IMI CONFIDENTIAL KLF4 regulates adult lung tumor-initiating cells and represses K-Ras-mediated lung cancer Cell Death and Differentiation (26 June 2015 ； IF= 8.184) 报告人：董浩

Background IMI CONFIDENTIAL 2 KLF4 A zinc finger transcription factor Enriched in the epithelium of various tissues In normal lung tissue (fibroblasts and airway epithelial cells) Development and cell differentiation Has role in cell fate and selfrenewal of embryonic stem cells Been identified as a tumor suppressor in many types of cancer In the lung and lung cancer are still not known

Background IMI CONFIDENTIAL 3 K-RAS GTP-binding protein Promotes proliferation and cell survival Mutations result in constitutive activation Mutations are linked to lung tumorigenesis, limited to non- small-cell lung cancer Mutations are found in more than one-third of human lung adenocarcinomas

Background IMI CONFIDENTIAL 4 Lung tumor-initiating cells Lung Bronchiolar epithelium Alveolar epithelium Clara cells (CC10) AT1 and AT2 cells (SP-C) functions as stem cells Clara and AT2 cells are the origin of lung tumorigenesis

Materials and Methods IMI CONFIDENTIAL 5 Clinical Date: TCGA (The Cancer Genome Atlas) Animal studies: Cre-inducible Klf4 fl/fl mice Cre-inducible K-ras LSL-G12D/+ mice Cell: Human lung cancer cell lines A549 and H460 HEK293T cells, Lung epithelial cell line BEAS-2B Proliferation assay (cell viability analyzer) Adenovirus transfection Western blotting RT-PCR real-time PCR H&E IHC Immunofluorescent Luciferase reporter assays

Materials and Methods IMI CONFIDENTIAL 6 诱导性基因敲除： Cre/LoxP 、 Flp/Frt 同源重组系统 Cre: 位点特异性重组酶，介导 LoxP 序列间同源重组 LoxP ：两个 13bp 反向重复序列 中间 8bp 序列

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Results IMI CONFIDENTIAL 9 1.Klf4 is a putative tumor suppressor for lung cancer 2.Klf4 ablation altered the differentiation pattern in mouse lung 3.Klf4 deletion facilitates lung tumor formation and progression 4.Multiple mechanisms of KLF4 downregulation in lung cancer

1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 10 1.KLF4 mutations ： 9 out of 488 patients Small-cell lung carcinoma Non-small-cell lung carcinoma (adenocarcinoma) <2%

1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 11 These mutations have no significant effect on KLF4 function in repressing cyclinB1 promoter activity.

1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL KLF4 mRNA in human lung tumor tissues Non-small-cell lung carcinoma (adenocarcinoma) Small-cell lung carcinoma KLF 1, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15 and 17 were also significantly lower in tumor

1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL The role of KLF4 in lung cancer cells Small-cell lung carcinoma KLF1, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15 and 17 were also significantly lower in tumor

1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL The role of KLF4 in lung cancer cells No significant effect on proliferation of these cells, probably because of the endogenous KLF4 expression

1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL Effects of KLF4 on target genes

1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL Effects of KLF4 on target genes KLF4 represses lung cancer cell growth by regulating cell cycle and cell proliferation.

2. Klf4 ablation altered the differentiation pattern in mouse lung IMI CONFIDENTIAL Cre-induced Klf4 deletion mouse

2. Klf4 ablation altered the differentiation pattern in mouse lung IMI CONFIDENTIAL Effects of Klf4 deletion on AT2 cells Klf4 deletion mouse lung WT mouse lung

2. Klf4 ablation altered the differentiation pattern in mouse lung IMI CONFIDENTIAL Effects of Klf4 deletion on other cells Klf4 deletion could be a driving force of lung tumorigenesis through stimulating AT2 cells in mouse lung. AT1 Clara cell Ciliated cell

3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL K-ras activation and Klf4 deletion in mouse Klf4 fl/fl mouse K-ras LSL-G12D/+ mouse Klf4 fl/fl /K-ras LSL-G12D/+ mouse

3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL Klf4 deletion contributed to tumor formation in mouse lung Klf4 fl/fl WT 8 weeks after Cre-induction

3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL Effect of Klf4 deletion and K-ras activation on lung tumor

3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL Effect of Klf4 deletion and K-ras activation on lung tumor

3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL Effect of Klf4 deletion and K-ras activation on lung tumor Klf4 deletion synergized with K-ras activation to facilitate lung tumor

4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL Correlation between KLF4 expression and methylation r=−0.03 Mutation Epigenetics TCGA

4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL Histone deacetylases (HDACs) expressed in lung cancer Class I HDACs Histone acetylation TCGA

4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL HDACs inhibit KLF4 expression

4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL HDACs inhibit KLF4 expression

4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL HDACs inhibitors repressed lung cancer cell proliferation

4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL HDAC3 has an inhibitory role on KLF4 promoter activity

S. KLF4 regulate lung homeostasis and tumorigenesis through multiple mechanisms IMI CONFIDENTIAL Bmi1 is overexpressed in lung tumor stem cell marker AT2 cell

S. KLF4 regulate lung homeostasis and tumorigenesis through multiple mechanisms IMI CONFIDENTIAL Myeloperoxidase (MPO) is overexpressed in lung tumor neutrophils marker inflammation Klf4 depletion triggered lung inflammation, which may contribute to lung tumorigenesis in the mice

Highlights IMI CONFIDENTIAL 33 Clinical Date: TCGA (The Cancer Genome Atlas) Proliferation assay (cell viability analyzer) Multiple mechanisms of KLF4 downregulation

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