A generalised enzyme kinetic model for predicting the behaviour of complex biochemical systems.

Quasi steady-state enzyme kinetic models are increasingly used in systems modelling. The Michaelis Menten model is popular due to its reduced parameter dimensionality, but its low-enzyme and irreversibility assumption may not always be valid in the in vivo context. Whilst the total quasi-steady state assumption (tQSSA) model eliminates the reactant stationary assumptions, its mathematical complexity is increased. Here, we propose the differential quasi-steady state approximation (dQSSA) kinetic model, which expresses the differential equations as a linear algebraic equation. It eliminates the reactant stationary assumptions of the Michaelis Menten model without increasing model dimensionality. The dQSSA was found to be easily adaptable for reversible enzyme kinetic systems with complex topologies and to predict behaviour consistent with mass action kinetics in silico. Additionally, the dQSSA was able to predict coenzyme inhibition in the reversible lactate dehydrogenase enzyme, which the Michaelis Menten model failed to do. Whilst the dQSSA does not account for the physical and thermodynamic interactions of all intermediate enzyme-substrate complex states, it is proposed to be suitable for modelling complex enzyme mediated biochemical systems. This is due to its simpler application, reduced parameter dimensionality and improved accuracy.

Overexpression of a key regulator of lipid homeostasis, Scap, promotes respiration in prostate cancer cells.

Prostate metabolism is unique, characterised by cholesterol accumulation and reduced respiration. Are these related? We modulated cholesterol levels and despite changes in mitochondrial cholesterol content, we saw no effects on lactate production or respiration. Instead, these features may be related via sterol regulatory element-binding protein 2 (SREBP-2), the master transcriptional regulator of cholesterol synthesis. SREBP-2 diverts acetyl-CoA into cholesterol synthesis and may thus reduce respiration. We examined LNCaP cells overexpressing the SREBP-2 regulator, Scap: although having higher SREBP-2 activity, these cells displayed higher respiration. This striking observation warrants further investigation. Given that SREBP-2 and Scap are regulated by factors driving prostate growth, exploring this observation further could shed light on prostate carcinogenesis.

Cholesterol accumulation in prostate cancer: a classic observation from a modern perspective.

Prostate cancer (PCa) is the most common cancer in men in developed countries. Epidemiological studies have associated high blood-cholesterol levels with an increased risk of PCa, whilst cholesterol-lowering drugs (statins) reduce the risk of advanced PCa. Furthermore, normal prostate epithelial cells have an abnormally high cholesterol content, with cholesterol levels increasing further during progression to PCa. In this review, we explore why and how this occurs. Concurrent to this observation, intense efforts have been expended in cardiovascular research to better understand the regulators of cholesterol homeostasis. Here, we apply this knowledge to elucidate the molecular mechanisms driving the accumulation of cholesterol in PCa. For instance, recent evidence from our group and others shows that major signalling players in prostate growth and differentiation, such as androgens and Akt, modulate the key transcriptional regulators of cholesterol homeostasis to enhance cholesterol levels. This includes adjusting central carbon metabolism to sustain greater lipid synthesis. Perturbations in cholesterol homeostasis appear to be maintained even when PCa approaches the advanced, 'castration-resistant' state. Overall, this provides a link between cholesterol accumulation and PCa cell growth. Given there is currently no cure for castration-resistant PCa, could cholesterol metabolism be a novel target for PCa therapy? Overall, this review presents a picture that cholesterol metabolism is important for PCa development: growth-promoting factors stimulate cholesterol accumulation, which in turn presents a possible target for chemotherapy. Consequently, we recommend future investigations, both to better elucidate the mechanisms driving this accumulation and applying it in novel chemotherapeutic strategies.

Does changing androgen receptor status during prostate cancer development impact upon cholesterol homeostasis?

BACKGROUND: Recent evidence associates prostate cancer with high cholesterol levels, with cholesterol being an important raw material for cell-growth. Within the cell, cholesterol homeostasis is maintained by two master transcription factors: sterol-regulatory element-binding protein 2 (SREBP-2) and liver X receptor (LXR). We previously showed that the androgen receptor, a major player in prostate cell physiology, toggles these transcription factors to promote cholesterol accumulation. Given that prostate cancer therapy targets the androgen receptor, selecting for cells with altered androgen receptor activity, how would this affect SREBP-2 and LXR activity? Using a novel prostate cancer progression model, we explored how this crosstalk between the androgen receptor and cholesterol homeostasis changes during prostate cancer development. METHODOLOGY/PRINCIPAL FINDINGS: Firstly, we characterised our progression model, which involved 1) culturing LNCaP cells at physiological testosterone levels to generate androgen-tolerant LNCaP-305 cells, and 2) culturing LNCaP-305 with the anti-androgen casodex to generate castration-resistant LNCaP-364 cells. This progression was accompanied by upregulated androgen receptor expression, typically seen clinically, and a reduction in androgen receptor activity. Although this influenced how SREBP-2 and LXR target genes responded to androgen treatment, cellular cholesterol levels and their response to changing sterol status was similar in all LNCaP sub-lines. CONCLUSION/SIGNIFICANCE: Overall cholesterol homeostasis is unaffected by changing androgen receptor activity in prostate cancer cells. This does not negate the relationship between androgens and cholesterol homeostasis, but rather suggests that other factors compensate for altered androgen receptor activity. Given that cholesterol regulation is maintained during progression, this supports the growing idea that cholesterol metabolism is a suitable target for prostate cancer.

