ADNP Is a Therapeutically Inducible Repressor of WNT Signaling in Colorectal Cancer

This article is featured in Highlights of This Issue, p. 2607

Colorectal cancer is a major cause of cancer morbidity and mortality, ranking third in cancer incidence among men and women (1). Most colorectal cancers are initiated by mutations in APC or β-Catenin that lead to overactivation of the WNT signaling pathway in these tumors (2). Despite this mutational activation, WNT signaling in colorectal cancer remains regulated on high levels, resulting in distinct tumor cell subpopulations with relatively low or high WNT activity (3). Colon cancer cell subpopulations with high WNT levels were attributed certain characteristics such as more mesenchymal phenotypes and putative cancer stem cell traits, express markers that are linked to tumor invasion, and therefore are thought to be crucial drivers of colon cancer progression (4, 5). These tumor cells are typically located at the infiltrative tumor edge where they invade the surrounding tissue, while those with lower WNT levels are frequently more central within the tumor and appear phenotypically more differentiated (6, 7). Because of these findings, high WNT signaling activity is assumed to be a driving force of colon cancer invasion and progression, making it an attractive potential target for therapeutic intervention. However, because WNT signaling is required for various physiologic processes including adult tissue and stem cell homeostasis, efforts in targeting this central pathway in clinical settings is complicated, and serious side effects may be anticipated (8).

Activity-dependent neuroprotector homeobox (ADNP) was initially identified in brain tissue and encodes a ubiquitously expressed zinc finger homeobox protein with transcription factor activity (9, 10). Most knowledge on ADNP function is related to the central nervous system where it is required for brain formation 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 genes involved in regulation of transcription, organogenesis, and neurogenesis, and is suggested to interact with chromatin-remodeling complexes that are associated with cellular differentiation (13). In regard to cancer, a previous report demonstrated overexpression of ADNP in proliferative tissues and several different malignancies, including colon cancer, and since ADNP depletion reduced the viability of certain cancer cells, it suggested a possible association with tumorigenesis and cell survival (10). However, the contribution of ADNP to human cancer and its functional role in malignancies is still poorly understood.

Focusing on transcription factors linked to WNT signaling, we here identify ADNP as a negative regulator of WNT in colorectal cancer and reveal its role as a tumor suppressor with effects on tumor cell proliferation, migration, invasion, as well as on tumor growth. Furthermore, we demonstrate that ADNP can be therapeutically induced in colon cancer in vivo, slowing tumor growth and progression, and highlight its potential as a prognostic predictor in human patients with colon cancer.

Three sets of differentially expressed genes from colon cancer cells with low and high WNT activity were screened for consistently deregulated genes (3, 5). From The Cancer Genome Atlas (TCGA) database (https://gdc.cancer.gov/), RNA-Seq data of 41 normal mucosa samples and 457 colon cancer samples were retrieved. Within the cancer sample data, Pearson correlations of ADNP expression and expression of 20,531 genes within this dataset were calculated and genes were ranked accordingly. GSEA analyses were conducted using this ranked gene list against curated sets of upregulated WNT targets derived from Nusse and colleagues (web.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes) and Herbst and colleagues (14). Gene sets are listed in Supplementary Table S1. Heatmaps for individual factors were drawn with GENE-E (Broad Institute).

HEK293 and HCT116 cell lines were obtained from ATCC, SW1222 from the Ludwig Institute for Cancer Research (New York, NY), and primary colon cancer cells from the Human Tissue and Cell Research Foundation. Cell lines were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Biochrom). For in vitro experiments, primary tumor cells were grown as spheroids as described previously (7). For WNT induction or inhibition, cells were treated with 20 ng/mL WNT3a (R&D Systems), 20 mmol/L LiCl, or 10 μmol/L XAV939 (both Sigma-Aldrich), respectively. In vitro ketamine treatment was done at concentrations of 200 μmol/L (Ratiopharm). Transfections and transductions for ADNP knockdown, overexpression, CRISPR/Cas9 genome editing, and luciferase assays are described in Supplementary Materials and Methods.

RNA was isolated from cell lines using TRIzol (Invitrogen) and used for quantitative real-time PCR, as described previously (3), using the primers listed in Supplementary Table S2. For RNA-Seq, libraries were constructed using the Encore Complete RNA-Seq library system (NuGEN) according to manufacturer's protocol, sequencing was done on a HiSeq 1500 instrument, and processed data were mapped to the hg19 human reference genome. Significantly upregulated genes were characterized according to signaling pathways using PANTHER version 10.0 (www.pantherdb.org). Expression data are accessible through GEO (GSE79395).

