Philip Lazarus - US grants

DESCRIPTION (Adapted from investigator's Abstract): Polymorphisms are associated with a number of enzymes involved in the metabolic activation or detoxification of tobacco carcinogens. By use of case-control molecular epidemiology studies, the association between genetic or phenotypic polymorphisms and cancer susceptibility has been investigated. The most well-defined correlation for susceptibility to tobacco-induced cancers is the null GSTM1 genotype and lung cancer. Some evidence also exists demonstrating a link between CYP1A1 polymorphisms and cancer susceptibility. As xenobiotic-metabolizing enzymes play an integral role in the activation and detoxification of many potent tobacco carcinogens such as polycyclic aromatic hydrocarbons (PAHs) like benxo(a)pyrene, and tobacco-specific nitrosamines (TSNAs) such as 4-(methyl-nitrosamino)-1-(3-pyridyl)-butanone (NNK), the applicant further hypothesizes that susceptibility to either PAH- or TSNA-induced oral cavity carcinogenicity will be reflected in the genotypes of these enzymes. Extensive epidemiological data have established that tobacco use is the major risk factor for squamous cell carcinoma of the oral cavity (OCSCC), with greater than 85% of oral cancer linked to the use of tobacco. Studies reporting on xenobiotic-metabolizing enzyme polymorphisms as markers for individual susceptibility to tobacco-induced OCSCC have not yet been performed. The applicant hypothesizes that polymorphic markers of xenobiotic-metabolizing enzymes can be used to assess individual susceptibility to OCSCC. In this application, studies are proposed to examine genetic polymorphic markers of xenobiotic-metabolizing enzymes involved in tobacco carcinogen metabolism in a case-control study of oral cavity cancer cases versus frequency-matched non-cancer controls at Memorial Sloan-Kettering Cancer Center, Temple University Hospital, and the New York Eye and Ear Infirmary, in order to assess individual susceptibility to OCSCC. The multi-institutional nature of this proposal will enable assessment of individual susceptibility to OCSCC in different racial groups (i.e. Caucasian versus African American) within the American population. The major goal of this application will be to elucidate highly susceptible genotypic profiles for OCSCC. In addition, studies will be done to elucidate new polymorphic markers of OCSCC risk, focusing on the CYP1A2 enzyme, which is a strong metabolizer of the potent tobacco specific nitrosamine, NNK. A third major aim will be to compare the polymorphism profile of xenobiotic-metabolizing enzymes from oral cancer patients with the p53 mutational profile of a subset of OCSCCs obtained from the same patients. These studies should enable accumulation of important data concerning the contribution of specific carcinogen groups to the carcinogenicity of tobacco and tobacco smoke.

DESCRIPTION: (Applicant's Description) UDP-Glucuronyltransferases (UGT) play an extremely important role as genoprotective enzymes by preventing the accumulation of carcinogenic compounds which could react with cellular macromolecules and the oxidation of xenobiotics into active carcinogenic electrophiles. For example, several major tobacco pro-carcinogens, such as metabolites of the polycyclic aromatic hydrocarbons(PAHs) like benz[a]pyrene (BaP) and tobacco-specific nitrosamines (TSNAs) like 4-(methylnitrosamino)-1- (3,l)-butanone (NNK), are detoxified via glucuronidation by increasing hydrophilicity of these agents, rendering them more water soluble, and therefore easily excreted and less active. The major goal of the present proposal is to examine detoxification by UGTs as a mechanism for differential susceptibility to lung cancer, specifically focusing on the glucuronidation of the major NNK metabolite, 4-(methylnitrosamino)- 1-(3-pyridyl)- l(NNAL). NNK and NNAL are considered to be major contributors to the induction of lung and other aerodigestive cancers. Large inter-individual variability in the ratio of the glucuronidated form of NNAL (NNAL-gluc):free NNAL suggests that individuals differ greatly in their ability to glucuronidate NNK metabolites and to detoxify NNK. This is consistent with recent studies suggesting that racial differences in risk for lung cancer may, in part, be explained by differences in the ability of individual subjects to detoxify NNK via NNAL glucuronidation. In our preliminary studies, we identified several human UGTs, including UGT1A9, which appear to possess NNAL-glucuronidating activity and demonstrate that this activity is inducible by phenobarbitol and phenolic antioxidants in rats. As the balance between activation and detoxification of carcinogens such as NNK may be influenced by the balance of host expression of enzymes involved in tobacco carcinogen activation or deactivation, we hypothesize that an individual's ability to glucoronidate NNAL will be correlated with that individual's risk for lung and potentially other aerodigestive tract cancers. The objective of this application will be to, (i) fully characterize the NNAL glucuronidation pathway in humans by determining the major UGT isoenzyme(s) responsible for the glucuronidation of NNAL, (ii) elucidate and functionally assess potentially important genetic polymorphisms in the human UGT gene which may reflect on an individual's capacity to convert NNAL-gluc as a measure of one's ability to detoxify NNK, and (iii) examine the importance of these polymorphic genotypes in a case control study of lung cancer susceptibility. These studies should enable us to elucidate potentially important biomarkers which may reflect upon an individual's risk for lung and potentially other tobacco-related cancers.

