Charles Roland Wolf

Mutations in APC, Kirsten-ras, and p53—alternative genetic pathways to colorectal cancer

Colorectal cancer is one of the most significant causes of cancer death. A genetic model for colorectal cancer has been proposed in which the sequential accumulation of mutations in specific genes, including adenomatous polyposis coli (APC), Kirsten-ras (K-ras), and p53, drives the transition from healthy colonic epithelia through increasingly dysplastic adenoma to colorectal cancer. We have characterized tumor mutation spectra in a large cohort of colorectal cancer patients. In marked contrast to the predictions of the sequential model of mutation accumulation, only 6.6% of tumors were found to contain mutations in APC, K-ras, and p53, with 38.7% of tumors containing mutations in only one of these genes. The most common combination of mutations was p53 and APC (27.1%), whereas mutations in both p53 and K-ras were extremely rare. Statistical analysis (two-sided Fishers exact test) confirmed that mutations in K-ras and p53 co-occurred less frequently than expected by chance (P < 0.01, Fishers exact test). This finding suggests that these mutations lie on alternate pathways of colorectal tumor development. The heterogeneous pattern of tumor mutations in our patient cohort suggests that multiple alternative genetic pathways to colorectal cancer exist and that the widely accepted genetic model of cancer development is not representative of the majority of colorectal tumors.

The prognostic significance of K-ras, p53, and APC mutations in colorectal carcinoma

Background: Accumulation of molecular alterations, including mutations in Kirsten-ras (K-ras), p53, and adenomatous polyposis coli (APC), contribute to colorectal carcinogenesis. Our group has previously characterised a panel of sporadic colorectal adenocarcinomas for mutations in these three genes and has shown that p53 and K-ras mutations rarely occur in the same colorectal tumour. This suggests that mutations in these genes are on separate pathways to colorectal cancer development and may influence patient prognosis independently. Aims: To correlate the presence or absence of mutations in K-ras, p53, and APC with survival in a cohort of colorectal cancer patients. Patients: A series of 107 inpatients treated surgically for colorectal cancer in Tayside, Scotland between November 1997 and December 1999. Methods: Colorectal tumours were characterised for mutations in K-ras, p53, and APC. Kaplan-Meier survival curves were constructed using overall survival and disease specific survival as the primary end points. Patient survival was analysed using the log rank test and Cox proportional hazards model. Results: Patients with K-ras mutations had significantly poorer overall survival than patients without K-ras mutations (p = 0.0098). Multivariate analysis correcting for Dukes’ stage, age, and sex confirmed this (hazard ratio 2.9 (95% confidence interval 1.4–6.2); p = 0.0040). K-ras mutations were also significantly associated with poorer disease specific survival. The presence of APC and p53 mutations did not affect survival in this cohort of patients (p = 0.9034 and p = 0.8290, respectively). Conclusions: Our data indicate that the presence of K-ras mutations predicts poor patient prognosis in colorectal cancer, independently of tumour stage.

Polymorphisms in the SLC6A4 and HTR2A genes influence treatment outcome following antidepressant therapy

The majority of antidepressant drugs act by increasing synaptic serotonin levels in the brain. Genetic variation in serotonin-related genes may therefore influence antidepressant efficacy. In this study, nine polymorphisms in four serotonin receptor genes (HTR1B, HTR2A, HTR5A and HTR6) and the serotonin transporter gene (SLC6A4) were analysed to investigate their influence on antidepressant response in a well-characterized unipolar depressive population (n=166) following a protocolized treatment regimen. 5-HTTLPR short-allele homozygotes were significantly associated with both remission (odds ratios (OR)=4.00, P=0.04) and response (OR=5.06, P=0.02) following second switch treatment, with a similar trend observed following initial treatment and paroxetine therapy. Following initial treatment, unipolar patients homozygous for the SLC6A4 intron 2 repeat polymorphism were significantly associated with lack of remission (OR=0.38, P=0.02) and lack of response (OR=0.42, P=0.01). Additionally, the HTR2A C1354T polymorphism showed an association with remission (OR=7.50, P=0.002) and response (OR=5.25, P=0.01) following paroxetine therapy. These results suggest that genetically determined variation in serotonin receptor genes makes a significant contribution to the efficacy of commonly prescribed antidepressant drugs.

Quantitative Prediction of Human Pregnane X Receptor and Cytochrome P450 3A4 Mediated Drug-Drug Interaction in a Novel Multiple Humanized Mouse Line

Cytochrome P450 (P450) 3A4 is the predominant P450 enzyme expressed in human liver and intestine, and it is involved in the metabolism of approximately 50% of clinically used drugs. Because of the differences in the multiplicity of CYP3A genes and the poor correlation of substrate specificity of CYP3A proteins between species, the extrapolation of CYP3A-mediated metabolism of a drug from animals to man is difficult. This situation is further complicated by the fact that the predictability of the clinically common drug-drug interaction of pregnane X receptor (PXR)-mediated CYP3A4 induction by animal studies is limited as a result of marked species differences in the interaction of many drugs with this receptor. Here we describe a novel multiple humanized mouse line that combines a humanization for PXR, the closely related constitutive androstane receptor, and a replacement of the mouse Cyp3a cluster with a large human genomic region carrying CYP3A4 and CYP3A7. We provide evidence that this model shows a human-like CYP3A4 induction response to different PXR activators, that it allows the ranking of these activators according to their potency to induce CYP3A4 expression in the human liver, and that it provides an experimental approach to quantitatively predict PXR/CYP3A4-mediated drug-drug interactions in humans.

