We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.

ABSTRACT:The elucidation of the individual contributions of the four CYP3A genes to the overall CYP3A activity has been hampered by similarities in their sequence and function. We investigated the expression of CYP3A mRNA species in the liver and in various other tissues using gene-specific TaqMan probes. CYP3A4 transcripts were the most abundant CYP3A mRNA in each of the 63 white European livers tested and accounted on average for 95% of the combined CYP3A mRNA pool. CYP3A5 and CYP3A7 each contributed on average 2%, whereas CYP3A43 contributed 0.3% transcripts to this pool. Fourteen percent of livers exhibited an increased share of CYP3A5 transcripts (range 4-20%). These livers were either heterozygous for the marker of the CYP3A5 polymorphism, the CYP3A5*1A allele, or expressed very low levels of CYP3A4 mRNA. The CYP3A7 expression was bimodal, and it was increased in 15% livers. CYP3A4 was the dominant CYP3A in the intestine, followed by CYP3A5. CYP3A5 and CYP3A7, but not CYP3A4, were also expressed in the adrenal gland and in the prostate, whereas only CYP3A5 was detected in the kidney. These three tissues were shown to express much lower levels of pregnane X receptor mRNA than the intestine, indicating possibly a different mode of regulation of CYP3A expression. Expression of CYP3A genes was undetectable in peripheral blood lymphocytes. In summary, these assays and results should aid in our efforts to further dissect the regulation and the physiological and pharmacological significance of CYP3A isozymes.

Human CYP3A enzymes play a pivotal role in the metabolism of many drugs, and the variability of their expression among individuals may have a strong impact on the efficacy of drug treatment. However, the individual contributions of the four CYP3A genes to total CYP3A activity remain unclear. To elucidate the role of CYP3A7, we have studied its expression in human liver and intestine. In both organs, expression of CYP3A7 mRNA was polymorphic. The recently identified CYP3A7*1C allele was a consistent marker of increased CYP3A7 expression both in liver and intestine, whereas the CYP3A7*1B allele was associated with increased CYP3A7 expression only in liver. Because of the replacement of part of the CYP3A7 promoter by the corresponding region of CYP3A4, the CYP3A7*1C allele contains the proximal ER6 motif of CYP3A4. The pregnane X and constitutively activated receptors were shown to bind with higher affinity to CYP3A4-ER6 than to CYP3A7-ER6 motifs and transactivated only promoter constructs containing CYP3A4-ER6. Furthermore, we identified mutations in CYP3A7*1C in addition to the ER6 motif that were necessary only for activation by the constitutively activated receptor. We conclude that the presence of the ER6 motif of CYP3A4 mediates the high expression of CYP3A7 in subjects carrying CYP3A7*1C.Cytochrome P450 enzymes play a pivotal role in the oxidative, peroxidative, and reductive metabolism of many endogenous compounds, procarcinogens, and drugs. The CYP3A subfamily composed of CYP3A4, CYP3A5, CYP3A7, and CYP3A43 in humans is of special importance because it accounts for as much as 30% of total liver cytochrome P450 content (1). At least 50% of all medicines are metabolized by enzymes of the CYP3A subfamily (2). The most abundant CYP3A isoform in liver and intestine is CYP3A4. Its interindividual hepatic expression varies 60-fold (3), and the in vivo function as assessed by clearance displays at least a 20-fold difference (4). Induction by xenobiotics (e.g. rifampin) and endogenous compounds (e.g. steroid hormones) further modulates the variability of CYP3A4 expression among individuals. The induction of CYP3A4 and most likely that of other CYP3A genes is mediated by the nuclear receptor NR1I2 (pregnane X receptor (PXR) 1 ) (reviewed in Ref. 5). CYP3A4-inducing compounds bind to PXR and stimulate the transcriptional activity of the receptor. Additional nuclear receptors such as NR1I3 (constitutively activated receptor (CAR)) and NR1I1 (vitamin D receptor) have also been implicated in the transcriptional regulation of CYP3A4 (6, 7). Although the substrate specificity of CYP3A5 is similar to that of CYP3A4, CYP3A5 has been regarded to be less important for drug elimination because it is expressed at much lower levels than CYP3A4 in most livers of Caucasian origin (8). CYP3A43 is expressed at very low levels in adult human livers, accounting for only 0.1-0.2% of CYP3A4 transcripts (9, 10). Therefore, its contribution to the elimination of CYP3A substrates is regarded to be negligible (10). This variability in CYP3A...

