Abstract 4214: Quinomycin A affects pancreatic cancer stem cells in part through suppression of notch signaling pathway
Authors: Subramaniam Dharmalingam , Ponnurangam Sivapriya , Sayed Afreen , Dhar Animesh , Dixon Dan A. , Tawfik Ossama , Parab Rajashri R. , Mishra Prabhu Dutt , Ranadive Prafull , Sharma Rajiv , Mahajan Girish , Sugumar Aravind , Weir Scott J. , Jensen Roy A. , Balakrishnan Arun , Anant Shrikant 
Published: August 2015

Abstract
Background: Pancreatic cancer (PCa) remains a leading cause of death in the United States. Cancer stem cells (CSC) are responsible for tumor behavior, and therapeutic resistance. Quinomycin (Qui) is an orally administered quinoxaline antibiotic that bifunctionally intercalates with double stranded DNA. As a first step for repurposing this drug, we determined whether Qui affects PCa growth, and if so whether this is by suppressing stem cells.
Method: Growth and apoptosis of PCa lines (MiaPaCa-2, PanC-1, BxPC-3) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, and caspase 3/7 assays, respectively. Pancosphere formation and FACS sorting were used for identifying effects on stem cells. For in vivo effects, MiaPaCa-2 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for stem cell markers and Notch signaling proteins.
Results: Qui treatment resulted in a dose- and time-dependent inhibition of proliferation and colony formation in all three PCa lines but not HPNE cells. Qui also induced PCa cells to undergo apoptosis. Qui also significantly reduced the number and size of pancospheres, suggesting effects on stem cells. In addition, flow cytometry and western blot analyses showed that Qui suppressed PCa stem cell marker proteins Doublecortin Calmodulin-like kinase 1 (DCLK1), CD44 and CD24. We next determined whether Qui affects the Notch signaling pathway, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human PCa tissues. Qui treatment significantly downregulated Notch-1, 2 and 3 expression and that of its ligand Jagged-1. Notch activation requires cleavage by the γ-secretase complex. Qui inhibits the expression of members of the complex Presenilin 1 and Nicastrin. Moreover, ectopic expression of the Notch Intracellular domain (NICD) rescued the cells from Qui -mediated growth suppression. These data demonstrate that Qui mediated effects of PCa stem cells is in part through downregulating Notch1 activation. To determine the effect of Qui on tumor growth in vivo, mice carrying MiaPaCa-2 tumor xenografts were administered the compound intraperitoneally (20 μg/kg bw) every day for 21 days. Qui treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, CD44 and CD24 and also the Notch signaling related proteins in the Qui-treated xenograft tissues.
Conclusion: Together, these data suggest that Qui suppresses PCa growth by inhibiting the Notch signaling pathway. Qui may therefore be a novel compound to target pancreatic cancers.
Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Afreen Sayed, Animesh Dhar, Dan A. Dixon, Ossama Tawfik, Rajashri R. Parab, Prabhu Dutt Mishra, Prafull Ranadive, Rajiv Sharma, Girish Mahajan, Aravind Sugumar, Scott J. Weir, Roy A. Jensen, Arun Balakrishnan, Shrikant Anant. Quinomycin A affects pancreatic cancer stem cells in part through suppression of notch signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4214. doi:10.1158/1538-7445.AM2015-4214