Published OnlineFirst March 1, 2017; DOI: 10.1158/0008-5472.CAN-16-2589 Cancer Molecular and Cellular Pathobiology Research Kruppel-like€ Transcription Factor KLF10 Suppresses TGFb-Induced Epithelial-to- Mesenchymal Transition via a Negative Feedback Mechanism Vivek Kumar Mishra1, Malayannan Subramaniam2, Vijayalakshmi Kari1, Kevin S. Pitel2, Simon J. Baumgart1, Ryan M. Naylor2, Sankari Nagarajan1, Florian Wegwitz1, Volker Ellenrieder3, John R. Hawse2, and Steven A. Johnsen1 Abstract TGFb–SMAD signaling exerts a contextual effect that sup- sequences in the promoter region of the EMT-promoting tran- presses malignant growth early in epithelial tumorigenesis but scription factor SLUG/SNAI2, repressing its transcription by promotes metastasis at later stages. Longstanding challenges in recruiting HDAC1 and licensing the removal of activating resolving this functional dichotomy may uncover new strate- histone acetylation marks. In clinical specimens of lung ade- gies to treat advanced carcinomas. The Kruppel-like€ transcrip- nocarcinoma, low KLF10 expression associated with decreased tion factor, KLF10, is a pivotal effector of TGFb/SMAD signaling patient survival, consistent with a pivotal role for KLF10 in that mediates antiproliferative effects of TGFb.Inthisstudy,we distinguishing the antiproliferative versus prometastatic func- show how KLF10 opposes the prometastatic effects of TGFb tions of TGFb. Our results establish that KLF10 functions to by limiting its ability to induce epithelial-to-mesenchymal suppress TGFb-induced EMT, establishing a molecular basis for transition (EMT). KLF10 depletion accentuated induction of the dichotomy of TGFb function during tumor progression. EMT as assessed by multiple metrics. KLF10 occupied GC-rich Cancer Res; 77(9); 1–14. Ó2017 AACR. Introduction onic development and is indispensable for tissue and organ development in multicellular organisms (4). During EMT, epi- Non–small cell lung carcinoma (NSCLC) is the leading cause of thelial cells lose apico-basolateral polarity and dissolution of cancer-related deaths in the United States and around the world adherence junctions occurs, resulting in the loss of cell–cell (1). Therapeutic resistance and tumor metastasis are the most contact and an opening of the basement membrane barrier (5). common causes of lung cancer-related death. Despite recent Cells attain front-to-rear polarity, become spindle shaped, and progress in early detection and therapeutic efficacy, most lung gain migratory and invasive capacity. Upon completion of EMT, cancers are diagnosed at an advanced or metastatic stage and, thus, expression of epithelial markers like E-cadherin (CDH1) and overall patient survival rates remain very poor (2). Zona Occludens 1 (ZO-1) is suppressed, whereas mesenchymal Epithelial-to-mesenchymal transition (EMT) is an evolutionary markers such as N-cadherin (CDH2), vimentin (VIM), and Fibro- conserved trans-differentiation process proposed to play a central nectin (FN1) are upregulated. Genes encoding various transcrip- role in cancer metastasis, therapeutic resistance, and cancer stem tion factors including Snail (SNAI1), Slug (SNAI2), ZEB1, and cell–like features (3). EMT plays an important role during embry- ZEB2 (collectively termed as EMT-TF) become activated following the extracellular stimuli and carry out the downstream nuclear regulation of EMT (6). EMT is proposed to play an important role 1 Department of General, Visceral and Pediatric Surgery, University Medical in driving cancer progression by enabling tumor cells to become Center Gottingen,€ Gottingen€ Center for Molecular Biosciences, Gottingen,€ Germany. 2Department of Biochemistry and Molecular Biology, Mayo Clinic, more invasive and migratory, which are essential features for Rochester, Minnesota. 3Department of Gastroenterology and Gastrointestinal promoting tumor metastasis (7). Oncology, University Medical Center Gottingen,€ Gottingen,€ Germany. One important EMT-TF is SNAI2 (also referred to as Slug), a Note: Supplementary data for this article are available at Cancer Research member of the Snail family of transcriptional repressors (8). Online (http://cancerres.aacrjournals.org/). SNAI2 promotes EMT, in part, by binding to an E-box element in the promoter proximal region of the CDH1 gene (encoding E- Corresponding Authors: Steven A. Johnsen, University Medical Center Gottingen,€ Justus-von-Liebig-Weg 11, Gottingen€ 37077, Germany. Phone: cadherin) and repressing its transcription (9). Reduced E-cadherin 4955-1391-3711; Fax: 4955-1391-2297; E-mail: expression has been shown to be associated with poorly differ- [email protected]; and John R. Hawse, Department of entiated and metastatic NSCLC. Furthermore, SNAI2 is frequently Biochemistry and Molecular Biology, 200 First St. SW, Mayo Clinic, 13-21B overexpressed in different types of metastatic cancers including Guggenheim Building, Rochester, MN 55905. Phone: 507-284-4268; Fax: lung adenocarcinoma (10). 