John R. Griffiths

Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a Mouse Model of Pancreatic Cancer

Its All in the Delivery Pancreatic cancer is almost universally associated with a poor prognosis, in part because the tumors are resistant to chemotherapeutic drugs. Working with a mouse tumor model that displays many features of the human disease, Olive et al. (p. 1457, published online 21 May; see the Perspective by Olson and Hanahan) found that the tumors were poorly vascularized, a factor likely to impede drug delivery. Treatment of the mice with the chemotherapeutic drug gemcitabine in combination with a drug that depletes tumor-associated stromal tissue led to an increase in tumor vasculature, enhanced delivery of gemcitabine, and a delay in disease progression. Thus, drugs targeting the tumor stroma may merit investigation as a way to enhance the efficacy of conventional chemotherapy for pancreatic cancer. Pancreatic tumors are unresponsive to chemotherapy because their limited vasculature precludes efficient drug delivery. Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.

SREBP Activity Is Regulated by mTORC1 and Contributes to Akt-Dependent Cell Growth

Summary Cell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). Here we show that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. We also show that silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of dSREBP in flies caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. Our results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth.

Causes and consequences of tumour acidity and implications for treatment

Tumour cells have a lower extracellular pH (pHe) than normal cells; this is an intrinsic feature of the tumour phenotype, caused by alterations either in acid export from the tumour cells or in clearance of extracellular acid. Low pHe benefits tumour cells because it promotes invasiveness, whereas a high intracellular pH (pHi) gives them a competitive advantage over normal cells for growth. Molecular genetic approaches have revealed hypoxia-induced coordinated upregulation of glycolysis, a potentially important mechanism for establishing the metabolic phenotype of tumours. Understanding tumour acidity opens up new opportunities for therapy.

Metabolic profiles of human brain tumors using quantitative in vivo 1H magnetic resonance spectroscopy

Proton spectroscopy can noninvasively provide useful information on brain tumor type and grade. Short‐ (30 ms) and long‐ (136 ms) echo time (TE) 1H spectra were acquired from normal white matter (NWM), meningiomas, grade II astrocytomas, anaplastic astrocytomas, glioblastomas, and metastases. Very low myo‐Inositol ([mI]) and creatine ([Cr]) were characteristic of meningiomas, and high [mI] characteristic of grade II astrocytomas. Tumor choline ([Cho]) was greater than NWM and increased with grade for grade II and anaplastic astrocytomas, but was highly variable for glioblastomas. Higher [Cho] and [Cr] correlated with low lipid and lactate (P < 0.05), indicating a dilution of metabolite concentrations due to necrosis in high‐grade tumors. Metabolite peak area ratios showed no correlation with lipids and mI/Cho (at TE = 30 ms), and Cr/Cho (at TE = 136 ms) best correlated with tumor grade. The quantified lipid, macromolecule, and lactate levels increased with grade of tumor, consistent with progression from hypoxia to necrosis. Quantification of lipids and macromolecules at short TE provided a good marker for tumor grade, and a scatter plot of the sum of alanine, lactate, and δ1.3 lipid signals vs. mI/Cho provided a simple way to separate most tumors by type and grade. Magn Reson Med 49:223–232, 2003.

Dynamic asymmetry of phosphocreatine concentration and O2 uptake between the on- and off-transients of moderate- and high-intensity exercise in humans

