NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells
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and suppresses PDAC initiation
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Isidoro Cobo,
1,2
Sumit Paliwal,
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Júlia Melià-Alomà,
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Ariadna Torres,
1,3
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Jaime Martínez-Villarreal,
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Fernando García,
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Irene Millán,
1,2
Natalia del Pozo,
1,2
Joo-
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Cheol Park,
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Ray J. MacDonald,
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Javier Muñoz,
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and Francisco X. Real
1-3
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Short title: NFIC in pancreatic homeostasis and cancer
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1
Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre-CNIO,
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Madrid, Spain
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2
CIBERONC, Madrid, Spain
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3
Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra,
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Barcelona, Spain
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4
Proteomics Unit, Spanish National Cancer Research Centre-CNIO, Madrid, Spain.
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ProteoRed - ISCIII
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Department of Oral Histology-Developmental Biology, School of Dentistry, Seoul
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National University, Seoul, Korea
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Department of Molecular Biology, University of Texas Southwestern Medical Center,
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Dallas, TX, USA
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Correspondence: Francisco X. Real
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Centro Nacional de Investigaciones Oncológicas-CNIO
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Melchor Fernández Almagro, 3
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28029-Madrid, Spain
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E-mail: preal@cnio.es
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Conflicts of interest: none to declare
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Funding: This work was supported, in part, by grants SAF2011-29530, SAF2015-70553-
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R, and RTI2018-101071-B-I00 from Ministerio de Ciencia, Innovación y Universidades
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(Madrid, Spain) (co-funded by the ERDF-EU) and RTICC from Instituto de Salud Carlos
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III (RD12/0036/0034) to FXR. IC was recipient of a Beca de Formación del Personal
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Investigador from Ministerio de Economía y Competitividad (Madrid, Spain). The
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research leading to these results has received funding from People Programme (Marie
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint
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Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-
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2013) (REA grant agreement n° 608765”). SP was supported by a Juan de la Cierva
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Programme fellowship from Ministerio de Ciencia, Innovación y Universidades. IM was
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supported by a Fellowship from Fundació Bancaria La Caixa (ID 100010434) (grant
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number LCF/BQ/ES18/11670009). CNIO is supported by Ministerio de Ciencia,
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Innovación y Universidades as a Centro de Excelencia Severo Ochoa SEV-2015-0510.
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Statement of author contributions
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IC: study concept and design; acquisition of data; analysis and interpretation of data;
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statistical analysis; drafting of the manuscript;
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SP: acquisition of data; analysis and interpretation of data; drafting of the manuscript
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JMA: acquisition of data; analysis and interpretation of data; drafting of the manuscript
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AT: acquisition of data; analysis and interpretation of data;
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JMV: analysis and interpretation of data;
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FG: acquisition of data; analysis and interpretation of data;
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IM: analysis and interpretation of data;
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NdP: technical support and acquisition of data;
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JCP: material support;
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RJM: critical revision of the data and important intellectual content;
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JM: acquisition of data; analysis and interpretation of data;
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FXR: study concept and design; analysis and interpretation of data; drafting of the
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manuscript; overall study supervision; obtained funding.
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All authors provided input about manuscript content.
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Accession numbers: RNA sequencing data have been deposited in GEO with
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accession number GSE126907 and NFIC ChIP sequencing data have been deposited
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in GEO with accession number GSE181098
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint
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ABSTRACT
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Tissue-specific differentiation is driven by specialized transcriptional networks.
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Pancreatic acinar cells crucially rely on the PTF1 complex, and on additional
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transcription factors, to deploy their transcriptional program. Here, we identify NFIC as a
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novel regulator of acinar differentiation using a variety of methodological strategies. NFIC
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binding sites are found at very short distances from NR5A2-bound genomic regions and
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both proteins co-occur in the same complex. Nfic knockout mice show reduced
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expression of acinar genes and, in ChIP-seq experiments, NFIC binds the promoters of
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acinar genes. In addition, NFIC binds to the promoter of, and regulates, genes involved
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in RNA and protein metabolism; in Nfic knockout mice, p-RS6K1 and p-IEF4E are down-
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regulated indicating reduced activity of the mTOR pathway. In 266-6 acinar cells, NFIC
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dampens the ER stress program through its binding to ER stress gene promoters and is
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required for complete resolution of Tunicamycin-mediated ER stress. Normal human
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pancreata from subjects with low NFIC mRNA levels display reduced epxression of
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genes down-regulated in Nfic knockout mice. Consistently, NFIC displays reduced
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expression upon induced acute pancreatitis and is required for proper recovery after
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damage. Finally, expression of NFIC is lower in samples of mouse and human pancreatic
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ductal adenocarcinoma and Nfic knockout mice develop an increased number of mutant
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Kras-driven pre-neoplastic lesions.
