Perspective on This Article from The Epidermal Growth Factor Receptor Inhibitor Gefitinib Prevents the Progression of Pancreatic Lesions to Carcinoma in a Conditional LSL-KrasG12D/+ Transgenic Mouse Model

Abstract Developing novel strategies to prevent the progression of pancreatic cancer, a devastating disease with dismal 5 year survival rate (<5%) is utmost important. Recent studies identified dual 5-LOX-COX inhibitor, licofelone, as a safer chemopreventive agent. Since 5-LOX and COX-2 enhance pancreatic carcinogenesis, we studied efficacy of licofelone on pancreatic intraepithelial neoplasms (PanINs) and their progression to ductal adenocarcinoma (PDAC) in conditional LSL-KrasG12D/+ mice. Six-week old male and female KrasG12D/+ (∼35/group) mice were fed (AIN-76A) diets containing 0, 250 and 500 ppm licofelone for 38 weeks. Pancreata were collected and evaluated histopathologically for PanINs and PDAC. To understand the mechanisms, we analyzed levels of proliferation, apoptosis, cell cycle; COX-2, 5-LOX, PCNA, p21, p2×7, cyclin D1, Bcl-2, β-catenin, FLAP, PGES-2, and modification in cancer stem cell markers (DCAMKL-1 and CD133); by Radiometric HPLC, IHC, IHF, WB and/or RT-PCR. Also, changes in miRNA expression levels with licofelone treatment were measured by miRNA array. Results suggest that control diet fed mice showed 64 and 82% incidence of PDAC in female and male mice, respectively. Dietary licofelone significantly inhibited incidence of PDAC in a dose-dependent manner in both male (60-94%; p<0.0001) and female (75-90%; p<0.0001) mice. Also, a significant suppression of PanIN 3 (35-56%; p<0.001) and adenocarcinoma (60-90%; p<0.0001) was observed in mice fed with licofelone. The pancreas of mice fed licofelone diets showed significantly reduced pancreatic cancer stem-cells positive for DCAMKL-1 and CD133 (45-62%; P<0.0001). A significant inhibition of COX-2, 5-LOX, PCNA, p21, p2X7, cyclin D1, Bcl-2, and β-catenin expression levels (p<0.05-0.0002) in licofelone treated pancreas was observed when compared to pancreatic cancer derived from the mice fed control diet. As expected, licofelone showed dose-dependent suppression of pancreatic tumor COX-1, COX-2 (29-41%; p<0.001) and 5-LOX activities (70-91%, p<0.0001). Importantly, licofelone treatment significantly upregulated miR122 (7.1 -28 fold), miR1 (3.0-7.2 fold), miR7a (2.6 fold) and miR148a (3.0 fold); key regulators of cyclin D1, FGF4, HDAC4, EGFR, ODC, MAPK, exportin 1, MMP-3, Bcl-2, EGFR, GAD45A and downregulation of miR150 that regulates frizzled (β-catenin), P2X, TGFβ, PI3-K; further correlating with PDAC suppression. In summary, licofelone prevents the progression of PanIN 1 and -2 lesions to carcinoma in situ (PanIN 3) and further to PDAC in a preclinical model by targeting COX-LOX and cancer stem cells, and modulating key miRNAs that regulate proliferation and apoptosis. These data highlight for the first time the promise of licofelone use for pancreatic cancer prevention in high-risk patients. {Supported by NCI-CN-53300} Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2839. doi:10.1158/1538-7445.AM2011-2839

