The role of insulin and IGF2 signalling on metabolic pathways in prostate cancer progression

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in Australia, North America, and Europe. If found early and locally confined, CaP can be treated with radical prostatectomy or radiation therapy; however, 25-40% patients will relapse and go on to advanced disease. The most common therapy in these cases is androgen deprivation therapy (ADT), which suppresses androgen production from the testis. Lack of the testicular androgen supply causes cells of the prostate to undergo apoptosis. However, in some cases the regression initially seen with ADT eventually gives way to a growth of a population of cancerous cells that no longer require testicular androgens. This phenotype is essentially fatal and is termed castrate resistant prostate cancer (CRPC). In addition to eventual regression, there are many undesirable side effects which accompany ADT, including development of a metabolic syndrome, which is defined by the U.S. National Library of Medicine as “a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes.” This project will focus on the effect of ADT induced hyperinsulinemia, as mimicked by treating androgen receptor positive CaP cells with insulin in a serum (hormone) deprived environment. While this side effect is not widely explored, in this thesis it is demonstrated for the first time that insulin upregulates pathways important to CaP progression. Our group has previously shown that during CaP progression, the enzymes necessary for de novo steroidogenesis are upregulated in the LNCaP xenograft model, total steroid levels are increased in tumours compared to pre castrate levels, and de novo steroidogenesis from radio-labelled acetate has been demonstrated. Because of the CaP dependence on AR for survival, we and other groups believe that CaP cells carry out de novo steroidogenesis to survive in androgen deprived conditions. Because (a) men on ADT often develop metabolic syndrome, and (b) men with lifestyle-induced obesity and hyperinsulinemia have worse prognosis and faster disease progression, and because (c) insulin causes steroidogenesis in other cell lines, the hypothesis that insulin may contribute to CaP progression through upregulation of steroidogenesis was explored. Insulin upregulates steroidogenesis enzymes at the mRNA level in three AR positive cell lines, as well as upregulating these enzymes at the protein level in two cell lines. It has also been demonstrated that insulin increases mitochondrial (functional) levels of steroid acute regulatory protein (StAR). Furthermore, insulin causes increased levels of total steroids in and induction of de novo steroid synthesis by insulin has been demonstrated at levels induced sufficient to activate AR. The effect of insulin analogs on CaP steroidogenesis in LNCaP and VCaP cells has also been investigated because epidemiological studies suggest that some of the analogs developed may have more cancer stimulatory effects than normal insulin. In this project, despite the signalling differences between glargine, X10, and insulin, these analogs did not appear to induce steroidogenesis any more potently that normal insulin. The effect of insulin of MCF7breast cancer cells was also investigated with results suggesting that breast cancer cells may be capable of de novo steroidogenesis, and that increase in estradiol production may be exacerbated by insulin. Insulin has also been long known to stimulate lipogenesis in the liver and adipocytes, and has been demonstrated to increase lipogenesis in breast cancer cells; therefore, investigation of the effect of insulin on lipogenesis, which is a hallmark of aggressive cancers, was investigated. In CaP progression sterol regulatory element binding protein (SREBP) is dysregulated and upregulates fatty acid synthase (FASN), acetyl CoA-carboxylase, and other lipogenesis genes. SREBP is important for steroidogenesis and in this project has been shown to be upregulated by insulin in CaP cells. Fatty acid synthesis provides building blocks of membrane growth, provides substrates for acid oxidation, the main energy source for CaP cells, provides building blocks for anti-apoptotic and proinflammatory molecules, and provides molecules that stimulate steroidogenesis. In this project it has been shown that insulin upregulates FASN and ACC, which synthesize fatty acids, as well as upregulating hormone sensitive lipase (HSL), diazepam-binding inhibitor (DBI), and long-chain acyl-CoA synthetase 3 (ACSL3), which contribute to lipid activation of steroidogenesis. Insulin also upregulates total lipid levels and de novo lipogenesis, which can be suppressed by inhibition of the insulin receptor (INSR). The fatty acids synthesized after insulin treatment are those that have been associated with CaP; furthermore, microarray data suggests insulin may upregulate fatty acid biosynthesis, metabolism and arachidonic acid metabolism pathways, which have been implicated in CaP growth and survival. Pharmacological agents used to treat patients with hyperinsulinemia/ hyperlipidemia have gained much interest in regards to CaP risk and treatment; however, the scientific rationale behind these clinical applications has not been examined. This thesis explores whether the use of metformin or simvastatin would decrease either lipogenesis or steroidogenesis or both in CaP cells. Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitor, which blocks synthesis of cholesterol, the building block of steroids/ androgens. It has also been postulated to down regulate SREBP in other metabolic disorders. It has been shown in this thesis, in LNCaP cells, that simvastatin inhibited and decreased insulin induced steroidogenesis and lipogenesis, respectively, but increased these pathways in the absence of insulin. Conversely, metformin, which activates AMP-activated protein kinase (AMPK) to shut down lipogenesis, cholesterol synthesis, and protein synthesis, highly suppresses both steroidogenesis and lipogenesis in the presence and absence of insulin. Lastly, because it has been demonstrated to increase steroidogenesis in other cell lines, and because the elucidation of any factors affecting steroidogenesis is important to understanding CaP, the effect of IGF2 on steroidogenesis in CaP cells was investigated. In patient samples, as men progress to CRPC, IGF2 mRNA and the protein levels of the receptors it may signal through are upregulated. It has also been demonstrated that IGF2 upregulates steroidogenic enzymes at both the mRNA and protein levels in LNCaP cells, increases intracellular and secreted steroid/androgen levels in LNCaPs to levels sufficient to stimulate the AR, and upregulated de novo steroidogenesis in LNCaPs and VCaPs. As well, inhibition of INSR and insulin-like growth factor 1 receptor (IGF1R), which IGF2 signals through, suggests that induction of steroidogenesis may be occurring predominantly through IGF1R. In summary, this project has illuminated for the first time that insulin is likely to play a large role in cancer progression, through upregulation of the steroidogenesis and lipogenesis pathways at the mRNA and protein levels, and production levels, and demonstrates a novel role for IGF-II in CaP progression through stimulation of steroidogenesis. It has also been demonstrated that metformin and simvastatin drugs may be useful in suppressing the insulin induction of these pathways. This project affirms the pathways by which ADT- induced metabolic syndrome may exacerbate CaP progression and strongly suggests that the monitoring and modulation of the metabolic state of CaP patients could have a strong impact on their therapeutic outcomes.