Proteins encoded by the human CYP3A genes metabolize every second drug currently in use. The activity of CYP3A gene products in the general population is highly variable and may affect the efficacy and safety of drugs metabolized by these enzymes. The mechanisms underlying this variability are poorly understood, but they include gene induction, protein inhibition and unknown genetic polymorphisms. To better understand the regulation of CYP3A expression and to provide a basis for a screen of genetic polymorphisms, we determined and analysed the sequence of the human CYP3A locus. The 231 kb locus sequence contains the three CYP3A genes described previously (CYP3A4, CYP3A5 and CYP3A7), three pseudogenes as well as a novel CYP3A gene termed CYP3A43. The gene encodes a putative protein with between 71.5% and 75.8% identity to the other CYP3A proteins. The highest expression level of CYP3A43 mRNA is observed in the prostate, an organ with extensive steroid metabolism. CYP3A43 is also expressed in several other tissues including liver, where it can be induced by rifampicin. CYP3A43 transcripts undergo extensive splicing. The identification of a new member of the CYP3A family and the characterization of the full CYP3A locus will aid efforts to identify the genetic variants underlying its variable expression. This, in turn, will lead to a better optimization of therapies involving the numerous substrates of CYP3A proteins.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/β-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3β inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/β-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/β-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/β-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage ( per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/β-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs. @ERSpublications Patient-derived 3D-LTCs are a powerful tool for preclinical drug validation and imaging of Wnt-induced lung repair

Induction of cytochrome P450 3A (CYP3A) by xenobiotics may lead to clinically relevant drug interactions. In contrast with other CYP3A family members, studies on the inducibility of CYP3A5 indicate conflicting results. We report the induction of CYP3A5 mRNA in 13 of 16 hepatocyte preparations exposed to rifampin. Furthermore, induction of CYP3A5 mRNA was observed in intestinal biopsies in three of eight probands following exposure to the antibiotic. The highest absolute levels of CYP3A5 transcripts were found following rifampin treatment in hepatocytes and intestines from carriers of CYP3A5*1 alleles. Elucidation of the mechanism involved in CYP3A5 induction revealed that constitutively activated receptor (CAR) and pregnane X receptor (PXR) transactivated the CYP3A5 promoter (؊688 to ؉49) and that the transactivation was dependent on an everted repeat separated by 6 bp (ER6-dependent). Treatment with the prototypical PXR ligand rifampin led to a 2-fold induction of the CYP3A5 promoter activity. In agreement with these observations, PXR and CAR bound specifically to the ER6 motif. Hepatic expression of PXR correlated with that of CYP3A5 mRNA levels in a bank of liver samples. Taken together, studies here revealed the presence of a functional ER6 motif in the CYP3A5 promoter located ؊100 bp upstream from the transcription start site, suggesting that CYP3A5 is inducible by mechanisms similar to those involved in CYP3A4 induction. Enhanced expression of CYP3A5 caused by exposure to inducers may phenocopy the effects of the high expression allele CYP3A5*1. In this manner, induction of CYP3A5 may contribute to the overall importance of this P450 in drug metabolism and drug interactions.Members of the human cytochrome 3A subfamily of P450 monooxygenases (CYP3A) 1 play an important role in drug metabolism because of their hepatic and intestinal abundance and because of the large substrate spectrum. CYP3A enzymes account for 50% of the total P450 in an average human liver, and these enzymes participate in the metabolism of up to 50% of the drugs currently in use (1, 2). Not surprisingly, CYP3A are frequently involved in drug-drug interactions. CYP3A inhibition by drugs such as ketoconazole or erythromycin is considered the most relevant mechanism. For example, the resulting increased levels of the co-administered nonsedating antihistamine terfenadine in the presence of ketoconazole inhibited a cardiac potassium channel, leading to serious and, in some cases, fatal cardiac arrhythmias (3). In addition, many drugs and other environmental substances increase the activity of CYP3A through transcriptional activation (induction) (4). CYP3A induction is frequently considered clinically less important than inhibition because it is expected to reduce the efficacy of co-administered CYP3A substrate drugs rather than to affect their safety. However, CYP3A inducers such as rifampin and rifabutin can reduce plasma concentrations of certain drugs up to 40-fold, effectively abolishing their efficacy (5, 6). For example, enhanced metabo...

The t(11;14)(q13;q32) is the most common translocation in multiple myeloma (MM), resulting in up-regulation of cyclin D1. We used a segregation fluorescence in situ hybridization (FISH)