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy with a dismal prognosis. Developing novel strategies to prevent or delay pancreatic cancer is currently of intense interest. The chemopreventive efficacy of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, was evaluated against the progression of pancreatic intraepithelial neoplasms (PanIN) to PDAC in conditional LSL-KrasG12D/+ transgenic mice. LSL-KrasG12D/+ and p48Cre/+ mice were bred, and offspring of activated KrasG12D/+ were generated. Six-week-old male KrasG12D/+ (20 per group) and C57BL/6 wild-type (12 per group) mice were fed (AIN-76A) diets containing 0, 100, and 200 ppm of gefitinib for 35 weeks. At termination, pancreases were evaluated histopathologically for PanINs and PDAC, and various biomarkers were measured by immunohistochemistry, immunofluorescence, immunoblotting, and/or reverse transcription-PCR. Dietary gefitinib at 100 and 200 ppm significantly suppressed PDAC incidence by 77% and 100%, respectively (P < 0.0001) when compared with control diet. Importantly, a significant inhibition of carcinoma and a dose-dependent suppression of PanINs [PanIN-1, 37-62% (P < 0.002); PanIN-2, 38-41 (P < 0.001); and PanIN-3, 7-34% (P < 0.0141)] were observed in mice treated with gefitinib. Furthermore, mice treated with 100 and 200 ppm of gefitinib exhibited 67.6% to 77.3% of the pancreas to be free from ductal lesions. Also, gefitinib reduced EGFR, proliferating cell nuclear antigen, cyclin D1, C2GNT, RhoA, β-catenin, p38, phospho-extracellular signal–regulated kinase, caveolin-1, and mucin and increased cyclin B1 in the pancreatic lesions/PDAC. In summary, these results show that gefitinib can prevent the progression of pancreatic cancer precursor lesions to PDAC in a preclinical model. The present study highlights the promise of chemoprevention and the potential usefulness of EGFR inhibitors in individuals at high risk for pancreatic cancer. Cancer Prev Res; 3(11); 1417–26. ©2010 AACR.

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic malignancy is a devastating disease with dismal prognosis (5-year survival rate of less than 5%), and is the fourth leading cause of cancer deaths in the USA. Developing novel strategies to prevent or delay the progression of pancreatic cancer is currently of intense interest. We studied chemopreventive efficacy of gefitinib, an EGFR inhibitor on pancreatic intraepithelial neoplasms (PanINs) and adenocarcinoma in conditional LSL-K-RasG12D transgenic mice. LSL-K-RasG12D and P48Cre mice were bred and offspring of activated K-rasG12D were generated. Six-week old male KrasG12D (20/group) and wild-type (12/group) mice were fed (AIN-76A) diets containing 0, 100, and 200 ppm gefitinib for 35 weeks. At the time of sacrifice, pancreas were collected, weighed, and were evaluated histopathologically for PanINs and PDAC. To understand the molecular mechanisms, expression levels of PCNA, p38, cyclin D1, C2GNT, RhoA, β catenin, pERK, EGFR and Cav1, were determined in pancreatic tissues by IHC, immuno-fluorescence, immunoblotting and/or RT-PCR. Results suggest that mice fed with 100 and 200 ppm gefitinib had significantly reduced (p<0.0001) ductal adenocarcinomas incidence than control diet fed mice. Importantly, significant reduction of pancreatic ducts with carcinoma (>94%, p< 0.0001) and suppression of PanIN 3 (∼34%) was observed in mice fed diet with gefitinib. We did find statistically significant dose dependent inhibition of PanIN 1 & 2 lesions between control and gefitinib fed groups. Also, mice fed gefitinib showed reduced pancreatic tumor weights (∼50%) compared to control diet fed mice. Furthermore, treated mice showed >75% of pancreas free from carcinoma. Also, pancreas of mice fed gefitinib diets showed significantly reduced expressions of PCNA, p38, EGFR, RhoA, C2GNT, β-catenin, pERK, caveolin-1, and a significant increase in cyclin B1 (p<0.005) levels compared to pancreas of control diet fed mice. In summary, our results suggest that gefitinib prevents the progression of pancreatic cancer precursor lesions in PanIN 1, 2 and 3 stages from further progressing to adenocarcinoma in a preclinical model. These data highlight the promise of chemoprevention in pancreatic cancer and administration of EGFR inhibitors represents an important strategy to prevent pancreatic cancer in high-risk patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2927.