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Metabolic Modulation for Enhanced Therapeutic Response
Kavindra Nath1, Stepan Orlovskiy1, Jeffrey Roman1, David S. Nelson1, Dennis B. Leeper2, Paul D. Smith3, Mary E. Putt4, Vivek Narayan5, and Jerry D. Glickson1

1Radiology, University of Pennsylvania, Philadelphia, PA, United States, 2Radiation Oncology, Thomas Jefferson University, Philadelphia, PA, United States, 3Cancer Biosciences, Cancer Research UK Cambridge Institute, AstraZeneca, Cambridge, United Kingdom, 4Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, PA, United States, 5Medicine, University of Pennsylvania, Philadelphia, PA, United States

Synopsis

SYNOPSIS: Metastatic prostate cancer is treated with androgen deprivation therapy (ADT), which involves the maximal suppression of circulating testosterone. Although initial responses are generally favorable, most cases progress to metastatic castration resistant prostate cancer (mCRPC), which is refractory to standard ADT. Our goal is to develop a treatment strategy to improve response of mCRPC by combining a clinically utilized metabolic modulator, AZD3965, with docetaxel. AZD3965 effects were measured in vivo by 31P and 1H MRS in PC3 prostate cancer xenografts. In vitro results suggest that AZD3965 enhances the growth inhibitory effect of docetaxel similarly both in PC3 and LNCaP cells although PC3 expresses both MCT1 & 4 and LNCaP only expresses MCT1.

Introduction

There is a critical need to develop methods to sensitize metastatic prostate cancer, the most common cancer in men and second most deadly, to taxane chemotherapy. While androgen deprivation therapy effectively treats prostate cancer, patients invariably progress to metastatic castration-resistant prostate cancer (mCRPC), which ultimately represents the lethal phenotype. MCT1 (monocarboxylate transporter1) is responsible for export of lactate by normoxic tumor cells. Hence, inhibiting lactate transport with AZD3965 (MCT1 inhibitor)1-3 increases the dependence of aerobic tumor cells on the MCT4, which provides an alternative route for export of lactic acid from tumor cells. These considerations provide the basis for our current study of androgen-dependent (LNCaP, expresses MCT1)4 and androgen-independent (PC3, expresses MCT1 & 4)4 human prostate cancer models to test the Overall Hypothesis that AZD3965 will sensitize mCRPC to docetaxel chemotherapy via modulation of the tumor microenvironment.

Methods

PC3 prostate cancer cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin. LNCaP cells were grown in RPMI 1640 with 10% fetal bovine serum and 0.5% penicillin/streptomycin. In vitro oxygen consumption and extracellular acidification rates (OCR and ECAR) were determined using the Seahorse XF-96 Extracellular Flux Analyzer on both prostate cancer cell lines. We inoculated 7×106 PC3 cells subcutaneously (s.c.) in each mouse (n=3) as a 0.1 mL suspension. AZD3965 (100 mg/kg) was administered orally twice daily up to 7 days.3 Tumors were allowed to grow until they reached 7-10 mm in diameter along the longest axis of the tumor. 31P and 1H MRS experiments were performed noninvasively after positioning the s.c. tumor in a dual-frequency slotted-tube resonator; the intracellular pH (pHi; n=3), extracellular pH (pHe; n=3), bioenergetic status (βNTP/Pi; n=3), and total lactate (n=3) were measured, respectively. MRS experiments were performed on a 9.4 T/31 cm horizontal bore Varian spectrometer. Physiological monitoring of temperature and respiration rate was maintained during an experiment. Procedures for data acquisition, post processing and parameter estimation were performed as previously described.5-7

In vitro growth inhibition study of docetaxel by AZD3965 was performed in PC3 and LNCaP cells as determined by BCA (bicinchoninic acid assay) protein assay. 5×103 cells were seeded in 96-well microplates. After attachment, the cells were treated with docetaxel (1000 nM) and/or AZD3965 (25 nM), with vehicle (DMSO) as control. After 3 days of treatment, BCA assay was performed. Student t-test was performed for statistical evaluation of both in vitro and in vivo results.

Results

Figure 1A compares PC3 and LNCaP prostate cancer cell lines with respect to OCR by a Seahorse standard cell energy phenotype experiment. Figure 1B displays a plot of OCR vs. extracellular acidification rate (ECAR) for these cell lines, also known as the Seahorse energy map. Table 1 summarizes the metabolic parameters derived for these two prostate cancer lines. Figure 2 shows the representative 31P MR spectra, and bar diagrams exhibiting variations in pHi, pHe, and bioenergetics (β-NTP/Pi) for subcutaneous PC3 xenografts (n=3) pre- and 7 days post-oral treatment twice daily with AZD3965. Figure 3 displays representative 1H MR lactate spectra measured by a Hadamard Multiquantum Coherence transfer pulse sequence of PC3 xenografts pre- and 7 days post-treatment with AZD3965 along with bar diagrams summarizing variations in tumor lactate levels. Figure 4 shows effects of docetaxel (1,000 nM), AZD3965 (25 nM), and their combination on PC3 and LNCaP cell growth. Using the Bicinchoninic Acid (BCA) protein assay, we determined that AZD3965 enhanced the growth inhibitory effect of docetaxel similarly both in the PC3 and LNCaP cells although PC3 expresses both MCT1 & 4 and LNCaP only expresses MCT1.

