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LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway Priya S

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LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway Priya S Published OnlineFirst February 13, 2019; DOI: 10.1158/1541-7786.MCR-18-0971 Chromatin, Epigenetics, and RNA Regulation Molecular Cancer Research LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway Priya S. Dalvi1,2, Iris F. Macheleidt1,2, So-Young Lim1,2, Sonja Meemboor1,2, Marion Muller€ 1, Hannah Eischeid-Scholz1, Stephan C. Schaefer1,3,4, Reinhard Buettner1,2,3,4, Sebastian Klein1,5,6, and Margarete Odenthal1,2 Abstract Lysine-specific demethylase 1 (LSD1) is a histone modifier Mechanistically, LSD1 directly regulates expression of PLK1 by that is highly overexpressed in lung adenocarcinoma, which binding to its promoter region that subsequently affects results in aggressive tumor biology. Tumor cell proliferation expression of its downstream target genes. Notably, using lung and migration analysis after LSD1 inhibition in the lung adenocarcinoma TCGA datasets a significant correlation adenocarcinoma cell line PC9, using the LSD1 inhibitor between LSD1 and PLK1 along with its downstream targets HCI-2509 and siRNA, demonstrated that LSD1 activity was was observed. Furthermore, the LSD1/PLK1 linkage was con- essential for proliferation and migration capacities of tumor firmed by IHC analysis in a clinical lung adenocarcinoma cells. Moreover, reduced proliferation rates after LSD1 inhibi- cohort (n ¼ 43). Conclusively, this is the first study showing a tion were shown to be associated with a cell-cycle arrest of direct transcriptional link between LSD1 and PLK1. the tumor cells in the G2–M-phase. Expression profiling fol- lowed by functional classification and pathway analysis Implications: These findings point to a role of LSD1 indicated prominent repression of the polo-like kinase 1 in regulating PLK1 and thus efficient G2–M-transition– (PLK1) pathway upon LSD1 inhibition. In contrast, transient mediating proliferation of tumor cells and suggest targeting overexpression of exogenous PLK1 plasmid rescued the LSD1 the LSD1/PLK1 axis as a novel therapeutic approach for lung inhibition–mediated downregulation of PLK1 pathway genes. adenocarcinoma treatment. Introduction that significantly affects transcriptomic changes by demethylating histone tails. LSD1 belongs to the amine oxidase superfamily of Lung cancer is ranked first among cancer-related deaths world- proteins, and catalyzes FAD-dependent oxidative demethyla- wide (1). The most common subtype of non–small cell lung tion (8). Specifically, it demethylates mono/di-methylated his- cancer (NSCLC), lung adenocarcinoma, is associated with a poor tone 3 lysine 4 (K4) and lysine 9 (K9) (H3K4 and H3K9) residues, 5-year survival rate of less than 20% after diagnosis, and is defined thereby altering the epigenetic marks and inducing gene expres- as a disease with genetic and cellular heterogeneity (2, 3). Com- sion changes (8, 9). In addition, LSD1 also demethylates lysine prehensive molecular characterization of lung adenocarcinoma residues of nonhistone proteins like E2F1, TP53, and DNMT1, has revealed a huge complexity of not only genetic alterations but resulting in a change in their cellular properties (10, 11). LSD1 also epigenetic alterations (4, 5). Importantly, tumor progression performs these functions in association with different histone and development in lung adenocarcinoma has been shown to be modifying complexes and transcription coactivating or corepres- linked with epigenetic silencing of crucial tumor suppressor sing complexes (12). LSD1 is also crucial for the development genes (6, 7). and maintenance of normal tissue homeostasis and for stem Posttranslational modification of histone tails is a complex cell differentiation, and has been shown to be involved in mechanism regulating gene expression changes. Lysine-specific embryogenesis (13–15). demethylase 1 (LSD1/KDM1A) is one such epigenetic modifier Furthermore, high expression of LSD1 has been linked to poor prognosis in various cancer types, including breast cancer, hepa- 1Institute of Pathology, University Hospital of Cologne, Cologne, Germany. tocellular carcinoma, esophageal cancer, lung cancer, neuroblas- 2Center for Molecular Medicine Cologne, Cologne, Germany. 3Center for Inte- toma, as well as acute myeloid leukemia (13). As a result, many grated Oncology Cologne Bonn, Cologne, Germany. 4Lung Cancer Group compounds targeting LSD1 are being employed in preclinical 5 Cologne, University Hospital of Cologne, Cologne, Germany. Department of studies (16, 17). Among these inhibitors, HCI-2509 a reversible Translational Genomics, University of Cologne, Cologne, Germany. 