507-284-2053; E-mail: [email protected]. EMT is controlled by various signaling pathways, with one of doi: 10.1158/0008-5472.CAN-16-2589 the most potent inducers being TGFb. TGFb is a pleiotropic Ó2017 American Association for Cancer Research. cytokine that exerts antiproliferative and tumor-suppressive www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst March 1, 2017; DOI: 10.1158/0008-5472.CAN-16-2589 Mishra et al. effects in normal cells and/or in early stages of cancer, but also MDA-MB-231 cells in DMEM (Invitrogen) and H1299 cells in elicits prometastatic functions during cancer progression (11). RPMI1640 (Invitrogen) supplemented with 10% FBS (Sigma) TGFb signaling occurs through a heteromeric complex containing and 1Â penicillin/streptomycin (Sigma) at 37C under 5% two each of the type 1 and type 2 transmembrane serine/threo- CO2. siRNA transfections were performed using Lipofectamine nine kinase receptors (TbRI and TbRII). Binding of TGFb ligand to RNAiMAX (Invitrogen) according to the manufacturer's instruc- TbRII facilitates interaction with and phosphorylation of TbRI, tions. SmartPool siRNA against KLF10 (Dharmacon) consisted which in turn phosphorylates the SMAD2 and SMAD3 proteins of the following sequences: 50-GAAGAACCCACCUAAAUG- (also known as R-Smad). SMAD2/3 proteins then form a hetero- U-30,50-GAUAAGGAGUCACAUCUGU-30,50-GAAGUGAGCAA- meric complex with SMAD4 (also known as a Co-Smad) and GCUAAAUG-30,50-CACCAGACCUGCCCAAUGA-30. siGENOME translocate to the nucleus. Subsequently, activated SMADs regu- nontargeting siRNA (Dharmacon; D-001206-13) was used as a late target genes either by directly binding to the DNA at so-called negative control. For plasmid transfections, cells were transfected Smad-binding elements or through interactions with other tran- at approximately 70% confluency with pEGFP-C1 or pEGFP-C1- scription factors (12). Numerous studies have documented both TIEG expression plasmids using Lipofectamine 2000 (Invitrogen) the anticancer and prometastatic effects of TGFb signaling; how- according to the manufacturer's instructions. For inducing EMT, ever, the mechanisms behind the switch between these two cells were treated with 5 ng/mL TGFb (R&D Systems) for 90 min- functions of TGFb signaling is not completely understood (13). utes or 72 hours. Kruppel-Like€ Factor-10 (KLF10) is a zinc finger-containing transcription factor originally identified as an early response gene Antibodies following TGFb treatment of human osteoblasts and thus initially The following commercially available antibodies were used for named TGFb-Inducible Early Gene-1 (TIEG1; ref. 14). Like other chromatin immunoprecipitation (ChIP), Western blotting, and KLF family members, the KLF10 protein contains three zinc immunofluorescence: vimentin (clone v9, Santa Cruz Biotech- fingers at its carboxy terminal end, which allow for the regulation nology, sc-6260, 1:400 for immunofluorescence), E-cadherin of gene transcription via direct binding to guanine-cytosine (GC)- (clone 24E10, Cell Signaling Technology, 3195, 1:200 for immu- rich DNA sequences at target genes (15). Overexpression of KLF10 nofluorescence, 1:1,000 for Western blotting), ZO-1 (clone was shown to mimic the antiproliferative and apoptotic effects of D7D12, Cell Signaling Technology, 8193, 1:200 for immunoflu- TGFb in various cell lines (16–19). Importantly, KLF10 serves as a orescence), SNAI2 (clone C19G7, Cell Signaling Technology, positive feedback loop for regulating TGFb signaling by inducing 9585, 1:200 for immunofluorescence, 1:1,000 for Western blot- the expression of SMAD2 and inhibiting the expression of the ting), KLF10 (clone EPR12102, Abcam, ab184182, 1:1,000 for inhibitory SMAD7 gene (20, 21). KLF10 functions primarily as a Western blotting, 1 mg for ChIP), H3K27ac (Diagenode, transcriptional repressor and regulates target gene expression by C15410196, 1 mg for ChIP), H3K9ac (Diagenode, C15410004, recruiting various epigenetic corepressors including Histone Dea- 1 mg for ChIP), HDAC1 (Diagenode, C15410053, 2 mg for ChIP), cetylases -1 and -2 (HDAC1 and -2) and Lysine Demethylase-5B HSC70 (clone B-6, Santa Cruz Biotechnology, sc-7298, 1:10,000 (KDM5B) to promoter regions of target genes (22, 23). Impor- for Western blotting), nonspecific IgG (ab46540, 1 mg for ChIP). tantly, multiple studies have demonstrated a putative tumor suppressor role for KLF10 in different types of cancer (24–26). Migration assay In this study, we demonstrate