0201

Androgen Independence Leads to Altered Metabolism in Prostate Cancer Cell and Murine Models
Jinny Sun1, Justin Delos Santos1, Robert Bok1, Romelyn Delos Santos1, Mark Van Criekinge1, Daniel B Vigneron1, Renuka Sriram1, and John Kurhanewicz1

1Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, United States

Synopsis

This study demonstrated significant increases in flux through glycolysis, oxidative metabolism, and glutaminolysis associated with androgen independence using patient-derived cell lines and a treatment-driven murine model. This data supports using a combination of hyperpolarized [1-13C]pyruvate, [2-13C]pyruvate and [5-13C]glutamine to noninvasively discriminate between androgen-dependent and androgen-independent prostate cancer in future patient studies using hyperpolarized 13C MRI.

Introduction

Mainstay treatment for patients with metastatic prostate cancer is androgen deprivation therapy (ADT). However, almost all patients eventually develop androgen-independent prostate cancer (ie. castration-resistant prostate cancer (CRPC)). Currently no reliable clinical or noninvasive imaging method can predict response to ADT, which is critical for treatment selection. While previous hyperpolarized 13C MRI studies have shown that pyruvate-to-lactate ratio and the rate of conversion of 13C-pyruvate to 13C-lactate increases as prostate tumors become more aggressive1,2, the precise metabolic profile of CRPC remained unknown. The goal of this research was to identify metabolic changes associated with resistance to ADT in order to determine the best combination of hyperpolarized 13C MRI probes that can discriminate between androgen-dependent and androgen-independent prostate cancer for future patient studies. The experimental approach was to characterize flux through glycolysis, oxidative metabolism, and glutaminolysis, initially using patient-derived cell lines that are androgen-dependent (LnCaP) and androgen-independent (PC3), and then using a transgenic adenocarcinoma of the mouse prostate (TRAMP) model that is known to mimic the development of therapeutic resistance in patients3.

Materials & Methods

Cell labeling: LnCaP and PC3 cells were incubated with either [U-13C]glucose or [U-13C]glutamine.

TRAMP treatment and labeling: Adult male TRAMP with a solid tumor mass between 0.1–1 cc underwent orchiectomy (equivalent to chemically-induced ADT in patients). Mice with <25% increase in tumor volume one-week post-orchiectomy were defined as androgen-dependent, and mice with ≥25% increase were defined as androgen-independent (Fig3AB). [U-13C]glucose or [U-13C]glutamine was injected via tail vein over 45 minutes4. Tissue collected immediately upon sacrifice was flash-frozen in LN2.

Metabolite extraction: Aqueous metabolites were extracted using cold methanol:water:chloroform5, then lyophilized and resuspended in D2O for NMR analysis.

NMR: High-resolution NMR spectra were acquired on a Bruker 800Mhz AvanceI spectrometer equipped with a multichannel cryo-probe (Fig2A, 3C).

Metabolite quantification: Fractional enrichment (FE) was quantified using the following equation: FE=[13C-metabolite]/[12C-metabolite+13C-metabolite]. Total metabolite concentration was quantified from 13C-decoupled 1H presaturation spectra, and 13C-labeled metabolite concentrations were quantified using 1H-13C HSQC. Glutathione fractional enrichment was quantified using 1H-1H TOCSY.

Results

First, no significant difference in glucose or glutamine uptake (Fig2B) or intracellular steady-state metabolite concentrations (Fig2C) was observed between androgen-dependent LnCaP and androgen-independent PC3 cells. In [U-13C]glucose labeling studies (Fig2D), PC3 cells had a significant increase in aspartate FE (88±4 vs 39±5, p<0.01) and glutamate FE (60±4 vs 41±3, p<0.05), indicating increased glucose contribution to oxidative metabolism, and a significant increase in lactate FE (74±3 vs 53±2, p<0.01), indicating upregulation of glycolysis, compared to LnCaP cells. Furthermore in [U-13C]glutamine labeling studies (Fig2E), PC3 cells had elevated aspartate FE (40±8 vs 65±5, p<0.05), glutamate FE (71±1 vs 57±2, p<0.01) and glutathione FE (74±1 vs 45±1, p<0.001) compared to LnCaP cells, indicating that glutaminolysis is upregulated in PC3 cells. Together these results indicate that the androgen-independent PC3 cells exhibit increased [U-13C]glucose flux through glycolysis and oxidative metabolism, and increased [U-13C]glutamine flux through glutaminolysis.