Cross-talk between the androgen receptor and the liver X receptor: implications for cholesterol homeostasis.

High cholesterol levels are associated with prostate cancer development. Androgens promote cholesterol accumulation by activating the sterol-regulatory element-binding protein isoform 2 (SREBP-2) transcription factor. However, SREBP-2 is in balance with the liver X receptor (LXR; NR1H2/NR1H3), a transcription factor that prevents cholesterol accumulation. Here, we show that LXR activity is down-regulated by the androgen receptor (AR; NR3C4). In turn, this reduces LXR target gene expression. This antagonism on LXR is also exerted by other steroid hormone receptors, including the estrogen, glucocorticoid, and progesterone receptors. This suggests a generalizable mechanism, but the AR does not affect LXR mRNA levels, protein degradation, or DNA binding. We also found that the AR does not require protein synthesis to influence LXR, suggesting a direct antagonism. However, the AR does not directly bind LXR. The AR N-terminal domain (involved in transactivation), but not its DNA-binding domain, is required to suppress LXR activity, suggesting coactivator competition. Overall, this androgen-mediated antagonism of LXR complements SREBP-2 activation, providing a more complete picture as to how androgens increase cellular cholesterol levels in a prostate cancer setting. Given the cross-talk between other steroid hormone receptors and LXR, hormonal regulation of cholesterol via LXR may occur in a variety of cellular contexts.

Cholesterol homeostasis in two commonly used human prostate cancer cell-lines, LNCaP and PC-3.

BACKGROUND: Recently, there has been renewed interest in the link between cholesterol and prostate cancer. It has been previously reported that in vitro, prostate cancer cells lack sterol-mediated feedback regulation of the major transcription factor in cholesterol homeostasis, sterol-regulatory element binding protein 2 (SREBP-2). This could explain the accumulation of cholesterol observed in clinical prostate cancers. Consequently, perturbed feedback regulation to increased sterol levels has become a pervasive concept in the prostate cancer setting. Here, we aimed to explore this in greater depth. METHODOLOGY/PRINCIPAL FINDINGS: After altering the cellular cholesterol status in LNCaP and PC-3 prostate cancer cells, we examined SREBP-2 processing, downstream effects on promoter activity and expression of SREBP-2 target genes, and functional activity (low-density lipoprotein uptake, cholesterol synthesis). In doing so, we observed that LNCaP and PC-3 cells were sensitive to increased sterol levels. In contrast, lowering cholesterol levels via statin treatment generated a greater response in LNCaP cells than PC-3 cells. This highlighted an important difference between these cell-lines: basal SREBP-2 activity appeared to be higher in PC-3 cells, reducing sensitivity to decreased cholesterol levels. CONCLUSION/SIGNIFICANCE: Thus, prostate cancer cells are sensitive to changing sterol levels in vitro, but the extent of this regulation differs between prostate cancer cell-lines. These results shed new light on the regulation of cholesterol metabolism in two commonly used prostate cancer cell-lines, and emphasize the importance of establishing whether or not cholesterol homeostasis is perturbed in prostate cancer in vivo.

Putative fat fighter hits the middle man.

In this issue, Kamisuki and colleagues characterize fatostatin. This compound inhibits the activity of SREBPs, the master transcription factors of lipid homeostasis. This useful laboratory tool also improved the lipid profile of obese mice; does this have clinical implications?

Are protein complexes made of cores, modules and attachments?

It has recently been proposed by Gavin et al. (Nature 2006, 440, 631-636) that protein complexes in the cell exist in different forms. The proteins within each complex were proposed to exist as three different classes, being core, module or attachment proteins. This study investigates whether the core-module-attachment classification of proteins within each complex is supported by other high-throughput protein data. Core proteins were found to have lower abundance, and shorter half-life as compared to attachment proteins, whilst the abundance and half-life of core and module proteins were similar. When the cell was perturbed, core proteins had smaller changes in abundance as compared to module and attachment proteins. Comparisons between six different pairwise interaction types of core, module and attachment proteins within a complex showed interaction types involving core or module proteins were more likely to be mediated by domain-domain interactions (DDIs) than interaction types involving attachment proteins. Interaction types that involve attachment proteins had a relatively higher ratio of abundance and ratio of half-life. So we conclude that, the core, module and attachment model of protein complexes is supported by data from these proteomic scale datasets, and describe a model for a typical protein complex that considers the above results.