Academic Background

Regulation of Nicotinic Receptor Gene Expression in Addiction and Lung Cancer

The highly addictive properties of nicotine have been appreciated for a number of years.  Nicotine addiction begins with the interaction of the drug, usually obtained from tobacco, with nicotinic acetylcholine receptors (nAChR) in the brain; the neurotransmitter acetylcholine being the endogenous ligand for the receptors.  The interaction of nicotine with nAChRs occurs within the dopamine reward pathway and leads to increased dopamine release within the brain.  The nicotine-mediated release of dopamine is critical for the onset and maintenance of dependence.  The health consequences of nicotine addiction are staggering.  Tobacco use is the primary etiological factor for the majority of lung cancer cases, the leading cause of cancer-related deaths in the world.  This is reflected in the fact that tobacco contains approximately 55 carcinogens and that nicotine contained in tobacco is one of the most addictive drugs known.  The research in my laboratory is aimed at elucidating the molecular mechanisms controlling expression of the genes encoding nAChRs under both normal and pathological conditions.  Working in close collaboration with the laboratory of Andrew Tapper ( www.umassmed.edu/bnri/faculty/Tapper.cfm), we use a multidisciplinary approach to address questions related to cholinergic signaling through nAChRs in the brain and the lungs.  This approach is a powerful combination of molecular, biophysical and behavioral analyses allowing us to pursue important issues that span the spectrum ranging from individual genes to whole organisms.

Nicotinic Acetylcholine Receptor Expression in the Nervous System

Nicotinic receptors are pentameric ion channels expressed in a variety of cell types.  Neuronal nAChRs are encoded by a family of genes, a2-a10 and b2-b4, and can be assembled as homomeric or heteromeric receptors.  In addition to nicotine addiction, these receptors have been implicated in a variety of brain processes and pathologies including neural development, learning and memory, ageing, anxiety, schizophrenia, Parkinson’s disease and epilepsy.  Thus, understanding how nAChR expression is regulated will have implications for normal development and differentiation as well as for therapies aimed at a variety of neurological pathologies.  Several recent genome-wide association studies have identified a chromosomal locus with genetic variations associated with tobacco use and lung cancer.  Interestingly, this locus maps to a cluster of three nAChR genes, those encoding the a3, a5 and b4 subunits.  My laboratory has studied the transcriptional mechanisms regulating expression of these three genes for a number of years.  Our approach has been to identify transcriptional regulatory elements within the subunit genes and to use these elements as molecular handles to identify and characterize the regulatory factors with which they interact to effect expression of the genes.  We have identified a number of elements and associated factors, some of which are broadly expressed while others have more restricted expression patterns.  In particular, we showed that the transcription factors Sp1 and Sp3 are critical players in nAChR gene expression.  In addition, we demonstrated that a member of the Sox family of regulatory proteins, Sox10, can activate transcription from the a3, a5 and b 4 gene promoters in a cell-type-specific manner.  This led to the hypothesis that Sox10-associated factors exist and are intimately involved in Sox10-mediated regulation of the nAChR genes.  We recently identified a number of candidate Sox10-interacting proteins and are currently characterizing their roles in nAChR gene expression and Sox10 function.  The identification of Sox10 as playing a role in receptor gene expression is intriguing in that mutations of Sox10 have been shown to be one cause of the congenital disorder, Waardenburg-Hirschsprung syndrome in humans.  Interestingly, a mouse model for the disease exhibits features similar to those seen in mice in which the genes encoding specific nAChR subunits have been inactivated.  We are using this mouse model to pursue these findings.

Nicotinic Receptors and Nicotine Addiction

It is clear that nicotine addiction begins with the activation of nAChRs.  However, exactly which specific nAChR subtypes are involved in the addiction process remains to be completely elucidated.  Furthermore, it is likely that specific nAChR subtypes mediate distinct dependence-related behaviors (e.g., reward vs. withdrawal).  In collaboration with the Tapper laboratory, we are attempting to identify nAChR subtypes involved in these processes with the goal of identifying potential therapeutic targets for nicotine cessation.

Nicotinic Receptors and Lung Cancer

Lung cancer is the leading cause of cancer-related deaths in the world.  To make matters worse, lung cancer has proven refractory to advances in cancer treatment with only 14% of the patient pool surviving longer than 5 years after diagnosis.  In addition to its role in the nervous system, it is now clear that nicotine promotes several cancer-related phenotypes including cell proliferation, transformation, angiogenesis and apoptotic inhibition.  Another goal of our research is to determine the specific nAChR subtypes that mediate these processes in lung cancer.  Using quantitative RT-PCR, we demonstrated differential expression of the various nAChR subunit genes across distinct lung cancer cell lines and patient samples.  Interestingly, many of these genes are overexpressed in small cell lung cancer (SCLC), the most aggressive form of lung cancer and one that is also tightly associated with cigarette smoking.

In line with our objective, we are studying the molecular mechanisms that govern the expression of nAChR subunit genes in lung cancer.  We are currently investigating the role of achaete-scute complex homolog 1 (ASCL1), a transcription factor that is overexpressed in SCLC.  ASCL1 is a basic helix-loop-helix transcription factor that binds to consensus recognition motifs known as E-boxes with the core sequence CANNTG.  The promoter regions of several nAChR subunit genes contain E-boxes.  To determine whether ASCL1 regulates expression of nAChR subunit genes, we used small interefering RNAs to knock-down ASCL1 expression.  This led to a corresponding decrease in the expression of specific nAChR subunit genes, suggesting that ASCL1 regulates their expression.  We are pursuing these observations using a variety of techniques to further elucidate the mechanisms by which ASCL1 exerts this regulation.  Our long-term goal is to identify novel therapeutic targets for the treatment of SCLC.