PMC

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.

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.

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.

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.

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.

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.