Inducible nitric oxide synthase enhances disease aggressiveness in pancreatic cancer

Pancreatic cancer is one of the most lethal malignancies and is refractory to the available treatments. Pancreatic ductal adenocarcinoma (PDAC) expresses high level of inducible nitric oxide synthase (NOS2), which causes sustained production of nitric oxide (NO). We tested the hypothesis that an aberrantly increased NO-release enhances the development and progression of PDAC. Enhanced NOS2 expression in tumors significantly associated with poor survival in PDAC patients (N = 107) with validation in independent cohorts. We then genetically targeted NOS2 in an autochthonous mouse model of PDAC to examine the effect of NOS2-deficiency on disease progression and survival. Genetic ablation of NOS2 significantly prolonged survival and reduced tumor severity in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice. Primary tumor cells isolated from NOS2-deficient KPC (NKPC) mice showed decreased proliferation and invasiveness as compared to those from KPC mice. Furthermore, NKPC tumors showed reduced expression of pERK, a diminished inactivation of Forkhead box transcription factor O (FOXO3), a tumor suppressor, and a decrease in the expression of oncomir-21, when compared with tumors in KPC mice. Taken together, these findings showed that NOS2 is a predictor of prognosis in early stage, resected PDAC patients, and provide proof-of-principle that targeting NOS2 may have potential therapeutic value in this lethal malignancy.


Human PDAC cell lines
Human pancreatic cancer cell lines, Capan2 and SU.86.86 were purchased from the American Type Culture Collection, ATCC (Rockville, MD) and authenticated by short tandem repeat (STR) analysis. Capan2 cells were grown in McCoy's 5A (modified) medium and SU.86.86 cells were grown in RPMI 1640, supplemented with 10% fetal bovine serum, penicillin-streptomycin (50 IU/ml and 50 mg/ml, respectively) (Sigma Aldrich, St. Louis, MO) and 2 mM L-glutamine in a humidified incubator containing 5% CO2 at 37°C. All products for cell culture were purchased from Life Technologies (Grand Island, NY).

Wound healing
Twenty-four hours prior to assay, KPC and NKPC primary tumor cells were seeded into 6-well plates to reach a final confluency of ~90%. Wounds were made by scratching the bottom of the plates with pipette tips. Pictures of the same region of the wounds were taken at 0 and 24 hours.

Transwell migration and invasion assay
Cell migration and invasion were measured by BD chamber migration and invasion system as described by manufacturer (BD Biosciences, San Jose, CA). KPC and NKPC primary tumor cells were made into single-cell suspension in DMEM-F12 medium by trypsin treatment and applied onto upper chamber, at a density of 2 × 10 5 cells/well. The complete medium (DMEM-F12 with 10% FBS) was loaded into lower chamber. Twenty-two hours after adding the cells onto upper chambers, the upper chambers were taken out and washed with PBS, followed by fixation in 100% methanol and staining with 0.1% crystal violate. Experiments were repeated at least 3 times.

Western blotting
Cells were washed in PBS and proteins were extracted with RIPA buffer (Life Technologies, Grand Island, NY). Electrophoresis was carried out on 4-20% SDS-PAGE gels (Life Technologies, Grand Island, NY), and then transferred on to a nitrocellulose membrane (Life Technologies, Grand Island, NY). To block nonspecific binding, the membrane was incubated for 60 min with 5% non-fat milk in PBST at room temperature. The membrane was then washed with PBST and incubated overnight with antibodies indicated and anti β-actin antibody (Sigma Aldrich: St. Louis, MO) as endogenous control in PBST. Secondary anti-mouse or -rabbit antibodies with HRP were purchased from Santa Cruz (Santa Cruz, CA). Specific protein was visualized using a Super-Signal protein detection kit (Pierce, Rockford, IL) according to manufacturer's instructions.

Immuno-fluorescence
A standard immunofluorescent method was used for staining. Cells were seeded into 6-well plates one day prior to the experiment with a glass coverslip placed in each well in advance. Cells were allowed to attach and grow under normal culture condition as mentioned earlier for 24 hours before being harvested. Prior to staining, coverslips with cells attached were washed with DPBS (Invitrogen, Carlsbad, CA) for 3 times and then fixed with 4% paraformaldehyde for 10 min. After fixation, cells were washed again with DPBS for 3 times and stained with 1:100 diluted Alexa Fluor555 pahlloidin (Cell Signaling Technology) for 30 min, and then washed with DPBS for 3 times. To mount the stained cover slip, a drop of DAPI (Sigma-Aldrich, St. Louis, MO) was dropped onto a slide and cover slips were placed onto DAPI drop. Actin filaments were observed under Zeiss fluorescent microscope (Carl Zeiss Inc, Thornwood, NY).