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 to identify effects on stem cells. For in vivo, MiaPaCa-2 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for stem cell markers and Hippo signaling proteins.

Results: Qui demonstrated a dose- and time-dependent inhibition of proliferation and colony formation in all three PCa lines but not HPNE cells. Qui induced PCa cells to undergo apoptosis. It also significantly reduced the number and size of pancospheres, and flow cytometry and western blot analyses confirmed that Qui suppressed PCa stem cell marker proteins DCLK1, CD44, CD24 and EPCAM. We next determined whether Hippo signaling pathway is affected. For this, we tested the effect of Qui on Hippo signaling pathway, which is an active pathway in CSCs including in PCa. The key effector protein, YAP1 has been shown to be oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm, and therefore no induction of downstream gene expression. Qui significantly decreased the phosphorylation oncogenic YAP1. Furthermore, Qui inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from Qui-mediated growth suppression. 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 in the expression of CSC marker proteins, oncogenic YAP1 phosphorylation and YAP1 interacting proteins TEAD1 in the Qui-treated xenograft tissues.

Conclusion: Together, these data suggest that Qui suppresses PCa growth that targets that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway.

Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Gaurav Fnu, Prabhu Ramamoorthy, Animesh Dhar, Ossama W. Tawfik, Shahid Umar, Scott J. Weir, Roy A. Jensen, Arun Balakrishnan, Shrikant Anant. Quinomycin A inhibits pancreatic cancer growth and affects stem cell viability by inhibiting oncogenic YAP1 function. [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 1727.