MERITS AND LIMITATIONS OF RECOMBINANT MODELS FOR THE STUDY OF HUMAN P450-MEDIATED DRUG METABOLISM AND TOXICITY: AN INTRALABORATORY COMPARISON

A wide variety of pharmacological and toxicological properties of drugs are determined by cytochrome P450-mediated metabolism. Characterization of these pathways and of the P450 isoenzymes involved constitutes an essential part of drug development. Similarly, because P450s are catalyzing the toxication and detoxication of environmental pollutants, an understanding of these reactions facilitates risk assessment in environmental toxicology. Recently, a variety of recombinant expression systems has been employed to study the role of human P450s in these reactions. These include insect, bacterial, yeast, and mammalian models. As these were developed and characterized by different laboratories, evaluation of their merits and limitations is inherently difficult. To resolve this problem, we have established and characterized the latter three systems and present the key results here. In general, the catalytic properties of P450 isozymes in the various models were rather similar. However, taking technical considerations into account as well as the high level of functional expression of P450s achieved in bacteria make this system ideally suited for drug metabolism research, including the generation of milligram quantities of metabolites for structural determinations. For toxicological studies, however, expression of P450s in mammalian cells was most appropriate. This is exemplified here by studies into the role of human P450s in the activation and inactivation of chemotherapeutic drugs.

Evidence that cytochrome b5 and cytochrome b5 reductase can act as sole electron donors to the hepatic cytochrome P450 system.

We previously described the development of genetic models to study the in vivo functions of the hepatic cytochrome P450 (P450) system, through the hepatic deletion of either cytochrome P450 oxidoreductase [POR; HRN (hepatic reductase null) line] or cytochrome b5 [HBN (hepatic cytochrome b5 null) line]. However, HRN mice still exhibit low levels of mono-oxygenase activity in spite of the absence of detectable reductase protein. To investigate whether this is because cytochrome b5 and cytochrome b5 reductase can act as the sole electron donor to the P450 system, we crossed HRN with HBN mice to generate a line lacking hepatic expression of both electron donors (HBRN). HBRN mice exhibited exacerbation of the phenotypic characteristics of the HRN line: liver enlargement, hepatosteatosis, and increased expression of certain P450s. Also, drug metabolizing activities in vitro were further reduced relative to the HRN model, in some cases to undetectable levels. Pharmacokinetic studies in vivo demonstrated that midazolam half-life, Cmax, and area under the concentration-time curve were increased, and clearance was decreased, to a greater extent in the HBRN line than in either the HBN or HRN model. Microsomal incubations using NADPH concentrations below the apparent Km of cytochrome b5 reductase, but well above that for POR, led to the virtual elimination of 7-benzyloxyquinoline turnover in HRN samples. These data provide strong evidence that cytochrome b5/cytochrome b5 reductase can act as a sole electron donor to the P450 system in vitro and in vivo.

Generation and Characterization of Novel Cytochrome P450 Cyp2c Gene Cluster Knockout and CYP2C9 Humanized Mouse Lines

Compared with rodents and many other animal species, the human cytochrome P450 (P450) Cyp2c gene cluster varies significantly in the multiplicity of functional genes and in the substrate specificity of its enzymes. As a consequence, the use of wild-type animal models to predict the role of human CYP2C enzymes in drug metabolism and drug-drug interactions is limited. Within the human CYP2C cluster CYP2C9 is of particular importance, because it is one of the most abundant P450 enzymes in human liver, and it is involved in the metabolism of a wide variety of important drugs and environmental chemicals. To investigate the in vivo functions of cytochrome P450 Cyp2c genes and to establish a model for studying the functions of CYP2C9 in vivo, we have generated a mouse model with a deletion of the murine Cyp2c gene cluster and a corresponding humanized model expressing CYP2C9 specifically in the liver. Despite the high number of functional genes in the mouse Cyp2c cluster and the reported roles of some of these proteins in different biological processes, mice deleted for Cyp2c genes were viable and fertile but showed certain phenotypic alterations in the liver. The expression of CYP2C9 in the liver also resulted in viable animals active in the metabolism and disposition of a number of CYP2C9 substrates. These mouse lines provide a powerful tool for studying the role of Cyp2c genes and of CYP2C9 in particular in drug disposition and as a factor in drug-drug interaction.

