Epigenetic Regulation of Anti-Cancer Efficacy of Selenium Agents in Lung Cancer

Abstract Lung cancer has the highest death rate in the U.S, with known risks from tobacco smoking and poor diet such as selenium (Se) deficiency, particularly in Appalachian Kentucky. Se supplement reduced lung cancer incidence in the heavy smoker/Se deficient cohort of a large national trial. Se forms are critical to their efficacy, with selenite and methylseleninic acid (MSA) far more toxic to lung cancer cells than selenomethionine (SeM) with molecular mechanism unknown. Using Stable Isotope Resolved Metabolomics (SIRM), we defined differential effects of selenite, MSA, and SeM on anaplerosis-fueled anabolic metabolism, relatable to variable toxicity in non-small cell lung cancer (NSCLC) cells and patient-derived tissues. Reverse Phase Protein Array (RPPA) analysis revealed variable suppression of many proteins in metabolism, mitogenesis, and cell cycle, including enhancer of Zeste homolog 2 (EZH2), which was suppressed by selenite and MSA. EZH2, a subunit of Polycomb Repressive Complex 2 (PRC2) is overexpressed in NSCLC. It silences tumor suppressor genes via trimethylation of K27 on histone H3 (H3K27me3) (PRC2-dependent) and can coactivate oncogene transcription (PRC2-independent). It is unknown if and how EZH2 modulates metabolic pathways other than glycolysis. We hypothesize that EZH2 loss mediates selenite or MSA toxicity by PRC2-dependent and/or -independent mechanism. We will study if and how EZH2 blockade disrupts tumor-promoting metabolism in lung cancer cells. Our proposal innovates by: (a) multiplexing SIRM tracers (mSIRM) for expanded tracing of metabolic network; (b) using tracer data to guide RPPA to verify EZH2- regulated metabolic targets and/or transcriptional regulators; and (c) using RPPA-verified targets to direct assay for gene-specific promotor binding to H3K27me3. Our SAs will rely on the Metabolism Core. SA1 will define metabolic dysregulation induced by EZH2 knockdown using mSIRM, RPPA, RNAseq and chromatin immunoprecipitation to map metabolic target genes and assay for H3K27me3 enrichment at their promotors. SA2 will define metabolic dysregulation induced by inhibitor of EZH2 enzyme activity to verify and help resolve PRC2-dependent from PRC2-independent mechanism of metabolic regulation. This work will acquire systematic understanding on epigenetic control of tumor-promoting metabolism, a paradigm shift from current focus of epigenetic research while providing strong preliminary data for 2 NIH proposals.