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Metastasis-Associated Protein 2 Represses NF-Ƙb to Reduce Lung Tumor Growth And

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Metastasis-Associated Protein 2 Represses NF-Ƙb to Reduce Lung Tumor Growth And Author Manuscript Published OnlineFirst on August 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1158 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Metastasis-associated protein 2 represses NF-ƙB to reduce lung tumor growth and 2 inflammation 3 Nefertiti El-Nikhely1#, Annika Karger1, Poonam Sarode1, Indrabahadur Singh1§, Andreas 4 Weigert2, Astrid Wietelmann1, Thorsten Stiewe3, Reinhard Dammann4, Ludger Fink5, 5 Friedrich Grimminger6, Guillermo Barreto1,7, Werner Seeger1,6,8, Soni S. Pullamsetti1,6, Ulf R. 6 Rapp1, Rajkumar Savai1,6,8* 7 Affiliations: 1Max Planck Institute for Heart and Lung Research, Member of the German 8 Center for Lung Research (DZL), Member of the Cardio-Pulmonary Institute (CPI), Bad 9 Nauheim, Germany; 2Institute of Biochemistry I, Goethe University Frankfurt, Frankfurt, 10 Germany; 3Institute of Molecular Oncology, Member of the DZL, Philipps-University 11 Marburg, Marburg, Germany; 4Institute for Genetics; member of the German Center for 12 Lung Research (DZL), Justus-Liebig-University, Giessen, Germany; 5Institute of Pathology and 13 Cytology, UEGP, Wetzlar, Germany; 6Department of Internal Medicine, Member of the DZL, 14 Member of CPI, Justus Liebig University, Giessen, Germany; 7Brain and Lung Epigenetics 15 (BLUE), Glycobiology, Cell Growth and Tissue Repair Research Unit (Gly-CRRET), Université 16 Paris-Est Créteil (UPEC), Créteil, France; 8Institute for Lung Health (ILH), Justus Liebig 17 University, Giessen, Germany; #present address: Department of Biotechnology, Institute of 18 Graduate Studies and Research, Alexandria University, Egypt; §present address: Emmy 19 Noether Research Group Epigenetic Machineries and Cancer, Division of Chronic 20 Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany. 21 Running title: MTA2/NuRD complex in lung cancer 22 Keywords: cRaf/Inflammation/ Lung cancer/ MTA2/ NF-ƙB/ NuRD 1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1158 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 23 *Correspondence: PD Dr. Rajkumar Savai, Molecular Mechanisms in Lung Cancer, 24 Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung 25 Research, Parkstrasse 1, D-61231 Bad Nauheim, Germany, Tel: +49 6032 705 420; Fax: +49 26 6032 705 471, Email: [email protected] 27 Financial support: R. Savai, S.S. Pullamsetti, W. Seeger and U.R. Rapp supported by the Max 28 Planck Society. R. Savai, S.S. Pullamsetti, T. Stiewe, F. Grimminger, R. Dammann, G. Barreto, 29 W. Seeger and U.R. Rapp supported by the UGMLC and Loewe Program. U.R. Rapp received 30 a grant from German Cancer Aid (Deutsche Krebshilfe; 109081). R. Savai and S.S. Pullamsetti 31 received Von-Behring-Röntgen-Stiftung (projects 66-LV06 and 63-LV01). R. Savai, S.S. 32 Pullamsetti, W. Seeger and F. Grimminger supported by Cardio-Pulmonary Institute (CPI). R. 33 Savai, S.S. Pullamsetti, T. Stiewe, R. Dammann, W. Seeger and F. Grimminger supported by 34 the German Center for Lung Research (DZL). R. Savai and S.S. Pullamsetti supported by the 35 German Research Foundation (DFG) by the CRC1213 (Collaborative Research Center 1213), 36 projects A01 (S.S. Pullamsetti), A05 (S.S. Pullamsetti) A10* (R. Savai). S.S. Pullamsetti 37 received European Research Council (ERC) Consolidator Grant (866051). 38 Author contributions: R.S., U.R.R, W.S. F.G. planned and initiated the project, and 39 supervised the entire project; N.E., S.S.P., I.S., P.S., G.B., R.S. designed experiments and 40 analyzed the data; N.E., A.H.K performed MTA2 cell culture experiments; P.S. and N.E. 41 performed ChIP experiments and Western blots; N.E., R.S., SSP., wrote the manuscript; T.S., 42 R.D., provided important constructs and reagents; N.E., As. W. performed MRI analysis. An. 43 W. performed FACS analysis. N.E., I.S., G.B. performed cell fractionation experiments and 44 analyzed data. 45 Competing financial interests: The authors declare no competing financial interests. 2 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1158 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 46 Supplementary data for this article are available at Cancer Research Online 45 47 (http://cancerres.aacrjournals.org/). 