We then measured the fluxes through these pathways using a treatment-driven murine model (Fig3A) that more closely mimics tumor progression and treatment response in patients. Androgen-independent TRAMP tumors had a significantly increased lactate concentration (0.13±0.003 vs 0.05±.015, p<0.01) compared to androgen-dependent TRAMP tumors (Fig3D). Following [U-13C]glucose infusion (Fig3E), androgen-independent TRAMP tumors had elevated aspartate FE (60±12 vs 21±5, p<0.05), glutamate FE (53±12 vs 16±1, p<0.05), and lactate FE (81±4 vs 46±9, p<0.05) compared to androgen-dependent TRAMP tumors. Preliminary [U-13C]glutamine infusion data (Fig3F) shows that androgen-independent TRAMP tumors also had elevated aspartate FE (26±8 vs 5), glutamate FE (25±5 vs 3), and lactate FE (31±3 vs 5) compared to androgen-dependent TRAMP tumors. These results support cell model findings that glycolysis, oxidative metabolism, and glutaminolysis are upregulated in androgen-independent prostate tumors.

Discussion & Conclusion

This study demonstrated alterations in glucose and glutamine metabolism associated with androgen dependence in human prostate cancer cell lines and a treatment-driven murine model. We first observed no difference in glucose or glutamine consumption, indicating that techniques like FDG-PET will not be able to discriminate between androgen-dependent and androgen-independent prostate tumors. While androgen-independent tumors had elevated lactate concentrations compared to androgen-dependent tumors, this region is difficult to quantify using 1H MRSI due to the presence of lipids. Androgen-independent prostate tumors had higher flux through glycolysis, oxidative metabolism, and glutaminolysis compared to androgen-dependent prostate tumors, which can be measured using hyperpolarized 13C MRI. Based on these metabolic differences, we propose using a combination of hyperpolarized [1-13C]pyruvate, [2-13C]pyruvate and [5-13C]glutamine to noninvasively discriminate between androgen-dependent and androgen-independent prostate tumors for future patient studies using hyperpolarized 13C MRI.

Acknowledgements

This work has been supported by the following grants: NIH R01CA166655, NIH R01CA215694, NIH P41EB013598, and DoD PC160630.

References

1. Albers MJ, Bok R, Chen AP, et al. Hyperpolarized 13C Lactate, Pyruvate, and Alanine: Noninvasive Biomarkers for Prostate Cancer Detection and Grading. Cancer Research. 2008;68(20):8607-8615. doi:10.1158/0008-5472.CAN-08-0749.

2. Chen H-Y, Larson PEZ, Bok RA, et al. Assessing Prostate Cancer Aggressiveness with Hyperpolarized Dual-Agent 3D Dynamic Imaging of Metabolism and Perfusion. Cancer Research. 2017;77(12):3207-3216. doi:10.1158/0008-5472.CAN-16-2083.

3. Gingrich JR, Barrios RJ, Kattan MW, Nahm HS, Finegold MJ, Greenberg NM. Androgen-independent Prostate Cancer Progression in the TRAMP Model. Cancer Research. 1997;57(21):4687-4691. http://cancerres.aacrjournals.org/content/57/21/4687.long.

4. Lane AN, Yan J, Fan TWM. 13C Tracer Studies of Metabolism in Mouse Tumor Xenografts. Bio-protocol. 2015;5(22).

5. Prasad Maharjan R, Ferenci T. Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. Analytical Biochemistry. 2003;313(1):145-154. doi:10.1016/S0003-2697(02)00536-5.

Figures

Figure 1. Labeling schematic shows the positional 13C labeling pattern in downstream metabolites resulting from either [U-13C]glucose or [U-13C]glutamine labeling.

Figure 2. (A) Representative 1D and 2D NMR spectra of prostate cancer cell extracts. 1H spectra: TD=24k, SW=15ppm, TR=12s, NS=32. 1H-1H TOCSY: TD=4096x512, SW=12x12ppm, TR=2s, AQ=0.212s, NS=8, tmix=60 ms. 1H-13C HSQC: TD=2048x4096, SW=6x120ppm, TR=1.5s, AQ = 0.212s, NS=8. JCH = 135Hz (average JCH of 127, 130, and 145 Hz for glutamate C2, C3, and C4). Comparison of (B) glucose and glutamine consumption rate and (C) steady-state intracellular metabolite concentrations indicate no significant differences between LnCaP and PC3 cells. (D) [U-13C]glucose labeling studies and (E) [U-13C]glutamine labeling studies indicate significantly elevated lactate FE, aspartate FE, glutamate FE, and glutathione FE. *p<0.05,**p<0.01, ***p<0.001 all N=3

Figure 3. (A) TRAMP treatment timeline (B) Box and whisker plot of tumor volume changes one-week post-orchiectomy (C) Representative 1D and 2D NMR spectra indicating intracellular metabolite regions of interest in prostate cancer cell extracts. (D) Steady-state metabolite concentrations indicated that androgen-independent TRAMP tumors had significantly elevated lactate concentration. (E) [U-13C]glucose infusion studies demonstrated that androgen-dependent TRAMP tumors had significantly elevated aspartate FE glutamate FE, and lactate FE. (F) Preliminary [U-13C]glutamine labeling studies showed that androgen-independent TRAMP tumors had elevated aspartate FE, glutamate FE, and glutathione FE. *p<0.05, **p<0.01, all N=3 except [U-13C]glucose infusion studies had androgen-dependent (N=1) and androgen-independent (N=2).

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)
0201