Epigenetic Mechanisms of Gene Expression in Thoracic Malignancies

Division Of Basic Sciences - Nci

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Abstract

Novel in-vitro models and correlative experiments with primary tumor/normal tissue specimens have been utilized to identify epigenomic alterations which contribute to initiation and progression of lung and esophageal cancers and malignant pleural mesotheliomas. A steady stream of manuscripts have been published by our group describing the epigenomic effects of cigarette smoke and other environmental carcinogens in normal aerodigestive tract epithelial cells and thoracic cancer cells in-vitro and in-vivo. Recently, a series of experiments have been conducted to examine the epigenenomic and transcriptomic effects of hookah smoke relative to conventional cigarette smoke in normal and immortalized human respiratory epithelial cells. Briefly, primary normal human small airway epithelial cells (SAEC) from three donors, as well as cdk4/hTERT-immortalized SAEC and human bronchial epithelial cells (HSAEC and HBEC, respectively) were cultured in normal media with or without cigarette smoke condensate (CSC) or water pipe condensate (WPC). CSC and WPC mediated dose-dependent growth inhibitory effects in cultured respiratory epithelia cells, which coincided with decreased global levels of H4K16Ac and H4K20Me3. CSC and WPC mediated distinct as well as overlapping transcriptome signatures, and pathway modulations that were cell line and dose-dependent. Transcriptome signatures uniquely associated with CSC or WPC exposures or common to both completely segregated a panel of lung cancer lines based on NSCLC vs SCLC histology. Conceivably, these signatures could prove useful for risk assessment or as biomarkers for chemoprevention trials in hookah and cigarette smokers. Epiregulin (EREG) encoding a master regulator of EGFR signaling which has been implicated in progression of lung cancers and maintenance of cancer stem cells was up-regulated, whereas Filamin A Interacting Protein 1-Like (FILIP1L) and API-3 binding protein (ABI3BP) encoding putative mediators of senescence were down-regulated in the five cell lines following CSC and WPC exposures. Induction of EREG and repression of FILIP1L by CSC and WPC coincided with DNA methylation changes within the respective promoters, whereas repression of ABI3BP by these condensates was mediated by Polycomb mechanisms. A comprehensive manuscript describing these findings has recently been published. Additional studies have focused on delineation of epigenetic mechanisms contributing to malignant pleural mesotheliomas (MPM), and identification of novel therapeutic targets in these neoplasms. Micro-array, qRT-PCR, immunoblot, and immunohistochemistry experiments demonstrated that expression of ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1)- a master regulator of DNA methylation was significantly higher in MPM lines/specimens relative to normal mesothelial cells/ normal pleura. Short term asbestos exposure induced UHRF1 expression in normal mesothelial cells. UHRF1 up-regulation correlated inversely with overall survival in MPM patients. Knockdown of UHRF1 down-regulated DNMT1, and reversed global DNA hypomethylation, inhibiting proliferation, invasion, and clonogenicity of MPM cells, as well as growth of MPM xenografts. These effects were phenocopied by exposure of MPM cells/xenografts to the repurposed chemotherapeutic agent, mithramycin, which we have previously shown to deplete the oncogenic transcription factor SP1, and up-regulate p53 in MPM cells, as well as HDM2 inhibitors currently in clinical development. Collectively, our findings which have recently been published provide the first evidence that UHRF1 is a novel, druggable epigenetic driver in MPM, and warrant further studies to optimize pharmacologic regimens that inhibit UHRF1 expression/activity for mesothelioma therapy. Additional efforts have been devoted to examining the prevalence and natural history of mesotheliomas arising in patients with BAP1 tumor predisposition syndrome (TPDS). A unique protocol evaluating the use of photon counting CT imaging, liquid biopsies, and minimally invasive surveillance has been initiated in our Branch. 18 different primary malignancies (all of which were subclinical) have been diagnosed in the first 6 patients enrolled on this trial. This protocol which is the only such protocol in the world will be used as a platform for window of opportunity trials evaluating if epigenetic therapies can prevent progression of mesotheliomas in patients with BAP1 TPDS.