DESCRIPTION (Adapted from the applicant's abstract): UDP glucuronosyltransferases (UGTs) may play important roles in cells as genoprotective enzymes by preventing the accumulation of carcinogenic compounds which could react with cellular macromolecules causing the oxidation of xenobiotics into active carcinogenic electrophiles. Several major tobacco procarcinogens, such as metabolites of the polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) and tobacco-specific nitrosamines (TSNAs) like 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), are detoxified via UGT-induced glucuronidation by increasing the hydrophilicity of these agents, thus rendering them more water-soluble, more easily excreted, and less bioactive. The major goal of the proposed work is to examine detoxification by UGTs as a mechanism for differential susceptibility to oral cancer, specifically focusing on the glucuronidation of the major NNK metabolite, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol (NNAL). NNK and NNAL are considered to be major contributors to the induction of cancers of the oral cavity and lung. Large inter-individual variability in the ratio of the glucuronidated form of NNAL (NNAL-Gluc):free NNAL suggests that individuals differ greatly in their ability to glucuronidate NNK metabolites and to detoxify NNK. This is consistent with recent studies which suggest that racial differences in morbidity and mortality of lung and potentially oral cancer may, in part, be explained by differences in the ability of individual subjects to detoxify NNK via NNAL glucuronidation. Preliminary studies have identified at least two human UGTs (1A9 and 2B7) which possess NNAL-glucoronidating activity and demonstrate that this activity is inducible by phenobarbitol and phenolic antioxidants in rats. As the balance between metabolic activation and detoxification of carcinogens such as NNK may be influenced by the balance of host expression of enzymes involved in tobacco carcinogen activation, the hypothesis was created that an individual's ability to glucuronidate NNAL is correlated with that individual's risk for oral cancer as well as for other aerodigestive tract cancers. Therefore, the objective of this proposed work is to (1) fully characterize the NNAL glucuronidation pathway in humans, (2) to elucidate, functionally assess, and determine the prevalence of potentially important genetic polymorphisms in the human UGT gene which may reflect an individual's capacity to convert NNAL to NNAL-gluc as a measure of one's ability to detoxify NNK, and (3) to examine the importance of these polymorphic genotypes in a case-control study of susceptibility to oral cancer. These studies may provide potentially important genetic biomarkers which may reflect upon an individual's risk for oral and potentially other tobacco-related cancers.

The p53 tumor suppressor gene has been strongly implicated in the process of carcinogenesis. One of the initial cellular responses observed following exposure to DNA-damaging agents is an up-regulation of p53 protein. In preliminary studies from our laboratory, we have demonstrated that there is differential usage of p53 transcriptional start sites in normal human tissues as compared to that observed in human tumor specimens and most cell lines. p53 transcripts initiated at the P1 transcription start site are the major p53 species observed in tumor specimens and cell lines. In contrast, the majority of human p53 gene transcription in normal tissue is initiated from sites upstream of the P1 start site, with a full length P0- initiated p53 transcript detected at levels that reach approximately 50 percent of total p53 mRNA. In addition, we have demonstrated that p53 5' UTR sequences upstream of the P1 start site (between P0 and P1) present only in P0/P2-initiated p53 mRNA significantly decrease mRNA translational efficiency in a cis- regulated manner. This decrease is not observed for mRNAs containing only P1-specific p53 5' UTR sequences. This phenomenon may be an important mechanism for controlling the expression of p53 and may be an important biomarker for cellular transformation and tumorigenesis. The goal of this proposal will be to examine the mechanisms underlying the translational regulation manifested by p53 5' UTR sequences as well as the mechanisms involved in the switching of transcriptional start sites in normal versus tumor cells. Outlined in this proposal are studies designed to elucidate the exact sequences necessary for the manifestation of P0-induced translational regulation and determine whether these sequences reside in P0- versus P2-initiated mRNA. We will elucidate potential inhibitory elements present within the p53 5' UTR such as stem-loop structures or upstream UG (uAUG) condons and upstream open reading frames (uORFs), and whether potential P0- specific RNA-binding proteins play a role in this inhibition. Also proposed are studies examining the p53 flanking promoter sequences necessary for P0/P2-transcription. We will establish an in vitro system where the switch from P0/P2- to P1-initiated p53 transcripts can be modulated. This will enable us to better elucidate differences in the transcription factors and/or transcriptional machinery involved in P0/P2- versus P1-initiated p53 gene transcription and to better evaluate the conditions necessary for P0/P2-P1 transcriptional switching in vitro. Finally, we will examine this process in multiple human tissues including normal tissues from tobacco smoke-exposed versus unexposed individuals, tumor specimens as well as premalignant lesions. These studies will provide us with a better understanding of the importance of this mechanism in the carcinogenic process and help us evaluate the potential of this phenomenon as a biomarker for tumor initiation and progression.

