Inhibition of Lipid Oxidation Increases Glucose Metabolism and Enhances 2-Deoxy-2-[(18)F]Fluoro-D-Glucose Uptake in Prostate Cancer Mouse Xenografts

Abstract

Purpose: Prostate cancer (PCa) is the second most common cause of cancer-related death among men in the United States. Due to the lipid-driven metabolic phenotype of PCa, imaging with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) is suboptimal, since tumors tend to have low avidity for glucose.

Procedures: We have used the fat oxidation inhibitor etomoxir (2-[6-(4-chlorophenoxy)-hexyl]oxirane-2-carboxylate) that targets carnitine-palmitoyl-transferase-1 (CPT-1) to increase glucose uptake in PCa cell lines. Small hairpin RNA specific for CPT1A was used to confirm the glycolytic switch induced by etomoxir in vitro. Systemic etomoxir treatment was used to enhance [(18)F]FDG-positron emission tomography ([(18)F]FDG-PET) imaging in PCa xenograft mouse models in 24 h.

Results: PCa cells significantly oxidize more of circulating fatty acids than benign cells via CPT-1 enzyme, and blocking this lipid oxidation resulted in activation of the Warburg effect and enhanced [(18)F]FDG signal in PCa mouse models.

Conclusions: Inhibition of lipid oxidation plays a major role in elevating glucose metabolism of PCa cells, with potential for imaging enhancement that could also be extended to other cancers.

Fig. 1

Lipid oxidation is abundant in prostate cancer cells and interrupted by etomoxir. a) The effect of etomoxir (150 μM) on the [¹⁴C]oleic acid oxidation rate at the indicated times. Benign cells are BPH-1 and WPMY-1. a Benign (n = 6) vs. PCa (n = 9) without treatment, t test—P = 0.004. b Effect of 4 h treatment on oleate oxidation in PCa cells. ANOVA P < 0.001, post hoc Tukey’s: LNCaP (P < 0.001), VCaP (P < 0.001), PC3 (P = 0.002). b) Effect of etomoxir on [¹⁴C]palmitate oxidation. c Benign (n = 6) vs. PCa (n = 9) with no treatment, t test—P = 0.006. d Effect of 4-h treatment on palmitate oxidation in PCa cells. ANOVA P < 0.001, post hoc Tukey’s: LNCaP (P = 0.001), VCaP (P = 0.001), PC3 (P < 0.001). c) CPT1 isoform expression in PCa cells and BPH-1 benign line. Post hoc tests, *P ≤ 0.03 compared with BPH-1. #P ≤ 0.002 compared with BPH-1. d) CPT1A Western blot of cell lines examined. αTUB = tubulin loading control.

Fig. 2

Knockdown of CPT1A results in decreased lipid oxidation and increased glucose uptake. a) Western blot of lysates of LNCaP CPT1A KD clones (#36279 and 36281) exposed to etomoxir for 24 h. C = control clone, V = vehicle-treated, E = etomoxir-treated. b) [¹⁴C]Palmitic acid oxidation rate of the control and CPT1A-KD clones, ANOVA P < 0.001. Post hoc tests, *P ≤ 0.01 compared with control clone, ^P ≤ 0.004 compared with vehicle treatment. c) Normalized 2-[³H] DG uptake in the LNCaP CPT1A KD clones, ANOVA P < 0.001. Post hoc tests, *P ≤ 0.001, compared with vehicle treatment, ^P ≤ 0.001 compared with control clone.

Fig. 3

Pharmacological block in fat oxidation results in glucose uptake in PCa cells. a) Normalized 2-[³H]DG uptake in patient-matched prostate-derived primary cells. Student’s t test: *vehicle-cancer compared with vehicle-benign P = 0.044. a  P = 0.011 compared with benign-vehicle. #P = 0.009 compared with vehicle-cancer. b) Effect of 150 μM etomoxir on the 2-[³H]DG uptake of prostate-derived cell lines over 48 h. Bracket points to PCa cells. *Etomoxir effect at 24 h between cells lines, ANOVA P < 0.001. Post hoc tests: BPH-1 vs. LNCaP (P = 0.001), PC3 (P < 0.001), VCaP (P = 0.017), WPMY-1 (P = 0.03). Benign WPMY-1 vs. PC3 (P = 0.043). **Etomoxir effect at 48 h between cell lines, ANOVA P = 0.005. Post hoc tests: BPH-1 vs. LNCaP (P = 0.011), PC3 (P = 0.007).

Fig. 4

[¹⁸F]FDG uptake is enhanced in VCaP xenografts after systemic treatment with etomoxir. a) Representative axial (left) and coronal images of a subcutaneous xenograft mouse model before (basal, top) and after etomoxir injection (bottom). Right tumor is indicated with white arrow. Note the increased uptake of [¹⁸F]FDG in the heart with etomoxir treatment. This systemic effect was observed in all the mouse models. The scale bar represents signal activity as a function of radioactivity. b) Normalized uptake values (NUV) fold change for xenografts examined by FDG-PET, *P = 0.035 t test between etomoxir and water-treated tumors. c) Representative western blot of VCaP tumor lysates from mice treated with vehicle (water 1, 2) or etomoxir 20 mg/kg (3–8) systemically for 24 h. d) Radioactive counts from [¹⁴C]palmitate oxidation in VCaP tumor homogenates incubated ex vivo with water or etomoxir (150 μM) for 45 min.

Fig. 5

[¹⁸F]FDG uptake is enhanced in PC3-LUC orthotopic xenografts and TRAMP mouse models after systemic treatment with etomoxir. a) Coronal (left) and sagittal PET images of representative PC3-LUC orthotopic xenografts. Bioluminescence image on the right indicates where the tumor cells were growing; red and blue indicate high and low luciferase expression, respectively. In the left panels (coronal views), the arrows point to the primary tumor above the bladder (strong, circular white signal). In the right panels (sagittal views), arrows point to the ventral metastasis below the heart. This metastasis corresponds to upper-small signal in the adjacent bioluminescence image. b) Axial (left) and sagittal PET images of representative 24-week-old TRAMP mouse. Magnetic resonance images (sagittal and coronal) were used to anatomically visualize the increased prostate cancer growth, which extended into the seminal vesicles. White arrows point to tumor growth located posterior to the bladder. Bladder cannot be seen in the sagittal views, but it is visible (strong, circular white signal) in the axial views. MRI photographs: B (bladder), P (prostate), SV (seminal vesicles), T (testis).