Targeting Mitochondrial Function and Biogenesis in KRAS ‐Dependent Pancreatic Cancer

Pancreatic cancer (PCa) is a highly aggressive disease with a very low 5‐year survival rate, high mortality, and substantial chemoresistance. Although 90% of PCa cases occur due to mutations in the human homolog of the Kirsten rat sarcoma (KRAS) viral oncogene, targeting the KRAS pathway is challenging. Our goal was to identify “synthetic lethal” mitochondrial proteins, which when targeted in the context of KRAS mutations would induce apoptosis of KRAS ‐mutant PCa cells. We screened drugs approved by the Food and Drug Authority for their ability to alter expression of mitochondrial proteins of either the apoptotic family or enzymes needed for the synthesis of the mitochondrial phospholipid cardiolipin, with the aim of identifying drugs that could compromise the mitochondria in KRAS ‐mutant PCa cells. From this screen, we identified the selective estrogen receptor modulator raloxifene, approved for the treatment of osteoporosis and breast cancer in postmenopausal women, as a candidate drug. Our data show that raloxifene treatment compromises mitochondrial viability as tested by MTT assay in both mutant and Wild‐type (Wt) KRAS PCa cells. However, this effect was more pronounced for the KRAS ‐mutant cell line. To investigate mechanisms for the loss of mitochondrial integrity, we measured reactive oxidative species (ROS) production. ROS production dramatically increased within 20 minutes of raloxifene treatment and persisted upon longer treatment (24, 48, and 72 hours). This increase in ROS correlated with loss in mitochondrial membrane potential, which continued to fall with raloxifene treatment over 24, 48, and 72 hours. Further, alkali treatment of mitochondria isolated from untreated mutant and Wt KRAS PCa cells showed the differential release of proteins suggesting differences in protein integration into the mitochondrial membrane in KRAS ‐mutant versus Wt PCa cells. Additionally, proteins regulating mitochondrial biogenesis, including proteins involved in mitochondrial fusion and fission were differentially regulated between the KRAS ‐mutant and Wt PCa cells. Western blot data showed low protein expression of mitochondrial fusion (Optic Atrophy‐1, OPA1) and fission (Dynamin‐Related Protein‐1, Drp‐1) proteins in both KRAS ‐mutant and Wt PCa. However, both OPA1 and Drp1 expression were higher in KRAS ‐mutant PCa but much lower in Wt cells, while another fusion protein, mitofusin‐2 was equally expressed in both PCa cell lines. These data suggest inherent differences in mitochondrial biogenesis in KRAS ‐mutant versus Wt PCa cells. Finally, raloxifene also increased the expression of PGC‐1α, known to regulate mitochondrial biogenesis, in mutant but not Wt PCa, while decreasing the expression of the general transcription factor Estrogen Related Receptor α which regulates various mitochondrial proteins. Taken together these data suggest that raloxifene alters mitochondrial regulation and response in KRAS ‐mutant PCa cells. The impact of our research will be to establish raloxifene as a drug that can target the mitochondria in KRAS ‐mutant PCa and determine the role of mitochondrial regulation in the context of KRAS mutations. Support or Funding Information Elsa U. Pardee Foundation Grant; Research Incentive Grant, Pacific University School of Pharmacy