Susan G Komen  
I've Been Diagnosed With Breast Cancer Someone I Know Was Diagnosed Share Your Story Join Us And Stay Informed Donate To End Breast Cancer
    Home > Research & Grants > Grants Program > Research Grants > Research Grants Awarded > Abstract

    Research Grants Awarded

    Inhibition Of Lysine Specific Demethylase 1 (Lsd1) As A Strategy For Re-Expression Of Epigenetically Silenced Genes In Breast Cancer

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    RATIONALE The primary purpose of the studies detailed in this application is to develop a new series of novel agents and strategies that target aberrant epigenetic silencing of genes in breast cancer. Epigenetic silencing results from a combination of promoter DNA hypermethylation and modification of histone proteins that favor closed chromatin conformation thus inhibiting transcription. Aberrant epigenetic silencing of tumor suppressor genes is critically important in the initiation and progression of breast cancer. Histone modifications, including acetylation, phosphorylation, and methylation, result in a combination of histone marks collectively known as the histone code. These modifications act in concert with gene promoter CpG DNA methylation status to determine whether specific genes are transcriptionally active. Recently, several laboratories including our own, have investigated means by which aberrant epigenetically silenced genes may be reactivated for therapeutic or chemopreventive advantage. This approach is based on the fact that unlike gene mutations, epigenetic modifications are reversible and reversing these modifications has the potential to beneficially alter the breast cancer phenotype through differentiation and/or restoration of growth control. Histone modifications are now known to be a result of dynamic processes; a prime example is the balance of histone acetylation maintained by the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Recently a similar dynamic process has been demonstrated for histone methylation. The first lysine demethylase discovered, lysine specific demethylase 1 (LSD1), targets mono- and dimethyl-lysine 4 histone 3 (H3K4me1 & H3K4me2) and works through a FAD-dependent oxidation reaction. Promoter region H3K4me2 is associated with transcriptionally active genes and thus is considered an activating mark. Demethylation of this mark by LSD1 may broadly repress gene expression and dysregulated LSD1 activity may play a role in the inactivation of specific tumor suppressor genes. Further, emerging data indicate its activity is associated with abnormal gene silencing and is thus a target for therapeutic manipulation to facilitate the re-expression of aberrantly silenced genes. The catalytic domain of the recently discovered LSD1 is closely related to the FAD-dependent spermine oxidase (SMO). Our laboratory first cloned and characterized SMO and aided in the discovery of LSD1. Based on our expertise in polyamine catabolism, we hypothesized that certain polyamine analogues would effectively inhibit LSD1 and thus have the potential of reactivating epigenetically silenced genes. We have discovered specific polyamine analogues are potent inhibitors of LSD1, leading to re-expression of aberrantly silenced genes important in tumorigenesis. Gene re-expression is subsequent to increased promoter chromatin activating marks (H3K4me1, H3K4me2, & H3K9-acetyl), decreased repressive chromatin marks, (H3K9me1 & H3K9me2), and in some genes, decreased promoter CpG methylation. HYPOTHESES TO BE TESTED Our general hypothesis is that our novel polyamine analogue inhibitors of LSD1, when used alone or in combination with other agents targeting epigenetic silencing, will more be efficacious in re-activating aberrantly silenced genes in breast cancer cells than current strategies and thus have the potential to improve treatment of breast cancer. RESEARCH AIMS are to: 1) determine the most efficacious analogue and treatment protocols in our in vitro human breast cancer cell models using lines representative of the major forms of breast cancer, with respect to gene re-expression, chromatin changes, and growth inhibition; 2) advance the most promising LSD1 inhibitors into our animal model of human breast cancer for in vivo evaluation of biological response and efficacy; 3) use microarray analysis to define the changes in gene expression profile of breast cancer cells treated with agents targeting epigenetic gene silencing with the goal of identifying new targets for therapy. The ultimate goal of the studies detailed in this application is to provide sufficient data to determine if clinical trials of this strategy are warranted. UNIQUE ADVANCES IN OUR KNOWLEDGE OF BREAST CANCER The basic understanding of epigenetic regulation of gene expression in normal and neoplastic breast cells is rapidly advancing and thus offers a unique opportunity to exploit breast tumor-specific defects in this regulatory pathway for therapeutic advantage. Our recent discovery of LSD1 as a rational target for intervention coupled with our identification of novel polyamine analogue inhibitors provide an entirely unique and exciting new avenue with substantial clinical potential. As epigenetic therapies are currently being testing clinically in breast cancer, the addition of LSD1 inhibitors to such regimens has considerable promise in improving treatment outcomes. Thus the results of the studies proposed here should provide a solid foundation for adding novel agents to the current clinical armamentarium in the fight against breast cancer.

