The Phospholipid Transacylase Tafazzin is a Drug Target for Overcoming Chemoresistance in Pancreatic Cancer

Pancreatic cancer has a high mortality, and low 5‐year prognosis. Discovery of novel therapeutic targets is an urgent need. Tafazzin remodels the inner mitochondrial membrane phospholipid cardiolipin. We hypothesized that depleting tafazzin would induce apoptosis or autophagy in pancreatic cancer. We treated the human pancreatic cancer cell line MIA PaCa‐2 with 0.05, 0.5, 5, and 20 μM Raloxifene, a Selective Estrogen Receptor Modulator. The rationale for raloxifene use was that 1) it causes apoptosis of cancer cells, and 2) that its natural homologue, estrogen, regulates mitochondrial biogenesis. Our data show that raloxifene reduced tafazzin expression in a dose‐dependent manner. To investigate the mechanism, we first tested the effect of raloxifene on the half‐life of tafazzin. Briefly, we treated MIA PaCa‐2 cells with either 5 μM or 20 μM raloxifene and prepared whole cell lysates at 0, 3, 6, and 9 hours, since the half‐life of tafazzin is known to be roughly 3 hours. Our data show that raloxifene reduced the half‐life of tafazzin and that this was a dose‐dependent effect, which was more pronounced at the 20 μM dose. Next, we pre‐incubated MIA PaCa‐2 cells with the global translation inhibitor cycloheximide and by Western Blot analyzed the effect of this treatment on tafazzin expression. We did not observe any difference in tafazzin expression between control and cycloheximide and raloxifene‐treated cells, suggesting the involvement of a protease in raloxifene‐mediated tafazzin depletion, the absence of which stabilized tafazzin expression. To confirm this, we pre‐incubated our cells with the proteasome inhibitor, MG‐132, followed by raloxifene treatment. Western Blot data showed an increase in tafazzin expression when the proteasome was inhibited prior to raloxifene treatment, confirming that the raloxifene‐mediated depletion of tafazzin required proteasomal activity. Further, the decrease in tafazzin, was correlated with an increase in the autophagy markers Beclin‐1 and LCIII‐B. Since, tafazzin regulates cardiolipin remodeling, it may have an indirect effect on total cellular phospholipids by regulating equilibrium levels of free fatty acids. Correspondingly, our data showed an increased in the lipidated form of the LCIII‐B protein, which is a late stage autophagy marker. These data suggest an increased incorporation of membrane‐forming phospholipids into the LCII‐B protein complex, perhaps dependent on tafazzin. Since tafazzin deficiency is known to altering the ATP/AMP and to activate AMP‐Kinase, we measured the expression of phosphorylated AMP‐Kinase compared to total AMP‐Kinase in raloxifene treated cells. Raloxifene induced the expression of AMP‐Kinase phosphorylated at threonine 172 compared to total AMP‐Kinase. Lastly, since falling ATP levels induce compensatory mitochondrial biogenesis by activating the ATP‐sensing PGC‐1α protein, we used Western Blot to measure effect of raloxifene‐mediated tafazzin depletion on PCG‐1α expression. Our data show an increase in the expression of the PGC‐1α in response to raloxifene treatment. Taken together, our data provide the first evidence for a direct link between tafazzin and pancreatic cancer. Support or Funding Information This research was funded in part by the Pacific University Research Incentive Grant and by the Collins Medical Trust of Oregon.