Published OnlineFirst November 30, 2016; DOI: 10.1158/1078-0432.CCR-16-1604 Cancer Therapy: Preclinical Clinical Cancer Research ADNP Is a Therapeutically Inducible Repressor of WNT Signaling in Colorectal Cancer Cristina Blaj1, Agnes Bringmann1, Eva Marina Schmidt1, Manuela Urbischek1, Sebastian Lamprecht1, Thomas Frohlich€ 2, Georg J. Arnold2, Stefan Krebs2, Helmut Blum2, Heiko Hermeking1,3,4, Andreas Jung1,3,4, Thomas Kirchner1,3,4, and David Horst1,3,4 Abstract Purpose: Constitutively active WNT signaling is a hallmark Results: ADNP was overexpressed in colon cancer cells with of colorectal cancers and a driver of malignant tumor high WNT activity, where it acted as a WNT repressor. Silencing progression. Therapeutic targeting of WNT signaling is dif- ADNP expression increased migration, invasion, and prolifera- ficult due to high pathway complexity and its role in tissue tion of colon cancer cells and accelerated tumor growth in homeostasis. Here, we identify the transcription factor xenografts in vivo. Treatment with subnarcotic doses of ketamine ADNP as a pharmacologically inducible repressor of WNT induced ADNP expression, significantly inhibited tumor growth, signaling in colon cancer. and prolonged survival of tumor-bearing animals. In human Experimental Design: We used transcriptomic, proteomic, patients with colon cancer, high ADNP expression was linked to and in situ analyses to identify ADNP expression in colo- good prognosis. rectal cancer and cell biology approaches to determine its Conclusions: Our findings indicate that ADNP is a tumor function. We induced ADNP expression in colon cancer suppressor and promising prognostic marker, and that ketamine xenografts by low-dose ketamine in vivo.Clinicalassocia- treatment with ADNP induction is a potential therapeutic approach tions were determined in a cohort of 221 human colorectal that may add benefit to current treatment protocols for patients cancer cases. with colorectal cancer. Clin Cancer Res; 23(11); 2769–80. Ó2016 AACR. Introduction tissue, while those with lower WNT levels are frequently more central within the tumor and appear phenotypically more differ- Colorectal cancer is a major cause of cancer morbidity and entiated (6, 7). Because of these findings, high WNT signaling mortality, ranking third in cancer incidence among men and activity is assumed to be a driving force of colon cancer invasion women (1). Most colorectal cancers are initiated by mutations and progression, making it an attractive potential target for in APC or b-Catenin that lead to overactivation of the WNT therapeutic intervention. However, because WNT signaling is signaling pathway in these tumors (2). Despite this mutational required for various physiologic processes including adult tissue activation, WNT signaling in colorectal cancer remains regulated and stem cell homeostasis, efforts in targeting this central pathway on high levels, resulting in distinct tumor cell subpopulations in clinical settings is complicated, and serious side effects may be with relatively low or high WNT activity (3). Colon cancer cell anticipated (8). subpopulations with high WNT levels were attributed certain Activity-dependent neuroprotector homeobox (ADNP) was characteristics such as more mesenchymal phenotypes and puta- initially identified in brain tissue and encodes a ubiquitously tive cancer stem cell traits, express markers that are linked to tumor expressed zinc finger homeobox protein with transcription factor invasion, and therefore are thought to be crucial drivers of colon activity (9, 10). Most knowledge on ADNP function is related to cancer progression (4, 5). These tumor cells are typically located at the central nervous system where it is required for brain formation the infiltrative tumor edge where they invade the surrounding and cranial neural tube closure (11). It also assumes protective roles against cognitive defects in neurodegenerative disease (12). Moreover, ADNP has been shown to reduce the expression of 1Pathologisches Institut, Ludwig-Maximilians-Universitat€ Munchen,€ Germany. genes involved in regulation of transcription, organogenesis, and 2Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Lud- neurogenesis, and is suggested to interact with chromatin-remo- 3 wig-Maximilians-Universitat€ Munchen,€ Germany. German Cancer Consortium deling complexes that are associated with cellular differentiation 4 (DKTK), Heidelberg, Germany. German Cancer Research Center (DKFZ), Hei- (13). In regard to cancer, a previous report demonstrated over- delberg, Germany. expression of ADNP in proliferative tissues and several different Note: Supplementary data for this article are available at Clinical Cancer malignancies, including