Combination of MEK-ERK inhibitor and hyaluronic acid has a synergistic effect on anti-hypertrophic and pro-chondrogenic activities in osteoarthritis treatment

We hypothesised that a potentially disease-modifying osteoarthritis (OA) drug such as hyaluronic acid (HA) given in combination with anti-inflammatory signalling agents such as mitogen-activated protein kinase kinase–extracellular signal-regulated kinase (MEK-ERK) signalling inhibitor (U0126) could result in additive or synergistic effects on preventing the degeneration of articular cartilage. Chondrocyte differentiation and hypertrophy were evaluated using human OA primary cells treated with either HA or U0126, or the combination of HA + U0126. Cartilage degeneration in menisectomy (MSX) induced rat OA model was investigated by intra-articular delivery of either HA or U0126, or the combination of HA + U0126. Histology, immunostaining, RT-qPCR, Western blotting and zymography were performed to assess the expression of cartilage matrix proteins and hypertrophic markers. Phosphorylated ERK (pERK)1/2-positive chondrocytes were significantly higher in OA samples compared with those in healthy control suggesting the pathological role of that pathway in OA. It was noted that HA + U0126 significantly reduced the levels of pERK, chondrocyte hypertrophic markers (COL10 and RUNX2) and degenerative markers (ADAMTs5 and MMP-13), however, increased the levels of chondrogenic markers (COL2) compared to untreated or the application of HA or U0126 alone. In agreement with the results in vitro, intra-articular delivery of HA + U0126 showed significant therapeutic improvement of cartilage in rat MSX OA model compared with untreated or the application of HA or U0126 alone. Our study suggests that the combination of HA and MEK-ERK inhibition has a synergistic effect on preventing cartilage degeneration.

Prognostic significance of IGF and ECM induced signalling proteins in breast cancer patients

Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM). Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome. To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins. This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2). Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models. The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.