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Word count: 211
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Keywords: NFIC, pancreas, acinar differentiation, ribosome, endoplasmic reticulum
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stress, unfolded protein response, transcriptional networks, pancreatitis, pancreatic
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cancer
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Abbreviations: ChIP, chromatin immunoprecipitation; DEG, differentially expressed
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genes; EMT, epithelial-mesenchymal transition; ER, endoplasmic reticulum; GSEA,
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Gene set enrichment analysis; IF, immunofluorescence; IHC; immunohistochemistry;
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PDAC, pancreatic ductal adenocarcinoma; TF, transcription factor; TM, tunicamycin;
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UPR, unfolded protein response.
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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INTRODUCTION
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Pancreatic acinar cells are highly specialized protein synthesis factories that
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have a well-developed rough endoplasmic reticulum (ER), a prominent Golgi complex,
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and abundant secretory granules
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. Acinar differentiation is contingent on the activity of a
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master regulator, the adult PTF1 complex, composed of the pancreas-specific
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transcription factors (TFs) PTF1A and RPBJL and the ubiquitous protein E47
2,3
. PTF1
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binds the proximal promoter of genes coding for digestive enzymes, secretory proteins
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and other TFs, and activates their expression. The PTF1 complex is the main driver of
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acinar differentiation but additional TF with tissue-restricted expression patterns are
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implicated in the fine-tuning of this process, including GATA6
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, MIST1
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, and
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NR5A2/LRH-1
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. Acinar cells play a crucial role in acute and chronic pancreatitis, two
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common and disabling conditions. Recent work using genetic mouse models has shown
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that, upon expression of mutant KRas, acinar cells can be the precursors of Pancreatic
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Intraepithelial Neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC)
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.
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Our laboratory and others have shown that the acinar differentiation program acts
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as a tumor suppressor in the pancreas. Monoallelic or homozygous inactivation of
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several acinar transcriptional regulators in the germline, the embryonic pancreas, or the
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adult pancreas can result in compromised acinar function that favors loss of cellular
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identity and poises acinar cells for transformation upon activation of mutant KRas
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.
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The tumor suppressive function of these TF is not obvious because the exocrine
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pancreas has a large functional reserve, i.e. massive alterations in cellular function need
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to occur in order to be reflected in histological or clinical changes.
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Here, we use bioinformatics tools to identify NFIC as a novel acinar regulator.
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NFIC is a member of the nuclear factor I family of TFs that regulate both ubiquitous and
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tissue-restricted genes
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. In the mammary gland, NFIC activates the expression of milk
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genes involved in lactation
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. Furthermore, it acts as a breast cancer tumor suppressor,
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as it directly represses the expression of Ccnd1 and Foxf1, a potent inducer of epithelial-
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mesenchymal transition (EMT), invasiveness, and tumorigenicity. Additional roles have
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been proposed through the regulation of Trp53
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. The physiological role of NFIC has
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been best studied in dentinogenesis, since Nfic
-/-
mice develop short molar roots and
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display aberrant odontoblast differentiation and dentin formation
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. NFIC regulates
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odontoblast-related genes, including Dssp
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, Wnt
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, and hedgehog signaling
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.
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint
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Using a combination of omics analyses and studies in knockout mice and cultured
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cells, we now uncover novel roles of NFIC as a regulator of acinar function whose major
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impact is at the level of the ER stress response in murine and human pancreas. Unlike
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most other TFs previously identified as required for full acinar function, NFIC belongs to
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a novel family of acinar regulators with tissue-wide expression. NFIC dysregulation
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sensitizes the pancreas to damage and neoplastic transformation.
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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