1. Lysyl-Like Oxidase 2 (LOXL2) is an Oncogenic Driver of Malignancy Regulated by miR-145 in Tobacco- Associated Esophageal Adenocarcinomas Shakirat O. Oyetunji, M.D., Sichuan Xi, M.D. Ph.D, David Straughan, M.D., Saïd Azoury, M.D., Julie A. Hong, M.S., Mary Zhang, M.S., David S. Schrump, M.D. Thoracic Epigenetics Section, Thoracic and GI Oncology Branch, CCR/NCI

2. Esophageal Adenocarcinomas (EAC) Increasing prevalence in Western societies: attributed to GERD and Barrett’s esophagus Additional risk factors: Barrett's Esophagus and Esophageal Adenocarcinoma Consortium (BEACON) – 10 population-based case–control studies and 2 cohort studies – OR of 1.96 between cigarette smoking and EA – OR of 2.18 between cigarette smoking and esophagogastric junctional adenocarcinoma – OR of 2.08 between cigarette smoking and all adenocarcinoma – Statistically significant dose–response relationship between pack-years and each studied outcome These studies suggest that smoking increases the risk of esophageal adenocarcinogenesis

3. Micro-RNAs Are Diverse Regulators of Gene Expression Traditional “central dogma of genetics” DNA RNA Protein

4. Hypothesis Cigarette smoke enhances esophageal adenocarcinogenesis via alteration of microRNA (miR) expression

5. Experimental Strategy Immortalized esophageal epithelial cell lines: Het1a, CP-A, CP-C EAC lines: NCI-EsC-1, 2, 3; OE-19; OE-33 5 day culture +/- cigarette smoke condensate (CSC): ~ 1ppd Micro-array identification of miRs consistently modulated by CSC Targets identified and confirmed Phenotypic characterization of effects of differentially expressed miRs and selected targets

6. miR-145 is Repressed in EAC Cells and Down-regulated by Cigarette Smoke Endogenous miR-145 Expression Modulation of miR-145 by CSC

7. miR-145 Inhibits Proliferation, Migration and Invasion of EAC Cells OE 19 vector ctrl OE 19 145KD 24 hrs 72 hrs48 hrs 0 hrs OE 19 145KD OE 19 vector control 28% 76% 100% 10%20%24% Cell Invasion * p-value <0.05 Cell Migration

8. MiR-145 Targets LOXL2 in EAC Cells LOXL2 qRT-PCR 3' UTR Luciferase Assay Percentage of control

9. miR-145KD and LOXL2 overexpression increased tumorigenicity in xenograft models miR-145 KD LOXL2 overexpression * p-value <0.05 n = 10 * p-value <0.005 n = 5

10. Summary miR-145 LOXL2 Tobacco Adenocarcinoma ✕