6Else Kroner€ LSD1 inhibitor identified by extensive screening approaches has Forschungskolleg Cologne, University Hospital of Cologne, Cologne, Germany. been shown to be highly specific for LSD1 (18). This compound Note: Supplementary data for this article are available at Molecular Cancer has demonstrated to significantly inhibit tumor growth in pro- Research Online (http://mcr.aacrjournals.org/). state and Ewing sarcoma tumor cell lines and is expected to enter Corresponding Author: Margarete Odenthal, Institute of Pathology, University phase I clinical trials within the near future (19–21). Hospital of Cologne, Kerpener Str. 62, Cologne 50937, Germany. Phone: 221- In this study, we used HCI-2509 to disrupt LSD1 activity 478-6367; Fax: 221-478-6360; E-mail: [email protected] in lung adenocarcinoma cells. Inhibition of LSD1 reduced doi: 10.1158/1541-7786.MCR-18-0971 cell proliferation, downregulated major cell growth regulatory Ó2019 American Association for Cancer Research. pathways, and most predominantly led to downregulation of www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst February 13, 2019; DOI: 10.1158/1541-7786.MCR-18-0971 Dalvi et al. the polo-like kinase (PLK) mitotic pathway. We demonstrate (Applied Biosystems). GoTaq QPCR Master Mix (Promega) was that PLK1 is a direct transcriptional target of LSD1, and that used to perform real-time PCR. Primers used are listed in Sup- depletion of LSD1 activates G2–M arrest. Moreover, we show plementary File S1; Supplementary Table S2. The real-time PCR that LSD1 binds to the PLK1 promoter region and thereby was run on CFX96 Thermo Cycler (Bio-Rad) or Roche Lightcycler regulates its expression and tumor cell proliferation. In sum- 480 (Roche). mary, we propose that therapeutic targeting of LSD1 offers a mechanistic rationale for the treatment of lung adenocarcino- Microarray ma and other cancers that exhibit PLK1 addiction. Gene expression–profiling analysis was performed on untreat- ed PC9 cells and PC9 cells treated with the LSD1 inhibitor HCI- Materials and Methods 2509. RNA isolation from cells was performed using the Ambion- PureLink RNA Mini Kit (Life Technologies) following the man- Cell culture ufacturer's instructions. The Human Gene 2.0 ST Array Platform Information about all cell lines used in this study is listed in (Affymetrix) was used for performing the microarray analysis. Supplementary File S1; Supplementary Table S1. Cell lines were Significance parameters used: P < 0.05 and fold change > 1.5. All either cultured in DMEM or RPMI media, supplemented with details regarding the Gene Ontology and pathway analysis 10% FCS, in presence or absence of L-Glutamine. All cell cultures can be found in the Supplementary File S1; Supplementary were maintained in 5% CO2 at 37 C. Cells were passaged when Methods section. The microarray data generated are available in 80% confluency was reached. the Gene Expression Omnibus repository under accession num- Cell lines used in this study are not listed in the International ber GSE117702. Cell Line Authentication Committee's misidentified cell lines database. All cell lines used in the study were tested for Protein isolation and Western blot analysis Mycoplasma using Venor GeM OneStep Kit (Minerva Biolabs; Protein lysates were extracted from cells using the Pierce RIPA last test, November 2017). On an average all the cell lines used Buffer (Thermo Fisher Scientific) and blotted as described previ- were in between 15–40 passage number and were used for ously (23). For studying phosphorylated proteins by Western blot cellular assays after 5–8 passages postthawing. analysis, protein lysates were extracted from cells using Cell Lysis Buffer 10X (New England Biolabs) as per the manufacturer's Cell treatment with the LSD1 inhibitor instructions. The membranes were incubated overnight with 2 One day prior to treatment, cells were plated on 20 cm plates primary antibodies in blocking solution (5% milk powder in and then supplemented with 2 mmol/L HCI-2509 (Xcess Bio- PBST) at 4C using the antibodies listed in Supplementary File S1; sciences). For microarray analysis and the corresponding valida- Supplementary Table S3. Developed blots were imaged using tion experiments, and for the expression analysis in different ChemiDoc XRsþ System (Bio-Rad) and processed using the cancer lines, HCI-2509 treatments were performed for 48 hours, associated Image Lab software v 4.0. while for all other experiments it was for 72 hours. Cells were harvested after 48 or 72 hours for either protein or RNA isolation. Cell-cycle analysis CycletestPlusDNAKit(BDBiosciences) was used to deter- Transfection with silencing RNA and plasmids mine percentage of cells in different phases of the cell cycle Transfections were performed using control siRNA (SIC001; following the manufacturer's instructions. Cell-cycle measure- Sigma) and three different siRNA sequences
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