ATM is required for the cellular response to thymidine induced replication fork stress

Genetically distinct checkpoints, activated as a consequence of either DNA replication arrest or ionizing radiation-induced DNA damage, integrate DNA repair responses into the cell cycle programme. The ataxia-telangiectasia mutated (ATM) protein kinase blocks cell cycle progression in response to DNA double strand breaks, whereas the related ATR is important in maintaining the integrity of the DNA replication apparatus. Here, we show that thymidine, which slows the progression of replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, depends on both ATM and ATR. Thymidine induces ATM-mediated phosphorylation of Chk2 and NBS1 and an ATM-independent phosphorylation of Chk1 and SMC1. AT cells exposed to thymidine showed decreased viability and failed to induce homologous recombination repair (HRR). Taken together, our results implicate ATM in the HRR-mediated rescue of replication forks impaired by thymidine treatment.

Integrative genomic profiling reveals conserved genetic mechanisms for tumorigenesis in common entities of non-Hodgkin's lymphoma

Recent developments in genomic technologies have resulted in increased understanding of pathogenic mechanisms and emphasized the importance of central survival pathways. Here, we use a novel bioinformatic based integrative genomic profiling approach to elucidate conserved mechanisms of lymphomagenesis in the three commonest non-Hodgkin's lymphoma (NHL) entities: diffuse large B-cell lymphoma, follicular lymphoma, and B-cell chronic lymphocytic leukemia. By integrating genome-wide DNA copy number analysis and transcriptome profiling of tumor cohorts, we identified genetic lesions present in each entity and highlighted their likely target genes. This revealed a significant enrichment of components of both the apoptosis pathway and the mitogen activated protein kinase pathway, including amplification of the MAP3K12 locus in all three entities, within the set of genes targeted by genetic alterations in these diseases. Furthermore, amplification of 12p13.33 was identified in all three entities and found to target the FOXM1 oncogene. Amplification of FOXM1 was subsequently found to be associated with an increased MYC oncogenic signaling signature, and siRNA-mediated knock-down of FOXM1 resulted in decreased MYC expression and induced G2 arrest. Together, these findings underscore genetic alteration of the MAPK and apoptosis pathways, and genetic amplification of FOXM1 as conserved mechanisms of lymphomagenesis in common NHL entities. Integrative genomic profiling identifies common central survival mechanisms and highlights them as attractive targets for directed therapy.

High rates of muscle glycogen resynthesis after exhaustive exercise when carbohydrate is coingested with caffeine

We determined the effect of coingestion of caffeine (Caff) with carbohydrate (CHO) on rates of muscle glycogen resynthesis during recovery from exhaustive exercise in seven trained subjects who completed two experimental trials in a randomized, double-blind crossover design. The evening before an experiment subjects performed intermittent exhaustive cycling and then consumed a low-CHO meal. The next morning subjects rode until volitional fatigue. On completion of this ride subjects consumed either CHO [4 g/kg body mass (BM)] or the same amount of CHO + Caff (8 mg/kg BM) during 4 h of passive recovery. Muscle biopsies and blood samples were taken at regular intervals throughout recovery. Muscle glycogen levels were similar at exhaustion [?75 mmol/kg dry wt (dw)] and increased by a similar amount (?80%) after 1 h of recovery (133 ± 37.8 vs. 149 ± 48 mmol/kg dw for CHO and Caff, respectively). After 4 h of recovery Caff resulted in higher glycogen accumulation (313 ± 69 vs. 234 ± 50 mmol/kg dw, P < 0.001). Accordingly, the overall rate of resynthesis for the 4-h recovery period was 66% higher in Caff compared with CHO (57.7 ± 18.5 vs. 38.0 ± 7.7 mmol·kg dw-1·h-1, P < 0.05). After 1 h of recovery plasma Caff levels had increased to 31 ± 11 ?M (P < 0.001) and at the end of the recovery reached 77 ± 11 ?M (P < 0.001) with Caff. Phosphorylation of CaMKThr286 was similar after exercise and after 1 h of recovery, but after 4 h CaMKThr286 phosphorylation was higher in Caff than CHO (P < 0.05). Phosphorylation of AMP-activated protein kinase (AMPK)Thr172 and AktSer473 was similar for both treatments at all time points. We provide the first evidence that in trained subjects coingestion of large amounts of Caff (8 mg/kg BM) with CHO has an additive effect on rates of postexercise muscle glycogen accumulation compared with consumption of CHO alone.

