Lipid catabolism via CPT1 as a therapeutic target for prostate cancer

Abstract

Prostate cancer is the most commonly diagnosed malignancy among Western men and accounts for the second leading cause of cancer-related deaths. Prostate cancer tends to grow slowly and recent studies suggest that it relies on lipid fuel more than on aerobic glycolysis. However, the biochemical mechanisms governing the relationships between lipid synthesis, lipid utilization, and cancer growth remain unknown. To address the role of lipid metabolism in prostate cancer, we have used etomoxir and orlistat, clinically safe drugs that block lipid oxidation and lipid synthesis/lipolysis, respectively. Etomoxir is an irreversible inhibitor of the carnitine palmitoyltransferase (CPT1) enzyme that decreases β oxidation in the mitochondria. Combinatorial treatments using etomoxir and orlistat resulted in synergistic decreased viability in LNCaP, VCaP, and patient-derived benign and prostate cancer cells. These effects were associated with decreased androgen receptor expression, decreased mTOR signaling, and increased caspase-3 activation. Knockdown of CPT1A enzyme in LNCaP cells resulted in decreased palmitate oxidation but increased sensitivity to etomoxir, with inactivation of AKT kinase and activation of caspase-3. Systemic treatment with etomoxir in nude mice resulted in decreased xenograft growth over 21 days, underscoring the therapeutic potential of blocking lipid catabolism to decrease prostate cancer tumor growth.

Figure 1. Lipid metabolic inhibitors reduce the viability of prostate cancer cell lines

A. Relative cell viability of prostate-derived cell lines exposed to Etomoxir (75μM) for 48 hours, *p<0.001 compared to vehicle. B, Viability of LNCaP cells exposed to inhibitors Etomoxir (75 μM), Orlistat (20 μM): ^p≤0.001, compared to vehicle, # p≤0.016, compared to single drug. C, Viability of VCaP cells: *p≤0.001, compared to vehicle, # p≤0.001, compared to single drug. D, MTS proliferation assay of LNCaP cells in FBS media. *p<0.02, **p=0.001 combination vs. single drug. E, CSS media *p<0.001 combination vs. single drug. F, MTS assay of VCaP cells in FBS: *p<0.001 combination vs. single drug. G, CSS media *p≤0.003, ^p= 0.026 combination vs. single drug. H–I, MTS assay of patient-matched prostate-derived benign (H) and cancer (I) cells exposed to inhibitors for 48 hours. Two-tailed t-tests: a, p<0.01 compared to Orlistat treatment in benign cells. b, p<0.05 compared to Etomoxir treatment in benign cells. c, p<0.05 compared to combinatorial treatment in benign cells. Combinatorial index is shown at bottom of the graph, where CI<1.0 indicates synergy.

Figure 2. Etomoxir and Orlistat decrease AR isoform expression and modify lipid oxidation and glucose uptake

Expression of full length AR (ARfl), variant 7 (ARv7), Total AR, PSA and NKX3.1 genes in LNCaP (A–B) and VCaP (C–D) cells treated with Etomoxir (75 μM) and/or Orlistat (20 μM). Post hoc tests compared to vehicle: A *p≤0.004, B *p≤0.05, C *p≤0.03, D *p≤0.05. E, Rate of ¹⁴C-palmitate oxidation in LNCaP (ANOVA, p=0.008), VCaP (ANOVA p=0.02) and BPH-1 cells exposed to inhibitors for 6 hours. *p<0.01, ^p≤0.02 compared to vehicle. $p<0.02 PCa cells compared to BPH-1. F, Glucose uptake of LNCaP (ANOVA, p<0.0001), VCaP (ANOVA p<0.001) and BPH-1(ANOVA, p=0.002) cells exposed to inhibitors for 6 hours. Post hoc tests: Comparisons to vehicle treatment: #p<0.05, *p<0.05, ^p≤0.007. VCaP and BPH-1 compared to LNCaP treated with etomoxir,ap<0.05.

Figure 3. Lipid catabolism blockade results in decreased mTOR signaling and increased apoptosis

AKT and BAD phosphorylation of LNCaP (A) and VCaP (B) lysates treated with Etomoxir (75 μM) and/or Orlistat (20 μM) for 6 hours. C, Expression of mTOR-S6K-BAD-Caspase3 axis after metabolic treatments for 16 hours in LNCaP cells. D, Blot of AMPK activation and ACC2 inactivation of LNCaP lysates E, Diagram of molecular pathway likely involved in the LNCaP cells. F, Expression of mTOR-S6K-BAD-Caspase3 axis after 16-hour treatments in VCaP cells. G, AMPK and phospho-ACC2 in VCaP lysates.

Figure 4. ER stress and apoptotic ceramides are increased after lipid metabolism blockade in LNCaP cells

A, Orlistat and Etomoxir treatments induce strong activation of the XBP-1 transcription factor after 16 hours of treatment. B, Relative qPCR analysis of the ER stress related factors after 16 hours. For each gene examined, ANOVA<0.001 across treatments. Post hoc tests: *p<0.01, a p=0.002 compared to vehicle. C–D, Western blots for phospho-eIF2a and LC3 fragments of LNCaP and VCaP lysates, respectively, treated with inhibitors. Total eIF2a bands were used as loading controls. E, Ceramide species in LNCaP cells were treated with Etomoxir for 24 hours and harvested for lipid extraction and ceramide analysis. Two-sided t test: *p≤0.05. Fatty acid composition of the ceramide molecules are indicated in the x-axis.

Figure 5. Downregulation of CPT1A decreases fat oxidation and leads to apoptosis

A, Western blots of CPT1A KD cells treated with vehicle (V), Orlistat (O), Orlistat+Etomoxir (OE) or Etomoxir alone (E) for 24 hours. B CPT1A expression in VCaP cells treated with inhibitors C, Trypan Blue viability assay of shRNA clones treated with 3 doses of Etomoxir for 2 days; *P<0.01 compared to control cells treated with vehicle. D, Palmitate oxidation rate in KD clones compared to control, *p≤0.01compared to control shRNA clone.

Figure 6. Systemic treatment with Etomoxir decreases xenograft tumor growth in nude mice

A, Tumor growth progression (mean ±SD) in mice treated with 40mg/Kg Etomoxir injections for 3 weeks. (*P<0.05 Tukey, compared to vehicle-treated tumors). B, Mouse body weight over the course of the experimental treatments. C, Representative CPT1A and phospho-S6 stains of tumor xenografts at the end of study.