The Polycomb Associated Protein JARID2 is a Novel Epigenetic Target for Lung Cancer Therapy Young Hong, Mary Zhang, Scott Atay, Vivek Shukla, Manish Raiji, Trevor Upham, Julie A. Hong, Mahadev Rao, David S. Schrump Thoracic Surgery Section, Thoracic and GI Oncology Branch, National Cancer Institute, Bethesda, MD No Disclosures Polycomb Group Proteins • Two major complexes: PRC1 and PRC2 • Critical mediators of stem cell pluripotency • Function to repress differentiation related genes • Aberrant expression/activity of Polycomb Group Proteins leads to epigenetic silencing of tumor suppressors in human cancers Polycomb Repressive Complex 2 (PRC2) • PRC2 is the major initiation complex • Major core components: EZH2, EED, and SUZ12 • Depletion of PRC2 components by biochemical or pharmacologic methods inhibits cancer growth • JARID2 is a novel protein that recruits PRC2 to DNA Hypothesis • Inhibition of JARID2 expression by biochemical or pharmacologic methods may decrease proliferation and tumorigenicity of lung cancer cells Methods • Quantitiative RT-PCR (qRT-PCR) and immunoblot analysis of JARID2 and PRC2 expression in lung cancer cell lines and primary lung cancers relative to normal respiratory epithelia • shRNA techniques and Mithramycin were used to deplete JARID2 • qRT-PCR, immunoblot, immunoflourescence, and murine xenograft experiments were performed to examine the effects of JARID2 inhibition JARID2 Is Over-expressed in Lung Cancers Tumor Specimens Cell Lines qRT‐PCR ** 10000 JARID2 mRNA Copy # ** * 10 5 10 4 * * * 1000 100 10 10 3 (n =6 ) et as ta se s M (n =3 4) Lu ng m al L or N NSCLC = Non small cell lung cancer SCLC = Small cell lung cancer Tu m or s (n =8 ) un gs SC LC SC LC N or m al 1 N JARID2 mRNA Copy # 10 6 Similar gene expression profiles were seen with EZH2, EED * P < 0.05 ** P < 0.005 and SUZ12 Expression of PRC2 Complex Proteins in Normal Respiratory Epithelia and Lung Cancer Cells JARID2 EZH2 EED SUZ12 H3K27Me3 ß‐Actin Up-regulation of JARID2 coincides with over-expression of other PRC2 proteins in lung cancer cells H526 H345 H69 H889 SCLC H358 H1299 H841 A549 NSCLC Calu‐6 Immunoblot SAEC NHBE Normal sh Co nt A5 ro 49 l sh JA A5 RI 49 D2 sh Co nt A5 ro l 49 sh Ca EZ lu H2 6 sh Co Ca nt ro lu l 6 sh JA RI D2 A5 49 Percent Cell Growth Knockdown of JARID2 or EZH2 Inhibits Proliferation of Lung Cancer Cells 120% 100% 80% 60% 40% 20% 0% Knockdown of JARID2 Inhibits Growth of A549 (wt P53) Lung Cancer Cells in vivo * shControl shJARID2 1.5 * 150 * Tumor Mass (g) Tumor volume (mm3) 200 * 100 * 50 0 0 10 20 30 40 50 1.0 * 0.5 0.0 shControl shJARID2 Day *p < 0.05 Knockdown of JARID2 Inhibits Growth of Calu6 (P53 null) Lung Cancer Cells in vivo 200 * shControl shJARID2 1.5 150 Tumor Mass (g) Tumor volume (mm3) 2.0 * * 100 * * 50 0.5 0 0 10 1.0 20 30 40 50 * 0.0 shControl shJARID2 