a lower expression of NOSTRIN is associated with poor survival in test cohort. (b) Validation in publically available data set. (c) Validation by qRT-PCR analysis in a second validation cohort showing that a lower expression of NOSTRIN is associated with poor survival. Association of AQP9, IGFBP3 and SLC16A3 with survival could not be validated by qRT-PCR in the second validation cohort. (d) A lower expression of NOSTRIN was found by qRT-PCR in tumors as compared to adjacent nontumor pancreas, which could be further validated in publically available datasets (e,f).

Figure 3. NOSTRIN suppresses invasiveness of pancreatic cancer cells by inhibiting eNOS-derived NO production

(a) Western blot showing NOSTRIN overexpression in 3 PDAC cell lines. (b) Overexpression of NOSTRIN reduced cell migration in Mia-paca 2 and CFPAC-1 pancreatic cancer cells. Cell migration index was recorded by Xcelligence system for up to 24 hr after cells were seeded in the upper wells. Histogram represents the migration index at 24 hr. Cell migration index was calculated by averaging the values in quadruplicate wells. Curves were compared using student t-test. P values less than 0.05 were considered significant. (c) Panc1 and CFPAC1 cells with NOSTRIN overexpression showed reduced invasion as determined by BD invasion assay. Representative picture showing cell invasion at 24 hr. Average cell number in 5 randomly selected fields for each cell lines is shown in the histogram. Results are compared using student t-test. (d) Western blot showing that overexpression of NOSTRIN reduced p-eNOS, while the knockdown of NOSTRIN by shRNA increased p-eNOS level. (e) NOSTRIN overexpression inhibited the production of nitric oxide in PANC1 cells. (f) Exposure to nitric oxide by treatment with NO-donor drug Deta/NO, at indicated concentration, increased cell migration in CFPAC and Panc10.05 cell lines. (g) shRNA-mediated knockdown of NOSTRIN and exposure to NO-donor increased cell migration to comparable levels. Experiments were repeated 3 times. Error bars indicate standard deviation (SD).

Figure 4. Overexpression of NOSTRIN sensitizes PDAC cell lines to chemotherapeutic drug, gemcitabine, by enhancing apoptosis

(a) NOSTRIN overexpression sensitized Mia-paca2 cells to Gemcitabine. Histogram showing the percentage of viable Mia-paca2 cells with or without NOSTRIN overexpression after 72 hours exposure to gemcitabine. (b) NOSTRIN overexpressing cells showed an enhanced apoptosis, as determined by determined by Capsase3/7 activity, following treatment with gemcitabine. (c) Representative pictures of colony formation assay in Mia-paca2 cells with or without NOSTRIN overexpression, following treatment with gemcitabine at 0.01 and 0.005uM concentration. All experiments were repeated 3 times. Error bars indicate standard deviation (SD).

Figure 5. NOSTRIN is regulated by miR-221

(a) The expression of NOSTRIN and miR-221 showed positive correlation in PDAC (N=37). (b) A higher expression of miR-221 (upper tertile, as determined by qRT-PCR) associated with poor survival in patients with PDAC. (c) Overexpression of miR-221 down regulated NOSTRIN and increased the expression of p-eNOS in ASPC-1 and SU86.86 cell lines. (d). miR-221 mediated decrease in NOSTRIN mRNA expression as determined by qRT-PCR. (e) Schema showing the predicted miR-221 binding site on 3′UTR of NOSTRIN gene. (f) NOSTRIN-Luciferase reporter activity assay showing reduced reporter activity in miR-221 expressing cells. miR-221 induced reduction in reporter activity was abolished when the miR-221 binding site was mutated in NOSTRIN 3′UTR. All experiments were repeated 3 times. Error bars indicate standard deviation (SD).

Figure 6

NOSTRIN inhibits disease aggressiveness by suppressing eNOS-mediated NO production and is post-transcriptionally regulated by miR-221 in pancreatic cancer. PECAM/Stat3 signaling is earlier shown to enhance the transcription of NOSTRIN (27). (NO: Nitric Oxide; eNOS: endothelial nitric oxide synthase, PECAM-1: Platelet-endothelial cell adhesion molecule-1).