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. 2015 Jun 20;6(17):15524-39.
doi: 10.18632/oncotarget.3499.

Targeting pancreatitis blocks tumor-initiating stem cells and pancreatic cancer progression

Altaf Mohammed  1 , Naveena B Janakiram  1 , Venkateshwar Madka  1 , Misty Brewer  1 , Rebekah L Ritchie  1 , Stan Lightfoot  1 , Gaurav Kumar  1 , Michael Sadeghi  1 , Jagan Mohan R Patlolla  1 , Hiroshi Y Yamada  1 , Zobeida Cruz-Monserrate  2 , Randal May  3 , Courtney W Houchen  3 , Vernon E Steele  4 , Chinthalapally V Rao  1
Affiliations

Targeting pancreatitis blocks tumor-initiating stem cells and pancreatic cancer progression

Altaf Mohammed et al. Oncotarget. .

Abstract

Recent development of genetically engineered mouse models (GEMs) for pancreatic cancer (PC) that recapitulates human disease progression has helped to identify new strategies to delay/inhibit PC development. We first found that expression of the pancreatic tumor-initiating/cancer stem cells (CSC) marker DclK1 occurs in early stage PC and in both early and late pancreatic intraepithelial neoplasia (PanIN) and that it increases as disease progresses in GEM and also in human PC. Genome-wide next generation sequencing of pancreatic ductal adenocarcinoma (PDAC) from GEM mice revealed significantly increased DclK1 along with inflammatory genes. Genetic ablation of cyclo-oxygenase-2 (COX-2) decreased DclK1 in GEM. Induction of inflammation/pancreatitis with cerulein in GEM mice increased DclK1, and the novel dual COX/5-lipoxygenase (5-LOX) inhibitor licofelone reduced it. Dietary licofelone significantly inhibited the incidence of PDAC and carcinoma in situ with significant inhibition of pancreatic CSCs. Licofelone suppressed pancreatic tumor COX-2 and 5-LOX activities and modulated miRNAs characteristic of CSC and inflammation in correlation with PDAC inhibition. These results offer a preclinical proof of concept to target the inflammation initiation to inhibit cancer stem cells early for improving the treatment of pancreatic cancers, with immediate clinical implications for repositioning dual COX/5-LOX inhibitors in human trials for high risk patients.