colon cancer, and since ADNP depletion Research Online (http://clincancerres.aacrjournals.org/). reduced the viability of certain cancer cells, it suggested a possible Corresponding Author: David Horst, Pathologisches Institut der LMU, Thal- association with tumorigenesis and cell survival (10). However, € kirchner Str. 36, Munchen 80337, Germany. Phone: þ4989-2180-73611; Fax: the contribution of ADNP to human cancer and its functional role þ4989-2180-73727; E-mail: [email protected] in malignancies is still poorly understood. doi: 10.1158/1078-0432.CCR-16-1604 Focusing on transcription factors linked to WNT signaling, we Ó2016 American Association for Cancer Research. here identify ADNP as a negative regulator of WNT in colorectal www.aacrjournals.org 2769 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst November 30, 2016; DOI: 10.1158/1078-0432.CCR-16-1604 Blaj et al. reference genome. Significantly upregulated genes were char- Translational Relevance acterized according to signaling pathways using PANTHER High WNT signaling activity is the main driver of tumor version 10.0 (www.pantherdb.org). Expression data are acces- invasion and progression in colorectal cancer. Here, we iden- sible through GEO (GSE79395). tify ADNP as a negative regulator of WNT signaling that can be pharmacologically induced by treatment with low-dose keta- Immunoblotting and proteome analysis mine. WNT suppression through ADNP induction may Immunoblotting of whole-cell lysates of colon cancer cells was improve treatment of patients with colorectal cancer. done as described previously (3). Antibodies are listed in Sup- plementary Table S3. For mass spectrometry (MS) proteome analysis, cells were lysed, sonicated, and centrifuged through QIA-Shredder devices (Qiagen). Ten micrograms of total protein was used for further cancer and reveal its role as a tumor suppressor with effects on reduction, alkylation, and trypsinization. For separation, an tumor cell proliferation, migration, invasion, as well as on tumor EASY-nLC 1000 chromatography system connected to an Orbi- growth. Furthermore, we demonstrate that ADNP can be thera- trap XL instrument (Thermo Scientific) was used. Raw data files in vivo peutically induced in colon cancer , slowing tumor growth were processed with the Homo sapiens subset of the UniProt and progression, and highlight its potential as a prognostic database and MaxQuant V1.5.1. Proteins with log2 fold changes predictor in human patients with colon cancer. of Æ0.6 at P values < 0.05 were considered relevant. Detailed methods for RNA-Seq and MS are described in Supplementary Materials and Methods Materials and Methods. Gene expression datasets, TCGA data, and GSEA Three sets of differentially expressed genes from colon cancer Proliferation, migration, and invasion assays cells with low and high WNT activity were screened for consis- To assess cell proliferation, 5 Â 104 cells per well were seeded on tently deregulated genes (3, 5). From The Cancer Genome Atlas conductive microtiter plates (E-Plate 16) and monitored for up to (TCGA) database (https://gdc.cancer.gov/), RNA-Seq data of 41 150 hours using an xCELLigence DP instrument (ACEA Biosci- normal mucosa samples and 457 colon cancer samples were ence). For transwell migration and invasion assays, 8 mm ThinCert retrieved. Within the cancer sample data, Pearson correlations of cell culture inserts (Greiner Bio-One) were used, which for inva- ADNP expression and expression of 20,531 genes within this sion were coated with 100 mL of 1 mg/mL growth factor–depleted dataset were calculated and genes were ranked accordingly. GSEA Matrigel (Corning). A total of 1 Â 105 cells/well were seeded in analyses were conducted using this ranked gene list against serum-free medium in the upper insert chambers and after 24 curated sets of upregulated WNT targets derived from Nusse and hours DMEM with 10% FBS was added to the bottom chambers of colleagues (web.stanford.edu/group/nusselab/cgi-bin/wnt/tar- the inserts. For HCT116 and SW1222 cells, inserts were removed get_genes) and Herbst and colleagues (14). Gene sets are listed after 1 or 3 days for migration, and 3 or 5 days for invasion, in Supplementary Table S1. Heatmaps for individual factors were respectively. Cells were fixed, stained with crystal violet, residual drawn with GENE-E (Broad Institute). cells from the top chamber were removed, and photomicrographs were taken. For quantification, staining was dissolved in 250 mLof Cell culture and in vitro treatments 30% acetic acid, and absorbance was measured at 590 nm on a HEK293 and HCT116 cell lines were obtained from ATCC, Varioskan instrument (Thermo Scientific). SW1222 from the Ludwig Institute for Cancer Research (New