    Lay Abstract:
    RATIONALE The transformation of normal breast epithelial cells into breast cancer cells requires a series of changes that result in the altered expression of proteins that prevent normal breast cells from undergoing uncontrolled division. The loss of normal expression of the growth regulating proteins can result from genetic changes, specifically gene mutations in the DNA sequence or loss of chromatin containing the growth regulatory genes. A second general mechanism by which growth regulatory gene expression can be lost is through epigenetic changes. Specifically, genes can be switched off or silenced by changes in how DNA is packaged on chromosomes through physical changes in chromatin proteins called histones as well as modifications of DNA that do not change the actual DNA sequence. These non-sequence changes in DNA, termed DNA methylation, normally function together with histone proteins to properly regulate gene expression. In breast cancer, these processes are dysregulated and this can lead to inappropriate silencing of important growth regulatory genes called tumor suppressor genes. Breast cancer can be the ultimate result of this faulty regulation of the expression of critical regulatory genes in previously normal breast tissue. STUDY HYPOTHESIS AND TESTING The intense interest in epigenetic changes leading to breast cancer is based on the fact that epigenetic changes, such as DNA methylation and changes in histone proteins, are potentially reversible unlike genetic changes such as mutations or loss of chromosomes. Therefore, it is possible through treatment to restore normal growth control by reversing epigenetic changes that result in abnormal gene silencing. In fact ongoing clinical trials with FDA-approved drugs that block DNA methylation (5-azacytidine) combined with drugs that block the changes in histone proteins (histone deacetylase inhibitors) that lead to gene silencing have demonstrated promising early results in a form of leukemia. Histone acetylation is an important histone modification that is generally correlated with the activity of a gene. Histone deacetylases (HDAC) are proteins that can modify histones, thereby contributing to the aberrant silencing of genes. This leads to the concept of using drugs to maintain the acetylation status of genes to prevent abnormal silencing characteristic of cancer. Another protein that is responsible for epigenetic silencing of genes is lysine specific demethylase 1 (LSD1). LSD1 functions by removing methyl groups from a specific lysine residue on a specific histone protein. The removal of this methyl group then contributes to the switch off of genes that are regulated by the histone that has been demethylated. If this demethylation occurs improperly, as is thought to occur in the transformation of a normal breast cell to a breast cancer cell, it can lead to silencing of growth regulatory genes, thus contributing to carcinogenesis. OUR HYPOTHESIS IS THAT TREATMENT OF BREAST CANCER CELLS WITH OUR UNIQUE AGENTS THAT ALTER CHROMATIN CAN BE USED TO REVERSE ABERRANT SILENCING OF GENESTO RESTORE NORMAL GENE FUNCTION. THESE AGENTS CAN THUS ULTIMATELY BE USED TO TREAT BREAST CANCER. We have very recently demonstrated that blocking LSD1 activity with our newly designed polyamine analogues results in the reactivation of epigenetically silenced genes whose inactivation is important in the genesis of cancer. Thus, we have demonstrated that LSD1 represents a rational target for the development of drugs that target its activity in cancer cells. The primary goal of the studies in this proposal is to determine if inhibition of LSD1 with our new compounds in breast cancer cells offers potential therapeutic advantages over existing therapies, including strategies targeting aberrant epigenetic gene silencing. We will test these new LSD1 inhibitors alone and in combination with other existing agents including inhibitors of DNA methylation and HDAC in our cell culture and animal models of human breast cancers. Thus, the studies detailed in this application have the overall goals of advancing a novel approach of targeting epigenetic changes in breast cancer that can be rapidly advanced into clinical testing. UNIQUE ADVANCES IN OUR KNOWLEDGE OF BREAST CANCER AND IMPORTANCE OF THIS RESEARCH TO PATIENTS WITH BREAST CANCER Understanding how epigenetic control of gene expression contributes to cancer has offered a unique opportunity to exploit cancer-specific defects in this regulatory pathway. This is particularly true in breast cancer as many genes important to normal breast tissue growth and development are under epigenetic control. We have discovered both an important new target for breast cancer drug development, LSD1, and a new series of agents, specific polyamine analogues, to attack this target. As epigenetic therapies are currently being clinically tested, the potential is great that this new strategy for reversing aberrant silencing of important genes will be rapidly translated into clinical trials. Thus the results of the studies proposed in this application have the potential to have considerable impact on the systemic treatment available to women with breast cancer. Finally our team of a molecular pharmacologist and physician-scientist is uniquely poised to take our new scientific discovery through the needed preclinical work with a goal of translation into patient testing in the near term.