Joan Boren

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin‐dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone‐dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Hyperpolarized [1-13C]-Ascorbic and Dehydroascorbic Acid: Vitamin C as a Probe for Imaging Redox Status in Vivo

Dynamic nuclear polarization (DNP) of 13C-labeled metabolic substrates in vitro and their subsequent intravenous administration allow both the location of the hyperpolarized substrate and the dynamics of its subsequent conversion into other metabolic products to be detected in vivo. We report here the hyperpolarization of [1-13C]-ascorbic acid (AA) and [1-13C]-dehydroascorbic acid (DHA), the reduced and oxidized forms of vitamin C, respectively, and evaluate their performance as probes of tumor redox state. Solution-state polarization of 10.5 ± 1.3% was achieved for both forms at pH 3.2, whereas at pH 7.0, [1-13C]-AA retained polarization of 5.1 ± 0.6% and [1-13C]-DHA retained 8.2 ± 1.1%. The spin–lattice relaxation times (T1s) for these labeled nuclei are long at 9.4 T: 15.9 ± 0.7 s for AA and 20.5 ± 0.9 s for DHA. Extracellular oxidation of [1-13C]-AA and intracellular reduction of [1-13C]-DHA were observed in suspensions of murine lymphoma cells. The spontaneous reaction of DHA with the cellular antioxidant glutathione was monitored in vitro and was approximately 100-fold lower than the rate observed in cell suspensions, indicating enzymatic involvement in the intracellular reduction. [1-13C]-DHA reduction was also detected in lymphoma tumors in vivo. In contrast, no detectable oxidation of [1-13C]-AA was measured in the same tumors, consistent with the notion that tumors maintain a reduced microenvironment. This study demonstrates that hyperpolarized 13C-labeled vitamin C could be used as a noninvasive biomarker of redox status in vivo, which has the potential to translate to the clinic.

Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation

A characteristic of apoptosis is the rapid accumulation of cytoplasmic lipid droplets, which are composed largely of neutral lipids. The proton signals from these lipids have been used for the non-invasive detection of cell death using magnetic resonance spectroscopy. We show here that despite an apoptosis-induced decrease in the levels and activities of enzymes involved in lipogenesis, which occurs downstream of p53 activation and inhibition of the mTOR signaling pathway, the increase in lipid accumulation is due to increased de novo lipid synthesis. This results from inhibition of mitochondrial fatty acid β-oxidation, which coupled with an increase in acyl-CoA synthetase activity, diverts fatty acids away from oxidation and into lipid synthesis. The inhibition of fatty acid oxidation can be explained by a rapid rise in mitochondrial membrane potential and an attendant increase in the levels of reactive oxygen species.

Comparison of the C2A domain of synaptotagmin-I and annexin-V as probes for detecting cell death.

The induction of apoptosis is frequently accompanied by the exposure of phosphatidylserine (PS) on the cell surface, which has been detected using radionuclide and fluorescently labeled derivatives of the PS-binding protein, Annexin V. The fluorescently labeled protein has been used extensively in vitro as a diagnostic reagent for detecting cell death, and radionuclide-labeled derivatives have undergone clinical trials for detecting tumor cell death in vivo following treatment. We show here that the C2A domain of Synaptotagmin-I, which had been fluorescently labeled at a single cysteine residue introduced by site-directed mutagenesis, detected the same levels of cell death as a similarly labeled Annexin-V derivative, in drug-treated murine lymphoma and human breast cancer cell lines in vitro. However, the C2A derivative showed significantly less binding to viable cells and, as a consequence, up to 4-fold more specific binding to apoptotic and necrotic cells when compared with Annexin-V. C2A offers a potential route for the development of a new generation of more specific imaging probes for the detection of tumor cell death in the clinic.

HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

Castrate‐resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c‐Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co‐factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up‐regulated in aggressive human prostate cancer and drives castration‐resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6‐associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient‐specific therapeutic strategies.

Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer.

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia‐inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome‐wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1‐induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

Carbonic Anhydrase Activity Monitored In Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors

Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH.

Late Imaging with (1- 11 C)Acetate Improves Detection of Tumor Fatty Acid Synthesis with PET

Tumors are often characterized by high levels of de novo fatty acid synthesis. The kinetics of acetate incorporation into tricarboxylic acid cycle intermediates and into lipids suggest that detection of tumors with [1-11C]acetate PET could be improved by imaging at later time points. Methods: The uptake and metabolism of [1-11C], [1-13C], and [1-14C]acetate were measured in mouse prostate and lung cancer models to investigate the time course of 11C label incorporation into tumor metabolites. Results: Radioactivity in the lipid fraction, as compared with the aqueous fraction, in extracts of C4-2B human prostate xenografts peaked at 90 min after [1-14C]acetate injection, which coincided with peak 13C label incorporation into the fatty acids palmitate and stearate. Contrast between the tumor and tissues, such as blood and muscle, increased in PET images acquired over a period of 120 min after [1-11C]acetate injection, and Patlak plots were linear from 17.5 min after injection. Similar results were obtained in a genetically engineered K-rasG12D; p53null lung cancer model, in which the mean tumor-to-lung ratio at 90 min after [1-14C]acetate injection was 4.4-fold higher than at 15 min. Conclusion: These findings suggest that when imaging de novo fatty acid synthesis with [1-11C]acetate it is preferable to measure uptake at later time points, when the effects of perfusion and 11C incorporation into tricarboxylic acid cycle intermediates and bicarbonate are declining. The data presented here suggest that future clinical PET scans of tumors should be acquired later than 30 min, when tracer accumulation due to de novo fatty acid synthesis prevails.

CARS based label-free assay for assessment of drugs by monitoring lipid droplets in tumour cells

Coherent anti-Stokes Raman scattering (CARS) is becoming an established tool for label-free multi-photon imaging based on molecule specific vibrations in the sample. The technique has proven to be particularly useful for imaging lipids, which are abundant in cells and tissues, including cytoplasmic lipid droplets (LD), which are recognized as dynamic organelles involved in many cellular functions. The increase in the number of lipid droplets in cells undergoing cell proliferation is a common feature in many neoplastic processes [1] and an increase in LD number also appears to be an early marker of drug-induced cell stress and subsequent apoptosis [3]. In this paper, a CARS-based label-free method is presented to monitor the increase in LD content in HCT116 colon tumour cells treated with the chemotherapeutic drugs Etoposide, Camptothecin and the protein kinase inhibitor Staurosporine. Using CARS, LDs can easily be distinguished from other cell components without the application of fluorescent dyes and provides a label-free non-invasive drug screening assay that could be used not only with cells and tissues ex vivo but potentially also in vivo.