Sumac‐Mediated Mitochondrial Targeting in KRAS‐dependent Pancreatic Cancer: A Possible Role for Sirtuin3?

Abstract

Pancreatic cancer is a devastating disease with a high mortality rate and a low 5-year survival rate of only 10% (2010-2016, SEER database). Each year about 57,000 Americans and 250,000 people worldwide are diagnosed with pancreatic cancer. It is difficult to treat due to a plethora of factors ranging from substantial chemoresistance, multiple genetic lesions, relative quiet progression, and ineffective targeted pharmacological intervention to a lack of understanding of the molecular events involved both in its etiology and in effective drug therapy. Our goal is to identify small molecule compounds that are synthetic lethal in cellular models of mutant KRAS-dependent pancreatic adenocarcinoma. Our preliminary experiments demonstrate that aqueous extracts of the Middle Eastern spice-plant sumac induce cytotoxicity in two cultured human pancreatic cancer cell lines with KRAS gene mutations; MIA PaCa-2 (G12C) and PANC-1 (G12V and G12D) but not in normal fibroblasts. Since gemcitabine or FOLFIRNOX therapy is largely ineffective in pancreatic cancer patients, 90% of whom have mutations in the KRAS oncogene, we posited that using sumac as an adjunct would skew the otherwise chemoresistant pancreatic cancer cells into an apoptotic phenotype. We hypothesized that sumac's cytotoxicity was a consequence of mitochondrial targeting, specifically a derangement of mitochondrial membrane potential, leading to mitochondrial compromise, and eventually apoptosis. To test our hypothesis, we treated MIA PaCa-2 and PANC-1 cells in vitro with increasing concentrations of aqueous extracts of sumac (0, 0.03, 0.3 and 3.0 mg/mL) for varying time points and measured 1) mitochondrial membrane potential using TMRE assays, 2) ROS production using DHT assays, 3) ATP levels using fluorimetry, and 4) activated caspases 3 and 7. Our data show that sumac treatment 1) ameliorated ROS production, 2) abrogated mitochondrial membrane potential, 3) activated caspases 3 and 7, and 4) reduced cellular ATP levels in a dose-dependent manner, all preferentially in PANC-1 compared to MIA PaCa-2 cells. These data suggest that sumac mediates “synthetic lethality” in pancreatic cancer cells based on type of KRAS gene mutation. We also hypothesized that the observed ATP reduction would alter levels of SIRT3, which is a major mitochondrial physiology regulating deacetylase enzyme. Immunoblot data show paradoxical increase in SIRT3 in a dose-dependent manner, suggesting other roles for SIRT-3 in this pathway. Furthermore, using HPLC-based NMR spectroscopy, we identified the active compound in the aqueous sumac extract to be [(2R, 3R, 4S, 5R)-3, 4, 5, 6-tetrakis [(2-deuterio-3, 4, 5-trihydroxybenzoyl)oxy]oxan-2-yl] methyl-2-deuterio-3, 4, 5-trihydroxybenzoate. In conclusion, sumac targets hitherto unknown mitochondrial signal transduction pathways and is synthetic lethal for the KRAS mutant G12V and G12D.