Immunoblotting of whole-cell lysates of colon cancer cells was done as described previously (3). Antibodies are listed in Supplementary 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 reduction, alkylation, and trypsinization. For separation, an EASY-nLC 1000 chromatography system connected to an Orbitrap XL instrument (Thermo Scientific) was used. Raw data files were processed with the Homo sapiens subset of the UniProt database and MaxQuant V1.5.1. Proteins with log2 fold changes of ±0.6 at P values < 0.05 were considered relevant. Detailed methods for RNA-Seq and MS are described in Supplementary Materials and Methods.

To assess cell proliferation, 5 × 10⁴ cells per well were seeded on conductive microtiter plates (E-Plate 16) and monitored for up to 150 hours using an xCELLigence DP instrument (ACEA Bioscience). For transwell migration and invasion assays, 8 μm ThinCert cell culture inserts (Greiner Bio-One) were used, which for invasion were coated with 100 μL of 1 mg/mL growth factor–depleted Matrigel (Corning). A total of 1 × 10⁵ cells/well were seeded in serum-free medium in the upper insert chambers and after 24 hours DMEM with 10% FBS was added to the bottom chambers of the inserts. For HCT116 and SW1222 cells, inserts were removed after 1 or 3 days for migration, and 3 or 5 days for invasion, respectively. Cells were fixed, stained with crystal violet, residual cells from the top chamber were removed, and photomicrographs were taken. For quantification, staining was dissolved in 250 μL of 30% acetic acid, and absorbance was measured at 590 nm on a Varioskan instrument (Thermo Scientific).

Mouse experiments were reviewed and approved by the Regierung von Oberbayern. To determine effects of ADNP depletion, 8 × 10⁶ SW1222 ADNP knockdown or control cells were suspended in 200 μL of a 1:1 mixture of PBS and growth factor–depleted Matrigel, and injected subcutaneously into age and gender matched 6- to 8-week-old NOD/SCID mice (Jackson Laboratory). Tumor growth was measured over time using calipers. Matched mice carrying ADNP knockdown and control xenografts were sacrificed when knockdown tumors reached volumes of 500–900 mm³. For in vivo treatment studies, subcutaneous xenografts were grown as described above using SW1222, HCT116, or primary colon cancer cells. Mice were randomly assigned to control or treatment groups when tumor volumes reached 100 mm³. Ketamine (20 mg/kg in PBS) or as control PBS were administered daily intraperitoneally until tumors reached volumes of 1,000–1,300 mm³.

IHC was done on 5-μm tissue sections, as described previously (7), using the antibodies listed in Supplementary Table S3. Scoring of ADNP in colon cancer cases was done in a manner blinded for clinical outcome, and cases were classified semiquantitatively for overall ANDP expression intensity, ranging from absent or barely detectable to strong overexpression. Scoring for nuclear β-Catenin was done as described previously (3). Staining intensities of ADNP in tumor cells with low or high β-Catenin were quantified with ImageJ (NIH, Bethesda, MD).

For immunofluorescence, cultured cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% TritonX100 for 15 minutes, blocked with 3% BSA in PBS for 30 minutes, and then incubated for 60 min at room temperature with ADNP Ab (1:50), AXIN2 Ab (1:100), or DNMT1 Ab (1:100). Secondary Alexa Fluor 488- or 555–conjugated antibodies (Invitrogen) were used for visualization and nuclei were counterstained with DAPI (Vector Laboratories). Confocal fluorescence images were taken on a LSM 700 laser scanning microscope using the ZEN software (Carl Zeiss).