Day *p < 0.05 Knockdown depletes JARID2 in Tumor Xenografts shControl T1 T2 T3 T4 shJARID2 T1 T2 T3 T4 JARID2 A549 B‐Actin shControl T1 T2 T3 T4 shJARID2 T1 T2 T3 T4 JARID2 Calu6 B‐Actin Anti-proliferative effects of JARID2 may coincide with p53 status Mithramycin A • Anti-neoplastic antibiotic used to treat Paget’s disease, chronic myeloid leukemia, testicular carcinoma, and malignant hypercalcemia • Produced by soil bacterium Streptomyces argillaceus • Inhibits binding of Sp1 to DNA • Gene expression mediated by Mithramycin may not be solely attributable to Sp1 inhibition Effects of Mithramycin in Lung and Esophageal Cancer Cells in-vitro and in-vivo Zhang M. Cancer Res.2012 Genes Modulated by Mithramycin in Lung Cancer Cells: Selected Microarray Results EHMT1 HDAC4 EIF2C2 EGFR MLL3 EIF2C2 ASH1L EIF2C2 JARID2 KDM6A KDM4B ARID2 MLL2 ARID4B PHF2 WDR4 KDM6A PHF15 GSK3B HDAC4 ARID1B BRD4 A549 A549 Calu6 Calu6 MM- MMMM50 200 MM-50 200 -49.5 -40.1 -44.2 -31.7 -15.4 -12.7 -24.9 -23.2 -13.2 -18.9 -11.7 -32.0 -12.3 -14.5 -12.9 -18.8 -10.6 -20.3 -10.7 -16.5 -10.1 -11.6 -12.1 -14.3 -9.9 -20.8 -6.5 -15.7 -8.8 -8.8 -6.9 -12.6 -8.7 -10.5 -8.2 -7.7 -7.1 -7.0 -6.9 -6.2 -5.9 -5.9 -5.8 -5.8 -5.5 -5.1 -5.1 -10.2 -6.5 -7.1 -7.6 -23.5 -11.7 -6.2 -9.1 -18.3 -5.8 -4.3 -5.3 -10.9 Zhang M. Cancer Res.2012 -8.2 -8.1 -10.0 -16.5 -4.7 -3.5 -5.6 -4.6 -4.1 -7.6 -5.2 -4.2 -10.9 -13.7 -5.3 -11.0 -13.0 -6.1 -4.6 -19.6 -8.0 -3.3 -12.1 -14.3 -11.1 -12.7 -14.5 -7.5 qRT‐PCR 1.0 Relative Fold Change Gene Symbol JARID2 0.8 0.6 0.4 0.2 0.0 Untreated 10 25 50 Mithramycin (nM) 100 Mithramycin Inhibits Expression of PRC2 Associated Proteins in Lung Cancer Cells EZH2 EED Relative Fold Change 0.8 0.6 0.4 0.2 qRT‐PCR SUZ12 1.0 Relative Fold Change qRT‐PCR EZH2 1.0 0.8 0.6 0.4 0.2 0.0 0.0 Untreated 10 25 50 Mithramycin (nM) 100 Untreated 10 25 50 Mithramycin (nM) Similar phenomenon observed for SUZ12 100 Mithramycin Inhibits Sp1 and Components of the PRC2 Complex Mithramycin (nM) 0 10 50 100 Sp1 JARID2 Immunoblot EZH2 EED B‐Actin A549 Microarray Analysis of Gene Expression in Lung Cancer Cells following JARID2 Knockdown or Mithramycin Treatment A549 Calu6 MM-50nM (7872) MM-50nM (6464) 2069 1225 5132 5055 190 481 5669 254 MM-200nM (11536) 38 146 742 shJARID2 (1667) 5886 93 MM-200nM (11180) 256 shJARID2 (533) Higher order analysis reveals numerous pathways regulating cell cycle progression, cancer cell signaling, DNA damage, and senescence Many of the commonly regulated genes are stem cell Polycomb targets Conclusion • JARID2 appears to be coordinately over-expressed with PRC2 components in lung cancer cells • Knockdown of JARID2 inhibits proliferation of lung cancer cells in-vitro and in-vivo • JARID2 is a novel epigenetic target for lung cancer therapy • Mithramycin depletes JARID2 and other PRC2 associated proteins in lung cancer cells