YEATS4 Is a Novel Oncogene Amplified in Non– Small Cell Lung Cancer That Regulates the p53 Pathway Speaker:Dai-Wei Hsuan Adviser:Dr. Guor-Mour, Her Data:2015/04/22 Larissa A. Pikor1, William W. Lockwood2, Kelsie L. Thu1, Emily A. Vucic1, Raj Chari3, Adi F. Gazdar4, Stephen Lam1, and Wan L. Lam1

Lung cancer

 In the world has a high mortality rate.  Until now is still a lack of effective treatment methods.  Mutated genes including TP53, CDKN2A,PTEN, NRAS, BRAF, PIK3CA, DDR2, KEAP1. Why we study Lung cancer?

Gene amplifications and cancer  Recurrent amplifications and deletions occur in almost cancers.  Recurrent amplifications of several regions activate known oncogenes.  DNA amplification directly contributes to oncogene activation and the promotion of tumorigenesis.

Lung cancer Classification  NSCLC(Non-small cell lung cancer): 85%-90% belong to non-small cell lung cancer, this cancer have three type: 1. Squamous-cell carcinoma(SCC, SqCC) 2. Adenocarcinoma 3. Anaplastic Carcinoma  SCLC(Small cell lung cancer) Lung cancer is about 10-15% are small cell lung cancer

Lung cancer survivorship curve

Schematic overview of potential tyrosine kinase inhibitor in non-small cell lung cancer

 YEATS -Yaf9,ENL,AF9,Taf14,Sas5. -High conserved protein domain. -Involved in establishing and recognizing different chromatin modifications.

YEATS4(YEATS domain-containing protein 4) -GAS41 -12q Component of the NuA4 histone acetyltransferase (HAT) complex. -This gene has been shown to be amplified in tumors. -Yaf9

YEATS4(YEATS domain-containing protein 4) YEATS4 Acetylation Nucleosome - DNA interaction Interaction of the other proteins. Associated with oncogene and proto-oncogene. Senescence, apoptosis, and DNA repair.

YEATS4 is expression in normal human tissues

YEATS4 and human disease YEATS4 Potent growth promoting human diseases Soft Tissue Sarcoma Brain Tumours Brain Stem Glioma Liposarcoma

Cisplatin and Nutlin  Cisplatin - Is a chemotherapy drug - Structure formation of chlorine molecules are substituted with a positively charged H2O actives.  Nutlin - Inhibit the interaction between mdm2 and tumour suppressor p53. P53 Mdm2 P53 Mdm 2 Nutlin

Purpose 14 Lung cancer cell growth YEATS4 HBEC Senescence Suppression P53 YEATS4 Drug resistant

Gene amplifications in lung cancer?  CGH(comparative genomic hybridization) - Checked on a chromosomal copy number changes in the genome of the entire tumor. - Small sample required for DNA test. - Compared with the traditional method has higher efficiency

What region which is amplified in NSCLC? 261NSCLC 92Sqcc 169AC CGH(comparative genomic hybridization) GISTIC is a tool to identify genes targeted by somatic copy-number alterations (SCNAs) that drive cancer growth

261NSCLC169AC92Sqcc 7p11.2 (EGFR) 8p11.23 (FGFR1) 8p12 (BRF2) 14q13.3 (NKX2-1) 20q13.3 (EEF1A) 12q15 (??) Recurrent amplifications in NSCLC

Why YEATS4 is important in 12q15? LYZ YEATS4 FRS2 CCT2 LRRRC10 BEST3 RAB31IP EDRN Normal tissue tumor YEATS4

Normal tissue Tumor YEATS4 is recurrently amplified and overexpressed in NSCLC and is the target of 12q15 amplification

Does Human NSCLC cells exhibit elevated YEATS4 expression YEATS4 NSCLC YEATS4 Normal human bronchial epithelial RT-qPCR Western blot Lung cancer

18 NSCLC cell line normal human bronchial epithelial cells YEATS4 AMP YEATS4 No AMP

How about YEATS4 in vitro and in vivo HBEC cell(HBEC-KT53) HBEC cell(HBEC-KT)  Transfect P53 knockdown Normal function P53 Control empty vector YEATS4 KT-EV KT53-EV HBEC-KT-YEATS4 HBEC-KT53-YEATS4

Normal function P53 P53 knockdown

Dose YEATS4 expression interaction HBEC cell survial? YEATS4 HBEC cell(HBEC-KT53) HBEC cell(HBEC-KT) colony formation

B-Gal staining for cellular senescence in EV and YEATS4 expressing HBECs Normal function P53 P53 knockdown

quantification of cellular senescence in YEATS4 and control cells Normal function P53 P53 knockdown

Does shRNA-mediated knockdown of YEATS4 inhibits the growth of NSCLC cells? Control PLKO YEATS4 shRNA YEATS4 Cancer cell YEATS4 Western blot RT-qPCR YEATS4

mRNA expression and protein levels in all cell lines relative to controls (PLKO) YEATS4 AMP YEATS4 No AMP

PLKO H1993 、 H1355 shRNA H1993 、 H1355

YEATS4 alters p21 and p53 protein levels

Cells treated with 2-fold dilutions of cisplatin for 72 hours

Immunoblot of PLKO and shY4 cell lines treated with 40 mm of cisplatin for 0, 24, or 48 hours

Disscussion Lung cancer cell growth Lung cancer cell Cisplatin resistent Lung cancer cell Cisplatin resistent 35 YEATS4 Supression p53

Conclusion 36 YEATS4 YEATS4 shRNA Provide a new way to study of Lung cancer Lung cancer cell Lung cancer Cisplatin resistent

37 Thank You For Listening