The on‐ and off‐transient (i.e. phase II) responses of pulmonary oxygen uptake (V̇O2) to moderate‐intensity exercise (i.e. below the lactate threshold, θL) in humans has been shown to conform to both mono‐exponentiality and ‘on‐off’ symmetry, consistent with a system manifesting linear control dynamics. However above θL the V̇O2 kinetics have been shown to be more complex: during high‐intensity exercise neither mono‐exponentiality nor ‘on‐off’ symmetry have been shown to appropriately characterise the V̇O2 response. Muscle [phosphocreatine] ([PCr]) responses to exercise, however, have been proposed to be dynamically linear with respect to work rate, and to demonstrate ‘on‐off’ symmetry at all work intenisties. We were therefore interested in examining the kinetic characteristics of the V̇O2 and [PCr] responses to moderate‐ and high‐intensity knee‐extensor exercise in order to improve our understanding of the factors involved in the putative phosphate‐linked control of muscle oxygen consumption. We estimated the dynamics of intramuscular [PCr] simultaneously with those of V̇O2 in nine healthy males who performed repeated bouts of both moderate‐ and high‐intensity square‐wave, knee‐extension exercise for 6 min, inside a whole‐body magnetic resonance spectroscopy (MRS) system. A transmit‐receive surface coil placed under the right quadriceps muscle allowed estimation of intramuscular [PCr]; V̇O2 was measured breath‐by‐breath using a custom‐designed turbine and a mass spectrometer system. For moderate exercise, the kinetics were well described by a simple mono‐exponential function (following a short cardiodynamic phase for V̇O2,), with time constants (τ) averaging: τV̇O2,on 35 ± 14 s (±s.d.), τ[PCr]on 33 ± 12 s, τV̇O2,off 50 ± 13 s and τ[PCr]off 51 ± 13 s. The kinetics for both V̇O2 and [PCr] were more complex for high‐intensity exercise. The fundamental phase expressing average τ values of τV̇O2,on 39 ± 4 s, τ[PCr]on 38 ± 11 s, τV̇O2,off 51 ± 6 s and τ[PCr]off 47 ± 11 s. An associated slow component was expressed in the on‐transient only for both V̇O2 and [PCr], and averaged 15.3 ± 5.4 and 13.9 ± 9.1 % of the fundamental amplitudes for V̇O2 and [PCr], respectively. In conclusion, the τ values of the fundamental component of [PCr] and V̇O2 dynamics cohere to within 10 %, during both the on‐ and off‐transients to a constant‐load work rate of both moderate‐ and high‐intensity exercise. On average, ≈90 % of the magnitude of the V̇O2 slow component during high‐intensity exercise is reflected within the exercising muscle by its [PCr] response.

Inferences from pulmonary O2 uptake with respect to intramuscular [phosphocreatine] kinetics during moderate exercise in humans

1 In the non‐steady state of moderate intensity exercise, pulmonary O2 uptake (V̇p,O2) is temporally dissociated from muscle O2 consumption (V̇m,O2) due to the influence of the intervening venous blood volume and the contribution of body O2 stores to ATP synthesis. A monoexponential model of V̇p,O2 without a delay term, therefore, implies an obligatory slowing of V̇p,O2 kinetics in comparison to V̇m,O2. 2 During moderate exercise, an association of V̇m,O2 and [phosphocreatine] ([PCr]) kinetics is a necessary consequence of the control of muscular oxidative phosphorylation mediated by some function of [PCr]. It has also been suggested that the kinetics of V̇p,O2 will be expressed with a time constant within 10 % of that of V̇m,O2. 3 V̇p,O2 and intramuscular [PCr] kinetics were investigated simultaneously during moderate exercise of a large muscle mass in a whole‐body NMR spectrometer. Six healthy males performed prone constant‐load quadriceps exercise. A transmit‐receive coil under the right quadriceps allowed determination of intramuscular [PCr]; V̇p,O2 was measured breath‐by‐breath, in concert with [PCr], using a turbine and a mass spectrometer system. 4 Intramuscular [PCr] decreased monoexponentially with no delay in response to exercise. The mean of the time constants (τPCr) was 35 s (range, 20–64 s) for the six subjects. 5 Two temporal phases were evident in the V̇p,O2 response. When the entire V̇p,O2 response was modelled to be exponential with no delay, its time constant (τ′V̇p,O2) was longer in all subjects (group mean = 62 s; range, 52–92 s) than that of [PCr], reflecting the energy contribution of the O2 stores. 6 Restricting the V̇p,O2 model fit to phase II resulted in matching kinetics for V̇p,O2 (group mean τV̇p,O2= 36 s; range, 20–68 s) and [PCr], for all subjects. 7 We conclude that during moderate intensity exercise the phase II τV̇p,O2 provides a good estimate of τPCr and by implication that of V̇m,O2 (τV̇m,O2).