Keywords: cancer stem cells; dual COX-5-LOX inhibition; inflammation; p48Cre/+-LSL-KrasG12D/+ mice; pancreatic cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Activation of inflammation and CSCs during progression of pancreatic cancer
A–C. Histopathological analysis of pancreas from 2-, 6- and 10-month-old GEM mice using H&E staining. Pancreas from animals showing PanIN lesions A. carcinoma B. and normal pancreas C. D–G. Expression of DclK1 in PanINs and PDAC. D. IHC for DclK1 in PanINs and PDAC, E. Western blotting of normal pancreas vs pancreatic tumor for DclK1 expression, F. IHF showing DclK1 (red) (left panel) and IHF showing DclK1 (red) merged with DAPI (blue) (right panel). G. Number of DclK1 positive cells in 2-, 6- and 10-month-old GEM. H. Whole genome transcriptome analysis by Solid sequencing showing increased mRNA expression of DclK1, COX-2 and 5-LOX in pancreas from GEM mice compared with wild type mice. I. IHF showing DclK1 (red) and COX-2 (green) expression in human normal pancreas (NP) and pancreatic ductal adenocarcinoma (PC). J. IHC showing DclK1 (brown) and IHF showing COX-2 (green) expression in human pancreatic ductal adenocarcinoma.
Figure 2
Figure 2. A–B. Effect of genetic ablation of COX-2 on DclK1 expression
Decreased expression of Dclk1 was observed in the COX-2 knock-out GEM mice (A, right panel) compared with GEM mice alone (A, left panel) as was a decreased number of DclK1-positive cells B. C–J. Histopathological analysis using H&E staining of pancreas from cerulean (C)-treated GEM mice with and without licofelone (L) in the diet. C. Effect of licofelone on pancreas weight at the termination of the experiment. Licofelone significantly reduced the pancreatic tumor weights. D–E. Effect of licofelone on the percentage of normal-appearing pancreas and on PanIN multiplicity. F–J. Compared with untreated mice, the licofelone-treated GEM mice showed decreased: F. pancreatitis, G. acinar destruction, H. inflammatory cell infiltration, I. stromal fibrosis, J. hyperplasia of ductules.
Figure 3
Figure 3. A–H. Effect of licofelone on cerulean-induced pancreatitis on inflammation, proliferation and DclK1 expression
Twelve week old p48Cre/+-LSL-KrasG12D/+ mice (N = 6/group) were injected with 5 μg/mice of cerulein every day for six consecutive days. Licofelone (500 ppm) was fed to a group of mice starting one day before cerulein injection to one day after the last cerulein injection. One day after cerulein injection, all mice were euthanized by CO2 asphyxiation and necropsied. Pancreata were collected from all groups, weighed and snap frozen in liquid nitrogen for further analysis. Immunohistochemical analyses were performed with paraffin-embedded and micro-sectioned pancreatic tissues as described in the Methods section. A significantly decreased expression of DclK1, COX-2, 5-LOX, PCNA and Ki67 was seen in licofelone-treated GEM. A significantly reduced number of DclK1 positive cells and proliferation index was seen in the licofelone-treated GEM compared with untreated mice.
Figure 4
Figure 4. Evaluation of licofelone efficacy in pancreatic cancer prevention
A. Experimental design for evaluation of licofelone efficacy in PC prevention in male and female p48Cre/+-LSL-KrasG12D/+ GEM mice. At 6-weeks of age, groups of mice (24–34/group for activated p48Cre/+-LSL-KrasG12D/+ or 24/group for wild-type) were fed AIN76-A diets containing 0, 250 or 500 ppm licofelone continuously for 38 weeks and each pancreas was evaluated histopathologically for marker expression as described in the text. B–D. Pancreatic tumor development in GEM. GEM (left) and wild type (right) pancreas B. PanIN lesions and carcinoma in GEM C. Pancreas weight at 44 weeks age in GEM compared to wild type (wt) mice (shown in parentheses) D. E. Effect of licofelone on mortality of GEM. F–G. Effect of licofelone on pancreas weight at the termination of the experiment in male F. and female G. mice. Both doses of licofelone significantly reduced the pancreatic tumor weights. H–I. Effect of licofelone on the incidence of PDAC in male H. and female I. mice.
Figure 5
Figure 5. Inhibition of PDAC incidence, PanIN lesions, carcinoma spread and arachidonic acid metabolites by licofelone
A–B. Effect of licofelone on PanIN multiplicity in male (panel A) and female (panel B) GEM (means ± SE). C–D. Effect of licofelone on the percentage of pancreas with carcinoma (C-male, D-female). E–F. Effect of licofelone on the percentage of normal appearing pancreas (E-male, F-female). The data in the panels were analyzed by unpaired ‘t’-test with Welch's correction; values are considered statistically significant at p < 0.05. G. Effects of licofelone on COX activity in pancreatic tumors from GEM as assessed with the radio-HPLC method. Values are means ± SEM, N = 6 per treatment group. A significant (P < 0.001) inhibition of AA metabolites (PGs and TXB2) was observed in pancreas of licofelone-treated mice compared with control mice. H. Effects of licofelone on 5-LOX activity in pancreatic tumors from GEM as assessed with the radio-HPLC method. Values are means ± SEM, N = 6 per treatment group. A significant (P < 0.0001) inhibition of the 5-LOX metabolite 5-HETE was observed in licofelone-treated mice compared with control mice.
Figure 6
Figure 6. Anti-inflammatory licofelone inhibits proliferation, CSCs and induces apoptosis
A. Immunohistochemical staining for PCNA in pancreatic tumors from GEM fed control diet or treated with licofelone. B. A significant difference was observed in the proliferative index between licofelone-treated and control group pancreas. C–D. TUNEL assay was done for apoptotic cells in pancreatic tumors from GEM fed control diet or treated with licofelone (N = 6 mice/group). A significant induction of apoptosis was observed in treated mice compared with untreated mice tumors. (E–J) Relative mRNA expression of apoptosis (caspase-3, p21 and p53) and CSC markers (DclK1, CD44, CD133) as determined by real time PCR. Licofelone treatment significantly increased expression of caspase-3 E. p21 F. and p53 G. and decreased expression of CSC markers DclK1 H. CD44 I. and CD133 J. K. Imunohistochemical staining showing a decrease in CD133 expression in the treatment groups.
Figure 7
Figure 7. Licofelone inhibits DclK1 and modulates miRNAs associated with inflammation and cancer stem cells
A–B. Effect of licofelone on DclK1. Immunohistochemical (brown-DclK1) and Immunofluorescence (green-DclK1, blue-DAPI) staining for DclK1 in pancreatic tumors from GEM fed control diet or treated with licofelone. A significant dose-dependent decrease was observed in Dclk1 expression in licofelone-treated group pancreas. C–G. Effect of licofelone on miRNAs including those related to inflammation and CSCs as determined by PCR arrays by real time pcr.
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