Jessica K.R. Boult

Model free approach to kinetic analysis of real-time hyperpolarized 13C magnetic resonance spectroscopy data.

Real-time detection of the rates of metabolic flux, or exchange rates of endogenous enzymatic reactions, is now feasible in biological systems using Dynamic Nuclear Polarization Magnetic Resonance. Derivation of reaction rate kinetics from this technique typically requires multi-compartmental modeling of dynamic data, and results are therefore model-dependent and prone to misinterpretation. We present a model-free formulism based on the ratio of total areas under the curve (AUC) of the injected and product metabolite, for example pyruvate and lactate. A theoretical framework to support this novel analysis approach is described, and demonstrates that the AUC ratio is proportional to the forward rate constant k. We show that the model-free approach strongly correlates with k for whole cell in vitro experiments across a range of cancer cell lines, and detects response in cells treated with the pan-class I PI3K inhibitor GDC-0941 with comparable or greater sensitivity. The same result is seen in vivo with tumor xenograft-bearing mice, in control tumors and following drug treatment with dichloroacetate. An important finding is that the area under the curve is independent of both the input function and of any other metabolic pathways arising from the injected metabolite. This model-free approach provides a robust and clinically relevant alternative to kinetic model-based rate measurements in the clinical translation of hyperpolarized 13C metabolic imaging in humans, where measurement of the input function can be problematic.

A role for iron in Wnt signalling

There is an emerging body of evidence implicating iron in carcinogenesis and in particular colorectal cancer, but whether this involves Wnt signalling, a major oncogenic signalling pathway has not been studied. We aimed to determine the effect of iron loading on Wnt signalling using mutant APC (Caco-2 and SW480) and wild-type APC (HEK-293 and human primary fibroblasts) containing cell lines. Elevating cellular iron levels in Caco-2 and SW480 cells caused increased Wnt signalling as indicated by increased TOPFLASH reporter activity, increased mRNA expression of two known targets, c-myc and Nkd1, and increased cellular proliferation. In contrast wild-type APC and β-catenin-containing lines, HEK 293 and human primary fibroblasts were not responsive to iron loading. This was verified in SW480 cells that no longer induced iron-mediated Wnt signalling when transfected with wild-type APC. The cell line LS174T, wild type for APC but mutant for β-catenin, was also responsive suggesting that the role of iron is to regulate β-catenin. Furthermore, we show that E-cadherin status has no influence on iron-mediated Wnt signalling. We thus speculate that excess iron could exacerbate tumorigenesis in the background of APC loss, a common finding in cancers.

Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease

Summary We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

The role of tumour-derived iNOS in tumour progression and angiogenesis

Background:Progressive tumour growth is dependent on the development of a functional tumour vasculature and highly regulated by growth factors and cytokines. Nitric oxide (NO) is a free radical, produced both by tumour and host cells, and functions as a signalling molecule downstream of several angiogenic factors. Both pro- and antitumourigenic properties have been attributed to NO.Methods:The expression of the inducible isoform of NO synthase (iNOS) was knocked down in the C6 glioma cell line using constitutive expression of antisense RNA, and the effect of tumour-derived NO on tumour progression and angiogenesis was investigated.Results:Tumours in which iNOS expression was decreased displayed significantly reduced growth rates compared with tumours derived from parental C6 cells. Quantitative non-invasive magnetic resonance imaging and fluorescence microscopy of tumour uptake of Hoechst 33342, and haematoxylin and eosin staining, revealed significantly impaired vascular development and function in antisense iNOS tumours compared with control in vivo, primarily associated with the more necrotic tumour core. Decreased iNOS expression had no effect on tumour VEGF expression.Conclusion:Nitric oxide derived from tumour iNOS is an important modulator of tumour progression and angiogenesis in C6 gliomas and further supports the therapeutic strategy of inhibiting iNOS for the treatment of cancer.

Dichloroacetate induces autophagy in colorectal cancer cells and tumours

Background:Dichloroacetate (DCA) has been found to have antitumour properties.Methods:We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, 1H and hyperpolarised 13C-magnetic resonance spectroscopy.Results:Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-13C] pyruvate-to-lactate exchange rate (kPL) were decreased in DCA-treated cells, along with increased NAD+/NADH ratios and NAD+. Steady-state lactate excretion and kPL returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD+ and NADH. Reduced kPL with DCA treatment was found in HT29 tumour xenografts in vivo.Conclusions:DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced kPL and increased NAD+/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment.

Chromosomal abnormalities and novel disease-related regions in progression from Barrett's esophagus to esophageal adenocarcinoma.

Barretts esophagus (BE) is a metaplastic condition caused by chronic gastroesophageal reflux which represents an early step in the development of esophageal adenocarcinoma (EAC). Single‐nucleotide polymorphism microarray (SNP‐chip) analysis is a novel, precise, high‐throughput approach to examine genomic alterations in neoplasia. Using 250K SNP‐chips, we examined the neoplastic progression of BE to EAC, studying 11 matched sample sets: 6 sets of normal esophagus (NE), BE and EAC, 4 of NE and BE and 1 of NE and EAC. Six (60%) of 10 total BE samples and 4 (57%) of 7 total EAC samples exhibited 1 or more genomic abnormalities comprising deletions, duplications, amplifications and copy‐number‐neutral loss of heterozygosity (CNN‐LOH). Several shared abnormalities were identified, including chromosome 9p CNN‐LOH [2 BE samples (20%)], deletion of CDKN2A [4 BE samples (40%)] and amplification of 17q12‐21.2 involving the ERBB2, RARA and TOP2A genes [3.1 Mb, 2 EAC (29%)]. Interestingly, 1 BE sample contained a homozygous deletion spanning 9p22.3–p22.2 (1.2 Mb): this region harbors only 1 known gene, basonuclin 2 (BNC2). Real‐time PCR analysis confirmed the deletion of this gene and decreased the expression of BNC2 mRNA in the BE sample. Furthermore, transfection and stable expression of BNC2 caused growth arrest of OE33 EAC cells, suggesting that BNC2 functions as a tumor suppressor gene in the esophagus and that deletion of this gene occurs during the development of EAC. Thus, this SNP‐chip analysis has identified several early cytogenetic events and novel candidate cancer‐related genes that are potentially involved in the evolution of BE to EAC.