Effect of high-frequency resistance exercise on adaptive responses in skeletal muscle

PURPOSE: Regulation of skeletal muscle mass is highly dependent on contractile loading. The purpose of this study was to examine changes in growth factor and inflammatory pathways following high-frequency resistance training. METHODS: Using a novel design in which male Sprague-Dawley rats undertook a "stacked" resistance training protocol designed to generate a summation of transient exercise-induced signaling responses (four bouts of three sets × 10 repetitions of squat exercise, separated by 3 h of recovery), we determined the effects of high training frequency on signaling pathways and transcriptional activity regulating muscle mass. RESULTS: The stacked training regimen resulted in acute suppression of insulin-like growth factor 1 mRNA abundance (P < 0.05) and Akt phosphorylation (P < 0.05), an effect that persisted 48 h after the final training bout. Conversely, stacked training elicited a coordinated increase in the expression of tumor necrosis factor alpha, inhibitor kappa B kinase alpha/beta activity (P < 0.05), and p38 mitogen-activated protein kinase phosphorylation (P < 0.05) at 3 h after each training bout. In addition, the stacked series of resistance exercise bouts induced an increase in p70 S6 kinase phosphorylation 3 h after bouts ×3 and ×4, independent of the phosphorylation state of Akt. CONCLUSIONS: Our results indicate that high resistance training frequency extends the transient activation of inflammatory signaling cascades, concomitant with persistent suppression of key mediators of anabolic responses. We provide novel insights into the effects of the timing of exercise-induced overload and recovery on signal transduction pathways and transcriptional activity regulating skeletal muscle mass in vivo.

Exercise-induced phosphorylation of the novel Akt substrates AS160 and filamin A in human skeletal muscle

Skeletal muscle contraction stimulates multiple signaling cascades that govern a variety of metabolic and transcriptional events. Akt/protein kinase B regulates metabolism and growth/muscle hypertrophy, but contraction effects on this target and its substrates are varied and may depend on the mode of the contractile stimulus. Accordingly, we determined the effects of endurance or resistance exercise on phosphorylation of Akt and downstream substrates in six trained cyclists who performed a single bout of endurance or resistance exercise separated by ?7 days. Muscle biopsies were taken from the vastus lateralis at rest and immediately after exercise. Akt Ser 473 phosphorylation was increased (1.8-fold; P = 0.011) after endurance but was unchanged after resistance exercise. Conversely, Akt Thr 308 phosphorylation was unaltered after either bout of exercise. Several exercise-responsive phosphoproteins were detected by immunoblot analysis with a phospho-Akt substrate antibody. pp160 and pp300 were identified as AS160 and filamin A, respectively, with increased phosphorylation (2.0- and 4.9-fold, respectively; P < 0.05) after endurance but not resistance exercise. In conclusion, AS160 and filamin A may provide an important link to mediate endurance exercise-induced bioeffects in skeletal muscle.

Mice selectively bred for High and Low fear behavior show differences in the number of pMAPK (p44/42 ERK) expressing neurons in lateral amygdala following Pavlovian fear conditioning

Individual variability in the acquisition, consolidation and extinction of conditioned fear potentially contributes to the development of fear pathology including posttraumatic stress disorder (PTSD). Pavlovian fear conditioning is a key tool for the study of fundamental aspects of fear learning. Here, we used a selected mouse line of High and Low Pavlovian conditioned fear created from an advanced intercrossed line (AIL) in order to begin to identify the cellular basis of phenotypic divergence in Pavlovian fear conditioning. We investigated whether phosphorylated MAPK (p44/42 ERK/MAPK), a protein kinase required in the amygdala for the acquisition and consolidation of Pavlovian fear memory, is differentially expressed following Pavlovian fear learning in the High and Low fear lines. We found that following Pavlovian auditory fear conditioning, High and Low line mice differ in the number of pMAPK-expressing neurons in the dorsal sub nucleus of the lateral amygdala (LAd). In contrast, this difference was not detected in the ventral medial (LAvm) or ventral lateral (LAvl) amygdala sub nuclei or in control animals. We propose that this apparent increase in plasticity at a known locus of fear memory acquisition and consolidation relates to intrinsic differences between the two fear phenotypes. These data provide important insights into the micronetwork mechanisms encoding phenotypic differences in fear. Understanding the circuit level cellular and molecular mechanisms that underlie individual variability in fear learning is critical for the development of effective treatment of fear-related illnesses such as PTSD.

