Re‐Purposing FDA‐approved Raloxifene to Target Apoptotic Proteins in KRAS‐mutant Pancreatic Cancer
Authors: Badejo Adeleke Modupe , Szlavich Ellyssa , McCreary Jordan , True Heather , Serrato Ana , Love Quintin , Malhotra Ashim 
Published: November 2023

Pancreatic cancer (PCa) has a very low 5‐year survival rate and is challenging to treat due to substantial chemoresistance. Changes in apoptotic protein regulation may account for PCa's chemoresistance. While 90% of PCa cases occur due to mutations in the human homolog of the Kirsten rat sarcoma (KRAS) viral oncogene, how this effects regulation of apoptotic proteins in PCa is unknown. We screened drugs approved by the Food and Drug Authority for their ability to induce apoptosis in both KRAS‐mutant and Wild‐type (Wt) human PCa cell lines. From this screen, we identified the selective estrogen receptor modulator raloxifene, which is approved for the treatment of osteoporosis and breast cancer in postmenopausal women. Our data show that raloxifene caused apoptosis in PCa cells. We hypothesized that raloxifene treatment compromised mitochondrial integrity either due to altered regulation of apoptotic proteins or due to changes in core mitochondrial membrane components like the phospholipid cardiolipin (CL). To investigate the effect of raloxifene treatment on mitochondrial CL production, we tested its effect on the expression of the CL‐synthesizing enzymes tafazzin and cardiolipin synthase 1. Our data show a raloxifene‐mediated dose‐dependent suppression of both enzymes. Interestingly, raloxifene does not affect the expression of other phospholipid synthesizing enzymes like glycerophosphate acyltransferase, which perhaps explains the lack of changes in the relative amount of phosphatidylcholine and lysophosphatidylcholine when KRAS‐mutant PCa cells were treated with increasing doses of raloxifene. However, suppression of tafazzin correlated with an inhibition of autophagy demonstrated by an increase in the autophagy marker proteins LCII and p62, suggesting that raloxifene prevents the autophagy‐based removal of damaged mitochondria in KRAS‐mutant PCa. To confirm that tafazzin suppression mediated the observed changes in autophagy regulating proteins, using CRISPR technology, we ablated the tafazzin gene. Western blot data show a 75% decrease in tafazzin expression over three days following CRISPR treatment. Interestingly, CRISPR‐based suppression of tafazzin also inhibited autophagy as suggested by changes in the autophagy markers, suggesting a central role for tafazzin in regulating mitochondrial viability in PCa. Additionally, raloxifene increased the expression of the pro‐apoptotic Bim, while having no effect on the expression of the anti‐apoptotic Bcl‐2 and the pro‐apoptotic Bax proteins. Further, there was a dose‐dependent decrease in the activation of caspases 3 and 7 over 72 hours. Western blot data confirmed a lack of the cleavage of procaspases 3 and 9, suggesting activation of caspase‐independent cell death mechanisms in KRAS‐mutant PCa. Raloxifene treatment increased MAP‐Kinase activation as an early event as evidenced by a sustained increase in phosphorylated ERK and p‐38 proteins within an hour of treatment. The impact of our research is to establish raloxifene as a drug that specifically targets the mitochondria in PCa which constitutes a novel strategy to combat this deadly disease by identifying mitochondrial proteins as synthetic lethal targets in the context of KRAS mutations.Support or Funding InformationElsa U. Pardee Foundation GrantResearch Incentive Grant, Pacific University School of Pharmacy