Studies from our group and others have demonstrated that the risk for tobacco-related cancers differs by race, gender and type of tobacco product consumed. These important public health differences cannot be fully explained by existing patterns of tobacco consumption. We hypothesize that risk is related to the type of cigarette smoked (e.g., low versus medium yield of carcinogens), the manner in which an individual's smoking habit regulates the dosage that reaches the lungs, metabolic capacity to activate and detoxify smoke-borne carcinogens, and susceptibility to cancer related to genetic factors that may affect metabolism or DNA repair. During the first three years of the study, the program focused on epidemiology, dosage and biomarkers of dose, and metabolic pathways of carcinogen activation and detoxification. In the coming period, the former Project (epidemiology) will be replaced by an epidemiological core facility (Core C) to provide appropriate study subjects for the two continuing projects and one new project. The current Project (Dosimetry of Lung and Bladder Cancer Risk among Cigarette Smokers) is about how smoking behavior affects the "delivered" carcinogen dose, and in turn how dose is related to biomarkers of carcinogen metabolites. Project (Metabolic Epidemiology of Tobacco-Related Cancers in Black and White Americans) is a study of differences between African Americans and Caucasians in metabolic activation and/or detoxification of an array of carcinogens derived from cigarette smoking, such as NNK (a potent lung carcinogen) and 4-aminobiphenyl (a bladder carcinogen). It utilizes metabolic and molecular techniques to study pathways of activation of tobacco-derived nitrosamines related to lung cancer, which is higher in African Americans compared to Caucasians, as well as detoxification of aromatic amines involved in bladder cancer, the rate of which is lower. Project (UDP Glucuronosyltransferases, Detoxification of NNK and Lung Cancer Risk) focuses on a family of detoxification enzymes that may be related to individual risk for developing lung or bladder cancer, and for which genetic polymorphisms exist that might explain variation in cancer risk. A broader understanding of these factors, both individually and comprehensively, will contribute greatly to our understanding of the causes of tobacco-related cancers in a way that can help improve our prevention strategies. The investigators are leaders in their respective fields with a strong history of collaboration. The program is supported by an Administrative Core with an Advisory Board of distinguished scientists and a community representative, by a Biostatistics and Computing Core Facility to provide efficient data management and statistical support, and by an Epidemiology Core Facility to manage accrual of subjects, interviews, acquisition of buccal cells, urine, and blood for biomarker assays, and pathological review.