MicroRNA-224 is associated with colorectal cancer progression and response to 5-fluorouracil-based chemotherapy by KRAS-dependent and -independent mechanisms

Background:Colorectal cancers arise from benign adenomas, although not all adenomas progress to cancer and there are marked interpatient differences in disease progression. We have previously associated KRAS mutations with disease progression and reduced survival in colorectal cancer patients.Methods:We used TaqMan low-density array (TLDA) qRT–PCR analysis to identify miRNAs differentially expressed in normal colorectal mucosa, adenomas and cancers and in isogeneic KRAS WT and mutant HCT116 cells, and used a variety of phenotypic assays to assess the influence of miRNA expression on KRAS activity, chemosensitivity, proliferation and invasion.Results:MicroRNA-224 was differentially expressed in dysplastic colorectal disease and in isogeneic KRAS WT and mutant HCT116 cells. Antagomir-mediated miR-224 silencing in HCT116 KRAS WT cells phenocopied KRAS mutation, increased KRAS activity and ERK and AKT phosphorylation. 5-FU chemosensitivity was significantly increased in miR-224 knockdown cells, and in NIH3T3 cells expressing KRAS and BRAF mutant proteins. Bioinformatics analysis of predicted miR-224 target genes predicted altered cell proliferation, invasion and epithelial–mesenchymal transition (EMT) phenotypes that were experimentally confirmed in miR-224 knockdown cells.Conclusions:We describe a novel mechanism of KRAS regulation, and highlight the clinical utility of colorectal cancer-specific miRNAs as disease progression or clinical response biomarkers.

Pyrosequencing-based methods reveal marked inter-individual differences in oncogene mutation burden in human colorectal tumours

Background:The epidermal growth factor receptor-targeted monoclonal antibody cetuximab (Erbitux) was recently introduced for the treatment of metastatic colorectal cancer. Treatment response is dependent on Kirsten-Ras (K-Ras) mutation status, in which the majority of patients with tumour-specific K-Ras mutations fail to respond to treatment. Mutations in the oncogenes B-Raf and PIK3CA (phosphoinositide-3-kinase) may also influence cetuximab response, highlighting the need for a sensitive, accurate and quantitative assessment of tumour mutation burden.Methods:Mutations in K-Ras, B-Raf and PIK3CA were identified by both dideoxy and quantitative pyrosequencing-based methods in a cohort of unselected colorectal tumours (n=102), and pyrosequencing-based mutation calls correlated with various clinico-pathological parameters.Results:The use of quantitative pyrosequencing-based methods allowed us to report a 13.7% increase in mutation burden, and to identify low-frequency (<30% mutation burden) mutations not routinely detected by dideoxy sequencing. K-Ras and B-Raf mutations were mutually exclusive and independently associated with a more advanced tumour phenotype.Conclusion:Pyrosequencing-based methods facilitate the identification of low-frequency tumour mutations and allow more accurate assessment of tumour mutation burden. Quantitative assessment of mutation burden may permit a more detailed evaluation of the role of specific tumour mutations in the pathogenesis and progression of colorectal cancer and may improve future patient selection for targeted drug therapies.

Altered Protein S-Glutathionylation Identifies a Potential Mechanism of Resistance to Acetaminophen-Induced Hepatotoxicity

Acetaminophen (APAP) is the most commonly used over-the-counter analgesic. However, hepatotoxicity induced by APAP is a major clinical issue, and the factors that define sensitivity to APAP remain unclear. We have previously demonstrated that mice nulled for glutathione S-transferase Pi (GSTP) are resistant to APAP-induced hepatotoxicity. This study aims to exploit this difference to delineate pathways of importance in APAP toxicity. We used mice nulled for GSTP and heme oxygenase-1 oxidative stress reporter mice, together with a novel nanoflow liquid chromatography–tandem mass spectrometry methodology to investigate the role of oxidative stress, cell signaling, and protein S-glutathionylation in APAP hepatotoxicity. We provide evidence that the sensitivity difference between wild-type and Gstp1/2−/− mice is unrelated to the ability of APAP to induce oxidative stress, despite observing significant increases in c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation in wild-type mice. The major difference in response to APAP was in the levels of protein S-glutathionylation: Gstp1/2−/− mice exhibited a significant increase in the number of S-glutathionylated proteins compared with wild-type animals. Remarkably, these S-glutathionylated proteins are involved in oxidative phosphorylation, respiratory complexes, drug metabolism, and mitochondrial apoptosis. Furthermore, we found that S-glutathionylation of the rate-limiting glutathione-synthesizing enzyme, glutamate cysteine ligase, was markedly increased in Gstp1/2−/− mice in response to APAP. The data demonstrate that S-glutathionylation provides an adaptive response to APAP and, as a consequence, suggest that this is an important determinant in APAP hepatotoxicity. This work identifies potential novel avenues associated with cell survival for the treatment of chemical-induced hepatotoxicity.