Neurons Activated During Fear Memory Consolidation and Reconsolidation are Mapped to a Common and New Topography in the Lateral Amygdala

A key question in neuroscience is how memory is selectively allocated to neural networks in the brain. This question remains a significant research challenge, in both rodent models and humans alike, because of the inherent difficulty in tracking and deciphering large, highly dimensional neuronal ensembles that support memory (i.e., the engram). In a previous study we showed that consolidation of a new fear memory is allocated to a common topography of amygdala neurons. When a consolidated memory is retrieved, it may enter a labile state, requiring reconsolidation for it to persist. What is not known is whether the original spatial allocation of a consolidated memory changes during reconsolidation. Knowledge about the spatial allocation of a memory, during consolidation and reconsolidation, provides fundamental insight into its core physical structure (i.e., the engram). Using design-based stereology, we operationally define reconsolidation by showing a nearly identical quantity of neurons in the dorsolateral amygdala (LAd) that expressed a plasticity-related protein, phosphorylated mitogen-activated protein kinase, following both memory acquisition and retrieval. Next, we confirm that Pavlovian fear conditioning recruits a stable, topographically organized population of activated neurons in the LAd. When the stored fear memory was briefly reactivated in the presence of the relevant conditioned stimulus, a similar topography of activated neurons was uncovered. In addition, we found evidence for activated neurons allocated to new regions of the LAd. These findings provide the first insight into the spatial allocation of a fear engram in the LAd, during its consolidation and reconsolidation phase.

The structure of Pavlovian fear conditioning in the amygdala

Do different brains forming a specific memory allocate the same groups of neurons to encode it? One way to test this question is to map neurons encoding the same memory and quantitatively compare their locations across individual brains. In a previous study, we used this strategy to uncover a common topography of neurons in the dorsolateral amygdala (LAd) that expressed a learning-induced and plasticity-related kinase (p42/44 mitogen-activated protein kinase; pMAPK), following auditory Pavlovian fear conditioning. In this series of experiments, we extend our initial findings to ask to what extent this functional topography depends upon intrinsic neuronal structure. We first showed that the majority (87 %) of pMAPK expression in the lateral amygdala was restricted to principal-type neurons. Next, we verified a neuroanatomical reference point for amygdala alignment using in vivo magnetic resonance imaging and in vitro morphometrics. We then determined that the topography of neurons encoding auditory fear conditioning was not exclusively governed by principal neuron cytoarchitecture. These data suggest that functional patterning of neurons undergoing plasticity in the amygdala following Pavlovian fear conditioning is specific to memory formation itself. Further, the spatial allocation of activated neurons in the LAd was specific to cued (auditory), but not contextual, fear conditioning. Spatial analyses conducted at another coronal plane revealed another spatial map unique to fear conditioning, providing additional evidence that the functional topography of fear memory storing cells in the LAd is non-random and stable. Overall, these data provide evidence for a spatial organizing principle governing the functional allocation of fear memory in the amygdala.

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI2/TXA2 ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer. © 2010 Elsevier B.V. All rights reserved.

The role of the molecular footprint of EGFR in tailoring treatment decisions in NSCLC

The majority of patients with non-small-cell lung cancer (NSCLC) present with advanced disease, with targeted therapies providing some improvement in clinical outcomes. The epidermal growth factor receptor (EGFR) tyrosine kinase (TK) plays an important role in the pathogenesis of NSCLC. Tyrosine kinase inhibitors (TKIs), which target the EGFR TK domain, have proven to be an effective treatment strategy; however, patient responses to treatment vary considerably. Therefore, the identification of patients most likely to respond to treatment is essential to optimise the benefit of TKIs. Tumour-associated activating mutations in EGFR can identify patients with NSCLC who are likely to have a good response to TKIs. Nonetheless, the majority of patients relapse within a year of starting treatment. Studies of tumours at relapse have demonstrated expression of a T790M mutation in exon 20 of the EGFR TK domain in approximately 50% of cases. Although conferring resistance to reversible TKIs, these patients may remain sensitive to new-generation irreversible/panerb inhibitors. A number of techniques have been employed for genotypic assessment of tumourassociated DNA to identify EGFR mutations, each of which has advantages and disadvantages. This review presents an overview of the current methodologies used to identify such molecular markers. Recent developments in technology may make the monitoring of changes in patients' tumour genotypes easier in clinical practice, which may enable patients' treatment regimens to be tailored during the course of their disease, potentially leading to improved patient outcomes.

EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer : analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 First-Line ErbituX in lung cancer (FLEX) study showed that the addition of cetuximab to first-line chemotherapy significantly improved overall survival compared with chemotherapy alone (hazard ratio [HR] 0·871, 95% CI 0·762-0·996; p=0·044) in patients with advanced non-small-cell lung cancer (NSCLC). To define patients benefiting most from cetuximab, we studied the association of tumour EGFR expression level with clinical outcome in FLEX study patients. Methods: We used prospectively collected tumour EGFR expression data to generate an immunohistochemistry score for FLEX study patients on a continuous scale of 0-300. We used response data to select an outcome-based discriminatory threshold immunohistochemistry score for EGFR expression of 200. Treatment outcome was analysed in patients with low (immunohistochemistry score <200) and high (≥200) tumour EGFR expression. The primary endpoint in the FLEX study was overall survival. We analysed patients from the FLEX intention-to-treat (ITT) population. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. Findings: Tumour EGFR immunohistochemistry data were available for 1121 of 1125 (99·6%) patients from the FLEX study ITT population. High EGFR expression was scored for 345 (31%) evaluable patients and low for 776 (69%) patients. For patients in the high EGFR expression group, overall survival was longer in the chemotherapy plus cetuximab group than in the chemotherapy alone group (median 12·0 months [95% CI 10·2-15·2] vs 9·6 months [7·6-10·6]; HR 0·73, 0·58-0·93; p=0·011), with no meaningful increase in side-effects. We recorded no corresponding survival benefit for patients in the low EGFR expression group (median 9·8 months [8·9-12·2] vs 10·3 months [9·2-11·5]; HR 0·99, 0·84-1·16; p=0·88). A treatment interaction test assessing the difference in the HRs for overall survival between the EGFR expression groups suggested a predictive value for EGFR expression (p=0·044). Interpretation: High EGFR expression is a tumour biomarker that can predict survival benefit from the addition of cetuximab to first-line chemotherapy in patients with advanced NSCLC. Assessment of EGFR expression could offer a personalised treatment approach in this setting. Funding: Merck KGaA. © 2012 Elsevier Ltd.

Phospho-Akt expression is associated with a favorable outcome in non-small cell lung cancer

Akt, a Serine/Threonine protein kinase, mediates growth factor-associated cell survival. Constitutive activation of Akt (phosphorylated Akt, P-Akt) has been observed in several human cancers, including lung cancer and may be associated with poor prognosis and chemotherapy and radiotherapy resistance. The clinical relevance of P-Akt in non-small cell lung cancer (NSCLC) is not well described. In the present study, we examined 82 surgically resected snap-frozen and paraffin-embedded stage I to IIIA NSCLC samples for P-Akt and Akt by Western blotting and for P-Akt by immunohistochemistry. P-Akt protein levels above the median, measured using reproducible semiquantitative band densitometry, correlated with a favorable outcome (P = 0.007). Multivariate analysis identified P-Akt as a significant independent favorable prognostic factor (P = 0.004). Although associated with a favorable prognosis, high P-Akt levels correlated with high tumor grade (P = 0.02). Adenocarcinomas were associated with low P-Akt levels (P = 0.039). Akt was not associated with either outcome or clinicopathologic variables. Cytoplasmic (CP-Akt) and nuclear (NP-Akt) P-Akt tumor cell staining was detected in 96% and 42% of cases, respectively. Both CP-Akt and NP-Akt correlated with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt also correlated with nodal metastases (P = 0.022) and squamous histology (P = 0.037). These results suggest P-Akt expression is a favorable prognostic factor in NSCLC. Immunolocalization of P-Akt, however, may be relevant as NP-Akt was associated with nodal metastases, a known poor prognostic feature in this disease. P-Akt may be a potential novel therapeutic target for the management of NSCLC. © 2005 American Association for Cancer Research.