Discussion and Conclusion

These results indicate that the PC3 has both higher basal ECAR and higher ECAR/OCR ratio, indicating that the PC3 is highly dependent on glycolysis for growth and expresses both MCT1 & 4.4 LNCaP on the other hand, is more dependent on oxidative metabolism and expresses only MCT1,4 so it was hypothesized that sensitization of LNCaP cells to taxanes will likely require a mitochondrial inhibitor (i.e., metformin or phenformin) in addition to AZD3965. We observed that AZD3965 alone affects PC3 growth less than LNCaP growth; so we believe that MCT4 expression in PC3 provides an escape mechanism from internal acidification in PC3 but not in LNCaP. MCT1 inhibition by AZD3965 induces cell death in Burkitt lymphoma cells and MCF7 breast cancer cells through disruption of lactate export, glycolysis, and glutathione synthesis.8 Although, these factors might sensitize metastatic prostate cancer to docetaxel chemotherapy, since AZD3965 sensitized both PC3 and LNCaP to docetaxel similarly, other targets of AZD3965 have to be sought.

Acknowledgements

McCabe Foundation Research Award

References

1. Bola BM, Chadwick AL, Michopoulos F, et al. Inhibition of monocarboxylate transporter-1 (MCT1) by AZD3965 enhances radiosensitivity by reducing lactate transport. Molecular cancer therapeutics. 2014;13(12):2805-16.

2. Hong CS, Graham NA, Gu W, et al. MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4. Cell reports. 2016;14(7):1590-601. PMCID: 4816454.

3. Polanski R, Hodgkinson CL, Fusi A, et al. Activity of the monocarboxylate transporter 1 inhibitor AZD3965 in small cell lung cancer. Clin Cancer Res. 2014;20(4):926-37.

4. Sanita P, Capulli M, Teti A, et al. Tumor-stroma metabolic relationship based on lactate shuttle can sustain prostate cancer progression. BMC cancer. 2014;14:154. PMCID: 3945608.

5. Nath K Romen J, Nelson DS, et al. Effect of Differences in Metabolic Activity of Melanoma Models on Response to Lonidamine plus Doxorubicin. Scientific Reports. 2018 8(14654):Epub ahead of Print.

6. Nath K, Nelson DS, Heitjan DF, et al. Lonidamine induces intracellular tumor acidification and ATP depletion in breast, prostate and ovarian cancer xenografts and potentiates response to doxorubicin. NMR in biomedicine. 2015;28(3):281-90.

7. Nath K, Nelson DS, Ho AM, et al. 31P and 1H MRS of DB-1 melanoma xenografts: lonidamine selectively decreases tumor intracellular pH and energy status and sensitizes tumors to melphalan. NMR in biomedicine. 2013;26(1):98-105. PMCID: 3465621.

8. Doherty JR, Yang C, Scott KE, et al. Blocking lactate export by inhibiting the Myc target MCT1 Disables glycolysis and glutathione synthesis. Cancer research. 2014;74(3):908-20. PMCID: 3946415.

Figures

Figure 1. Seahorse Cell Energy Phenotype Test (A) Basal oxygen consumption rates (OCR) vs. extracellular acidification rates (ECAR) (energy map) (B) on PC3 and LNCaP prostate cancer cells. Table 1. Summary of Seahorse in vitro OCR and ECAR obtained in PC3 and LNCaP prostate cancer cells using the Seahorse XF96 Extracellular Flux Analyzer. We performed the protocol as prescribed by the manufacturer and an initial seeding density of 20,000 cells per well – 3 wells of each PC3 and LNCaP cells. Data presented as mean ± s.e.m.*, p < 0.01, a significant difference between PC3 and LNCaP (t-test).

Figure 2. Representative in vivo localized 31P MRS of PC3 subcutaneous xenografts (lower) pre- and (upper) 7 days post- AZD3965 (100 mg/kg; oral; twice daily) treatment (A) intracellular pH (pHi) (B) extracellular pH (pHe) (C), bioenergetics (β-NTP/Pi) (D) profile of PC3 (n = 3) prostate cancer xenografts pre- and 7 days post- administration of AZD3965 (100 mg/kg; oral; twice daily) using 31P MRS. The values are presented as the mean ± s.e.m.

Figure 3. Representative spectra show the effect of AZD3965 (100 mg/kg; oral; twice daily up to 7 days) on tumor lactate production in PC3 subcutaneous prostate cancer xenografts (A). The lactate peak is at 1.33 ppm. Spectra were obtained before and 7 days post- treatment using 1H MRS with a slice selective double frequency Hadamard Selective Multiple Quantum Coherence (Had-Sel-MQC) pulse sequence on 9.4 T Varian magnet. Area under the peak is shown in figure B. The values are presented as the mean ± s.e.m.

Figure 4. Growth inhibition study of docetaxel and AZD3965 in (A) PC3 and (B) LNCaP prostate cancer cells as determined by BCA protein assay. 5×103 cells were seeded in 96-well microplates. After allowing to attach, the cells were treated with docetaxel (1000 nM) and/or AZD3965 (25 nM), with DMSO control. After 3 days of treatment, BCA assay was performed. Data shown as percentage protein amount relative to control, mean ± s.e.m. (n = 4). Note that AZD3965 alone had a greater effect on LNCaP cells however, the resulting effect of AZD3965 on docetaxel was similar in both cell lines.

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