Bayesian Estimation of Changes in Transverse Relaxation Rates

Although the biasing of R*2 estimates by assuming magnitude MR data to be normally distributed has been described, the effect on changes in R*2 (ΔR*2), such as induced by a paramagnetic contrast agent, has not been reported. In this study, two versions of a novel Bayesian maximum a posteriori approach for estimating ΔR*2 are described and evaluated: one that assumes normally distributed data and the other, Rice‐distributed data. The approach enables the robust, voxelwise determination of the uncertainty in ΔR*2 estimates and provides a useful statistical framework for quantifying the probability that a pixel has been significantly enhanced. This technique was evaluated in vivo, using ultrasmall superparamagnetic iron oxide particles in orthotopic murine prostate tumors. It is shown that assuming magnitude data to be normally distributed causes ΔR*2 to be underestimated when signal‐to‐noise ratio is modest. However, the biasing effect is less than is found in R*2 estimates, implying that the simplifying assumption of normally distributed noise is more justifiable when evaluating ΔR*2 compared with when evaluating precontrast R*2 values. Magn Reson Med, 2010.

The HIF-pathway inhibitor NSC-134754 induces metabolic changes and anti-tumour activity while maintaining vascular function

Background:Hypoxia-inducible factor-1 (HIF-1) mediates the transcriptional response to hypoxic stress, promoting tumour progression and survival. This study investigated the acute effects of the small-molecule HIF-pathway inhibitor NSC-134754.Methods:Human PC-3LN5 prostate cancer cells were treated with NSC-134754 for 24 h in hypoxia. Orthotopic prostate tumour-bearing mice were treated with a single dose of NSC-134754 for 6, 24 or 48 h. Treatment response was measured using magnetic resonance spectroscopy and imaging. Ex-vivo histological validation of imaging findings was also sought.Results:In vitro, NSC-134754 significantly reduced lactate production and glucose uptake (P<0.05), while significantly increasing intracellular glucose (P<0.01) and glutamine uptake/metabolism (P<0.05). Increased glutamine metabolism was independent of c-Myc, a factor also downregulated by NSC-134754. In vivo, a significantly higher tumour apparent diffusion coefficient was determined 24 h post-treatment (P<0.05), with significantly higher tumour necrosis after 48 h (P<0.05). NSC-134754-treated tumours revealed lower expression of HIF-1α and glucose transporter-1, at 6 and 24 h respectively, while a transient increase in tumour hypoxia was observed after 24 h. Vessel perfusion/flow and vascular endothelial growth factor levels were unchanged with treatment.Conclusion:NSC-134754 induces metabolic alterations in vitro and early anti-tumour activity in vivo, independent of changes in vascular function. Our data support the further evaluation of NSC-134754 as an anti-cancer agent.

Exploring R-2* and R-1 as imaging biomarkers of tumor oxygenation

To investigate the combined use of hyperoxia‐induced ΔR2* and ΔR1 as a noninvasive imaging biomarker of tumor hypoxia.

Active site mutant dimethylarginine dimethylaminohydrolase 1 expression confers an intermediate tumour phenotype in C6 gliomas

Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the endogenous inhibitor of nitric oxide synthesis, asymmetric dimethylarginine (ADMA). Constitutive over‐expression of DDAH1, the isoform primarily associated with neuronal nitric oxide synthase (nNOS) results in increased tumour growth and vascularization, and elevated VEGF secretion. To address whether DDAH1‐mediated tumour growth is reliant upon the enzymatic activity of DDAH1, cell lines expressing an active site mutant of DDAH1 incapable of metabolizing ADMA were created. Xenografts derived from these cell lines grew significantly faster than those derived from control cells, yet not as fast as those over‐expressing wild‐type DDAH1. VEGF expression in DDAH1 mutant‐expressing tumours did not differ from control tumours but was significantly lower than that of wild‐type DDAH1‐over‐expressing tumours. Fluorescence microscopy for CD31 and pimonidazole adduct formation demonstrated that DDAH1 mutant‐expressing tumours had a lower endothelial content and demonstrated less hypoxia, respectively, than wild‐type DDAH1‐expressing tumours. However, there was no difference in uptake of the perfusion marker Hoechst 33342. Non‐invasive multiparametric quantitative MRI, including the measurement of native T1 and T2 relaxation times and apparent water diffusion coefficient, was indicative of higher cellularity in DDAH1‐expressing xenografts, which was confirmed by histological quantification of necrosis. C6 xenografts expressing active site mutant DDAH1 displayed an intermediate phenotype between tumours over‐expressing wild‐type DDAH1 and control tumours. These data suggest that enhanced VEGF expression downstream of DDAH1 was dependent upon ADMA metabolism, but that the DDAH1‐mediated increase in tumour growth was only partially dependent upon its enzymatic activity, and therefore must involve an as‐yet unidentified mechanism. DDAH1 is an important mediator of tumour progression, but appears to have addition roles independent of its metabolism of ADMA, which need to be considered in therapeutic strategies targeted against the NO/DDAH pathway in cancer. Copyright