Abstract 271: Overexpression of hPaf1/PD2 in pancreatic cancer stem cells results in stemness and drug resistance: a novel target for pancreatic cancer therapy.

Abstract

Abstract Cancer stem cells (CSCs) or side-population (SP) constitute a sub-population of cells in a tumor which are responsible for the maintenance of several cancers, including pancreatic cancer (PC). In the process of identification of a CSC maintenance marker for PC, we have focused on the human polymerase association factor 1 (hPaf1) or pancreatic differentiation 2 (PD2) due to its critical role in self-renewal of mouse embryonic stem cells (mESCs) through the interaction with Oct3/4, a gatekeeper of pluripotency and self-renewal for ESCs. Furthermore, PD2 has been found to be highly overexpressed in poorly differentiated PC compared to well-differentiated PC due to gene amplification in the 19q13 locus. Additionally, ectopic overexpression of PD2 led to the oncogenic transformation of NIH3T3 immortalized cells. An essential property of stem cells is its ability to self-renew, which is also an important property of CSCs. Based on the aforementioned work, we hypothesize that PD2 is involved in the maintenance of self-renewal and drug resistance of pancreatic CSCs. To test our hypothesis, SP and Non-SP were isolated from pancreatic tumors as well as from cell lines using Hoechst 33342 exclusion assay followed by FACS analysis. SP and NSP were characterized using both in vitro and in vivo tumorigenicity assays. In vitro assays such as tumorsphere assay revealed that SP maintained its colony formation and formed large tumorspheres whereas the NSP cells were dispersed and formed smaller spheres. Both confocal and western bolt analysis revealed that SP overexpressed PD2 as well as CSC markers (CD133, ALDH1) and self-renewal markers (Shh and β-catenin). In vivo assays such as subcutaneous and orthotopic injection of 5000 SP and NSP cells into athymic nude mice were performed. SP injected mice produced a larger tumor as compared to NSP injected mice. Gemcitabine treatment enriched the SP cells thereby increasing PD2 expression compared to NSP cells. Furthermore, we carried out transient knockdown of PD2 by specific siRNA against PD2 in pancreatic CSCs and subsequently treated with gemcitabine which resulted in reduced viability thereby indicating a novel role of PD2 in CSC mediated drug resistance. Also, it was further corroborated by the downregulation of CD133 and multidrug resistance gene 2 expressions in SP. Further, analysis of PD2 expression along with CSC markers in PC progression mouse models revealed co-expression of PD2 and CSC markers in specific cells. Altogether, our results propose a novel role of PD2 in CSC maintenance as well as in drug resistance, which could be anticipated to be developed as a novel targeted therapy against pancreatic CSCs. Citation Format: Arokia Priyanka Vaz, Moorthy P. Ponnusamy, Satyanarayana Rachagani, Parama Dey, Surinder Batra. Overexpression of hPaf1/PD2 in pancreatic cancer stem cells results in stemness and drug resistance: a novel target for pancreatic cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 271. doi:10.1158/1538-7445.AM2013-271