Abstract 2490: Smoking enriches cancer stem cell population and activates Sox9 through NF-kB signaling in pancreatic ductal adenocarcinoma

Abstract

Abstract Introduction: Recent studies have demonstrated a clear association between smoking and the incidence of pancreatic ductal adenocarcinoma (PDAC); however, the effect of cigarette smoke in the activation of stem cell (SC) or cancer stem cell (CSC) genes and their involvement in the initiation and progression of PDAC have not yet been studied. It is well known that CSCs are responsible for the drug resistance and aggressiveness of the disease including PDAC. In this study, we investigated the effects of smoking on enrichment of SC/CSCs in pancreatic normal and ductal adenocarcinoma cells, and we also examined whether smoking can activate NF-kB signaling, which is in part leads to enrichment of CSC and induction of CSC markers in PDAC. Methods: Cigarette smoke extract (CSE) was prepared, and HPNE (Human pancreatic nestin positive cells) and Capan-1 pancreatic cancer (PC) cells were treated with CSE for up to ∼15 weeks. Side population (SP) were analyzed by Hoechst staining using Flow-cytomer, and various CSC markers such as PD2 (a stem cell maintenance marker), CD44, ALDH-1, SOX-9 (a multipotent SC marker) and Oct-3/4 (a pluripotent marker), and NF-kB signaling molecules were analyzed by western blotting. ALDH1+ cells, CD44+CD24+ CSCs and G0/G1 phase low cycling quiescent cells were analyzed by flow cytometer. An in-vitro sphere culture was also performed to further confirm the smoke induced CSC properties. Smoke exposed pancreatic tissues excised from unfloxed littermate control (LSL-K-Ras G12D) pancreatic tissue sections were immunostained for SOX-9 using immunohistochemistry (IHC), and for SOX9 and CD44 using immunofluorescence. Results: Our results showed increased SC/CSCs and more number of spheres by CSE treated cells as compared to their untreated controls and displayed elevated protein expressions of SC/CSC markers. We also observed an elevated CD44+CD24+ CSCs, increased ALDH1+ cells and increased G0/G1 low cycling quiescent cell population in CSE treated cells as compared to untreated controls. In addition, increased immunohistochemical staining for SOX9 and increased immunofluorescent signal for SOX-9 and CD44 were observed in smoke exposed animal tissues indicating that smoking may transform SOX-9+ multipotent SCs into SOX9+CD44+ CSCs. We also analyzed the expression levels of NF-kB signaling molecules in CSE treated HPNE and Capan-1 cells. As compared to their untreated controls, CSE treated cells showed elevated protein expression levels of phospho AKT, phospho RelA (SOX-9 promoter binding subunit of NF-kB complex) and phospho IKKα (a kinase that phosphorylates IKBα, an inhibitor of RelA) suggesting that smoking activates NF-kB signaling. Conclusion: Our results illustrate that smoking enriches SC/CSCs populations in normal pancreatic cells as well as in pancreatic cancer cells, and activates SOX9 through NF-kB signaling in PDAC. Citation Format: Ramakrishna Nimmakayala, Parthasarathy Seshacharyulu, Seema Chugh, Imayavaramban Lakshmanan, Satyanarayana Rachagani, Surinder K Batra, Moorthy P Ponnusamy. Smoking enriches cancer stem cell population and activates Sox9 through NF-kB signaling in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2490.