48 Abstract 49 Although NF-ƙB is known to play a pivotal role in lung cancer, contributing to tumor growth, 50 microenvironmental changes, and metastasis, the epigenetic regulation of NF-ƙB in tumor 51 context is largely unknown. Here we report that the IKK2/NF-ƙB signaling pathway 52 modulates metastasis-associated protein 2 (MTA2), a component of the nucleosome 53 remodeling and deacetylase complex (NuRD). In triple transgenic mice, downregulation of 54 IKK2 (Sftpc-cRaf-IKK2DN) in cRaf-induced tumors in alveolar epithelial type-II cells restricted 55 tumor formation, whereas activation of IKK2 (Sftpc-cRaf-IKK2CA) supported tumor growth; 56 both effects were accompanied by altered expression of MTA2. Further studies employing 57 genetic inhibition of MTA2 suggested that in primary tumor growth, independent of IKK2, 58 MTA2/NuRD corepressor complex negatively regulates NF-ƙB signaling and tumor growth, 59 while later dissociation of MTA2/NuRD complex from the promoter of NF-ƙB target genes 60 and IKK2-dependent positive regulation of MTA2 leads to activation of NF-ƙB signaling, 61 epithelial mesenchymal transition, and lung tumor metastasis. These findings reveal a 62 previously unrecognized biphasic role of MTA2 in IKK2/NF-ƙB-driven primary-to-metastatic 63 lung tumor progression. Addressing the interaction between MTA2 and NF-ƙB would provide 64 potential targets for intervention of tumor growth and metastasis. 65 Statement of significance 66 Findings strongly suggest a prominent role of MTA2 in primary tumor growth, lung 67 metastasis, and NF-ƙB signaling modulatory functions 68 3 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1158 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 69 70 Introduction 71 Lung cancer is a multifaceted disease in which inflammation plays an important role in 72 tumor progression. Mutation of KRas occurs in 30% lung adenocarcinoma cases. Blasco et al. 73 using KRas+/G12V mice concluded that cRaf expression was found to be essential for 74 mediating KRas signaling and tumor formation. Several subsequent studies revealed a 75 potential role for cRaf in lung development and carcinogenesis (1,2). 76 Previous studies have shown that cRaf activates not only the mitogenic cascade but also NF- 77 ƙB signaling either via an autocrine loop or even more direct through MEKK1 (3). NF-ƙB 78 transcription factor dependent signaling demonstrated as a key player in diverse immune 79 and inflammatory responses and regulates a wide range of genes (4,5), many of which play 80 roles in neoplastic transformation. Indeed, aberrant NF-ƙB expression and activity are 81 observed in many cancers (6). As to the NF-ƙB signaling, in unstimulated cells, the NF- 82 ƙB1/RelA (p50/p65)– heterodimers are sequestered in the cytoplasm by a family of 83 inhibitors called IƙBs (Inhibitor of ƙB). Whereas, upon stimulation, activation of the NF-ƙB is 84 initiated by the signal-induced degradation of IƙB proteins, which occurs primarily via 85 activation of a kinase called the IƙB kinase (IKK). IKK is composed of a heterodimer of the 86 catalytic IKKα and IKKβ (also known as IKK1 and IKK2, respectively) subunits and a regulatory 87 protein NEMO (NF-ƙB essential modulator) or IKKγ (7). Several studies have attempted the 88 use of selective IKK2 inhibitors to modulate NF-ƙB activity since targeting NF-ƙB sensitizes 89 cancer cells to chemotherapy in non-small-cell lung carcinoma (NSCLC) (8) and breast cancer 90 (9). In support of these data, Bay11-7085, a selective IKK2 inhibitor, increases the 4 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 14, 2020; DOI: 10.1158/0008-5472.CAN-20-1158 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 91 therapeutic effectiveness of bortezomib, a proteasome inhibitor, for ovarian cancer (10). 92 However, these attempts failed to reach final clinical trials owing to their off-target effects. 93 These studies emphasize the importance of acquiring more insight into the regulation of NF- 94 ƙB. Increasing evidence has shown that NF-ƙB is regulated epigenetically, especially in the 95 context of tumorigenesis. For example, members of NF-ƙB family can, themselves, act as 96 epigenetic regulators of NF-ƙB–dependent gene activation; the homodimer of p50 interacts 97 with HDAC1 to inhibit pro-inflammatory genes (11). This inhibition has also been reported 98 with respect to lipopolysaccharide (LPS) tolerance (12). A study concerning acute myeloid 99 leukemia suggests that C/EBP displaces HDACs from p50 homodimers and thus drives the 100 activation of anti-apoptotic genes (13). Another mechanism for epigenetic regulation is via 101 diverse chromosome regulatory machinery such as the nucleosome and remodelling 102 deacetylase complex (NuRD) complex. The NuRD complex was first discovered in 1998 in 103 different species. NuRD is critical for chromatin assembly, transcription, and genomic 104 stability (14). Its main structural components are metastasis-associated proteins 1/2 105 (MTA1/2), retinoblastoma protein associated protein 46/48 (RbAp46/48); its enzymatic 106 components are chromodomain helicase DNA binding protein 3/4 (CHD3/4) and HDAC1/2.
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