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.

PKB/Akt induces transcription of enzymes involved in cholesterol and fatty acid biosynthesis via activation of SREBP.

Protein kinase B (PKB/Akt) has been shown to play a role in protection from apoptosis, cell proliferation and cell growth. It is also involved in mediating the effects of insulin, such as lipogenesis, glucose uptake and conversion of glucose into fatty acids and cholesterol. Sterol-regulatory element binding proteins (SREBPs) are the major transcription factors that regulate genes involved in fatty acid and cholesterol synthesis. It has been postulated that constitutive activation of the phosphatidylinositol 3 kinase/Akt pathway may be involved in fatty acid and cholesterol accumulation that has been described in several tumour types. In this study, we have analysed changes in gene expression in response to Akt activation using DNA microarrays. We identified several enzymes involved in fatty acid and cholesterol synthesis as targets for Akt-regulated transcription. Expression of these enzymes has previously been shown to be regulated by the SREBP family of transcription factors. Activation of Akt induces synthesis of full-length SREBP-1 and SREBP-2 proteins as well as expression of fatty acid synthase (FAS), the key regulatory enzyme in lipid biosynthesis. We also show that Akt leads to the accumulation of nuclear SREBP-1 but not SREBP-2, and that activation of SREBP is required for Akt-induced activation of the FAS promoter. Finally, activation of Akt induces an increase in the concentration of cellular fatty acids as well as phosphoglycerides, the components of cellular membranes. Our data indicate that activation of SREBP by Akt leads to the induction of key enzymes of the cholesterol and fatty acid biosynthesis pathways, and thus membrane lipid biosynthesis.

Multiple Reaction Monitoring to Identify Sites of Protein Phosphorylation with High Sensitivity

Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.

Effects of prior exercise on oxygen uptake and phosphocreatine kinetics during high‐intensity knee‐extension exercise in humans

1 A prior bout of high‐intensity square‐wave exercise can increase the temporal adaptation of pulmonary oxygen uptake (V̇O2) to a subsequent bout of high‐intensity exercise. The mechanisms controlling this adaptation, however, are poorly understood. 2 We therefore determined the dynamics of intramuscular [phosphocreatine] ([PCr]) simultaneously with those of V̇O2 in seven males who performed two consecutive bouts of high‐intensity square‐wave, knee‐extensor exercise in the prone position for 6 min with a 6 min rest interval. A magnetic resonance spectroscopy (MRS) transmit‐receive surface coil under the quadriceps muscle allowed estimation of [PCr]; V̇O2 was measured breath‐by‐breath using a custom‐designed turbine and a mass spectrometer system. 3 The V̇O2 kinetics of the second exercise bout were altered compared with the first such that (a) not only was the instantaneous rate of V̇O2 change (at a given level of V̇O2) greater but the phase II τ was also reduced ‐ averaging 46.6 ± 6.0 s (bout 1) and 40.7 ± 8.4 s (bout 2) (mean ±s.d.) and (b) the magnitude of the later slow component was reduced. 4 This was associated with a reduction of, on average, 16.1 % in the total exercise‐induced [PCr] decrement over the 6 min of the exercise, of which 4.0 % was due to a reduction in the slow component of [PCr]. There was no discernable alteration in the initial rate of [PCr] change. The prior exercise, therefore, changed the multi‐compartment behaviour towards that of functionally first‐order dynamics. 5 These observations demonstrate that the V̇O2 responses relative to the work rate input for high‐intensity exercise are non‐linear, as are, it appears, the putative phosphate‐linked controllers for which [PCr] serves as a surrogate.