[unreadable] DESCRIPTION (provided by applicant): Glucuronidation plays an extremely important role in the metabolism and elimination of a variety of carcinogens and endogenous factors associated with increased risk for cancer. Our studies over the first five years of this award strongly suggest that two UGTs - UGT1A10 and UGT2B17 - play key roles in cancer susceptibility. We have identified prevalent deletion polymorphisms for both UGT1A10 and UGT2B17 that include either part of the proximal promoter region or a large part of the coding sequence, and that the whole-gene UGT2B17*2 deletion allele is associated with significant decreases in liver microsome glucuronidating activities and increased risk for lung adenocarcinoma. Both enzymes are, (1) present in target sites for tobacco-related cancers including the aerodigestive tract and lung and are active against many important metabolites of tobacco smoke carcinogens including BaP and NNK, (2) UGT1A10 is highly active against relevant C18 steroids like estradiol and is widely-expressed in hormone-related tissues, and (3) UGT2B17 is present in prostate and is highly active against relevant C19 steroids including testosterone and dihydrotestosterone. Therefore, both enzymes could potentially play a significant role in the detoxification of relevant substrates in all of these aforementioned sites, and UGT genetic variations like gene deletions could have a significant impact on cancer risk. We hypothesize that UGT1A10 and UGT2B17 polymorphisms that significantly alter activities against exogenous xenobiotics like tobacco carcinogens or endogenous compounds like C18 or C19 steroids are correlated with altered glucuronidation phenotypes, and that they play an important role in cancer risk. It is the goal of this proposal to, (1) characterize these and other potential polymorphisms in the two genes, (2) assess their effect on enzyme function or expression both in vitro and in genotype:phenotype assays, and (3) perform preliminary studies examining their role in cancer risk. These studies will be combined with a careful assessment of the overall importance of UGT1A10- and UGT2B17-glucuronidating activities against a variety of tobacco smoke carcinogens or their metabolites. These studies should significantly impact on the field of cancer genetics and epidemiology as they will enable us to better assess the role of variation in glucuronidation pathways and cancer induction. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Aromatase inhibitors (AIs) are widely used as adjuvant treatment for estrogen-receptor positive breast cancer in post-menopausal women. AIs have been demonstrated to have equal to or greater efficacy and less toxicity than tamoxifen (TAM), the drug of choice for many years. Exemestane (EXE) is a 3rd-generation AI that has demonstrated efficacy in the treatment of breast cancer patients, and as with TAM and other AIs, there has been considerable inter-individual variability in overall response to EXE and in the occurrence of toxicities, but the causes of this variability have not been elucidated. Differences in drug metabolism can be a source of variability between patients. Genetic variations occur in several of the enzymes involved in phase I and II metabolic reactions and many of these can lead to alterations in enzyme activity which in turn can alter therapeutic response to drugs. EXE is extensively metabolized as unchanged EXE and is found at less than 1% in urine and 10% in plasma. We have characterized the EXE metabolism pathway and have identified the enzymes most active in this process. One of the key metabolic steps is the reduction of the 17-keto group to form 17-dihydroexemestane (17-OH-EXE), a metabolite that exhibits significant anti-aromatase activity in vitro and which is extensively glucuronidated by UDP-glucuronosyltransferases (UGTs) for excretion in the urine. In preliminary studies, we have shown that a deletion polymorphism in UGT2B17 may have a significant impact on the disposition of EXE in liver and thus potentially on its therapeutic effect. In addition to 17-OH-EXE and its glucuronide, there are 4 other metabolites of EXE, two of which are derived from 17-OH-EXE. Considerable variability in EXE metabolite formation from different individuals was observed in these and previous studies. These data underscore the importance of understanding whether genetic variations may affect an individual's response to the drug. It is our hypothesis that EXE metabolism is an important source of the inter-individual variations in EXE metabolic profiles and those polymorphisms in EXE-metabolizing enzymes play a role in affecting EXE therapeutic efficacy and toxicity. The specific aims of this proposal are to, (1) characterize the EXE metabolism pathway and determine the in vitro effect of functional polymorphisms in enzymes active in EXE metabolism, (2) establish EXE metabolism profile kinetics and determine whether correlations exist in vivo between UGT2B17 deletion genotype and urinary EXE metabolite profiles, and (3) determine whether correlations exist between metabolizing enzyme genotypes, serum EXE metabolite profiles and EXE-induced toxicity and adverse events in a large population of women taking EXE, utilizing samples and clinical data from the NCIC CTG MAP.3 trial that is examining EXE in the chemoprevention and risk reduction setting. Together, these studies will allow us to fully characterize functionally-relevan polymorphisms in the EXE-metabolizing enzyme pathway that are potentially important in EXE clinical efficacy.

? DESCRIPTION (provided by applicant): Abstract Smoking and tobacco use are major risk factors for many diseases including lung and head and neck (H&N) cancer. A long term goal is to establish susceptibility markers for lung and H&N cancer to identify tobacco users at higher risk for these cancers. UDP-glycosyltransferase (UGT) enzymes play a critical role in the detoxification of many carcinogens abundant in tobacco and/or tobacco smoke including polycyclic aromatic hydrocarbons (PAHs) and tobacco-specific nitrosamines (TSNAs). Preliminary data suggest that members of the minimally-studied UGT2A and 3A sub-families of metabolizing enzymes are expressed in tobacco target tissues including lung and H&N, exhibit activity against tobacco carcinogens, and that polymorphic and splicing variants exist that alter their activity, thus potentially altering local detoxification of tobacco carcinogens at these targt sites. These characteristics suggest that UGT2A and 3A enzymes are potentially important susceptibility markers for tobacco-related cancers. The goal of this proposal is to test our hypothesis that UGT2A and 3A activity is important in the local detoxification of tobacco carcinogens in tobacco target tissues, and that UGT2A and 3A activities and expression are altered via genotypic and splicing mechanisms and play a role in risk of tobacco-related cancers. The specific aims of this proposal are to, (1) Characterize the expression and activity of UGT2A and 3A enzymes; (2) Examine the importance of UGT2A and 3A SNPs to risk for cancers of the lung and H&N; and (3) Examine UGT2A and 3A splicing mechanisms as a form of UGT2A and 3A regulation by determining whether UGT2A and 3A splice variants function to regulate UGT conjugating activities and potentially act to alter tobacco-related cancer risk. The proposed studies will enable us to better understand the potential role of the UGT2A and 3A enzymes as susceptibility markers for tobacco- related cancer induction, help us identify subjects for targeted prevention strategies, and enable us to evaluate the innovative concept that differential splicing may act as a form of gene regulation that plays a role in cancer risk.