Resection specimens of patients diagnosed with FAP as well as colorectal cancer specimens from patients that underwent intentionally curative surgical resection between 1994 and 2006 at the LMU were drawn from the archives of the institute of pathology. For colorectal cancer specimens, inclusion criteria were localized colorectal adenocarcinomas with absence of nodal (N0) or distant metastasis (M0) at the time of diagnosis (UICC stage I and II). Follow-up data were recorded prospectively by the Munich Cancer Registry. Specimens and data were anonymized, and the need for consent was waived by the Institutional Ethics Committee of the Medical Faculty of the LMU. Colorectal cancer tissues were assembled into tissue microarrays (TMA) with representative 1-mm cores, including tumor edges and tumor centers of each case. The final colorectal cancer collection consisted of 221 cases of which in 43 cases (19%), patients had died from their tumor within the follow-up period. For tumor-specific survival analysis, colorectal cancer–attributed deaths were defined as clinical endpoints. For analysis of disease-free survival, tumor progression after surgical resection was the clinical endpoint, documented as either tumor recurrence or metastasis. Survival was analyzed by the Kaplan–Meier method and groups were compared with the log-rank test. Cox proportional hazards model was used for multivariate analysis. Statistics were calculated using SPSS (IBM).

To identify transcription factors that are linked to WNT signaling in colon cancer, we comparatively analyzed three previously published gene expression profiles of colon cancer cell subpopulations with low and high WNT activity (3, 5). Among the few genes that were consistently upregulated in tumor cells with high WNT signaling, we identified ADNP as the only overexpressed gene encoding a transcription factor (Supplementary Table S4). Direct comparison confirmed that increased ADNP expression coincided with high WNT target gene expression and, conversely, with repression of genes linked to tumor cell differentiation (Fig. 1A). We then analyzed an independent data set of 41 normal mucosa samples and 457 colon cancers from The Cancer Genome Atlas (TCGA) and found that ADNP mRNA expression on average was 2.73-fold increased in colon cancer compared to normal mucosa (Supplementary Table S4). Gene Set Enrichment Analyses (GSEA) revealed that ADNP in colon cancer samples strongly correlated with genes enriched for WNT target gene signatures (Fig. 1B). On the protein level, ADNP was overexpressed at the infiltrative tumor edge of colorectal cancers, where it colocalized with strong nuclear β-Catenin expression, a marker for colon cancer cells with high WNT activity (Fig. 1C; Supplementary Fig. S1A and S1B). Moreover, in adenomas of patients with FAP (n = 23 adenomas), known to carry APC gene mutations (15), ADNP was overexpressed when compared with normal mucosa (Fig. 1C). In addition, even in normal mucosa, epithelial cells at the crypt base, where WNT activity is increased (16), showed slightly increased ADNP expression (Supplementary Fig. S1C). These findings demonstrated consistent upregulation of ADNP in colorectal cancer cells and precursor lesions with high WNT activity on mRNA and protein levels, and suggested a possible regulation of ADNP by WNT.

To test whether ADNP expression directly responds to WNT, we stimulated WNT signaling in HEK293 cells by the GSK3β inhibitor lithium chloride (LiCl) or by WNT3a, and then evaluated ADNP mRNA and protein levels. Surprisingly, although TOPflash luciferase assays and overexpression of β-Catenin and AXIN2 indicated strong induction of WNT signaling, there were no significant effects on ADNP expression (Fig. 1D and E). Also, suppressing WNT activity by silencing β-Catenin in colorectal cancer cell lines had no significant impact on ADNP protein levels (Fig. 1F). Hence, although ADNP expression strongly coincided with WNT signaling activity in colon cancer, it apparently is no direct WNT target gene.

To obtain initial insights into ADNP function in colorectal cancer, we next analyzed the effects of ADNP knockdown on gene expression in HCT116 colon cancer cells using RNA-seq. Depletion of ADNP by siRNA caused 1.4-fold or more deregulation of 4.82% of the transcriptome. Interestingly, upregulated genes were significantly more frequent than downregulated genes (3.69% vs. 1.13%, P < 0.0001), suggesting predominantly repressive functions of ADNP on the transcriptome (Fig. 2A). To identify potential targets of ADNP repression, we therefore focused on genes that were upregulated by ADNP knockdown and screened them for functional and pathway associations using the PANTHER analysis tool. Surprisingly, these analyses revealed that WNT signaling was the pathway most prominently related to genes affected by ADNP depletion (Fig. 2B). We therefore compared our gene expression dataset with WNT target gene signatures using GSEA, and found that indeed upregulated WNT target genes were significantly enriched upon ADNP depletion (Fig. 2C), with overexpression of typical WNT targets such as DNMT1, CD44, AXIN2, and TCF7 (Fig. 2D). These findings suggested repression of WNT signaling by ADNP.

Next, to determine effects on the proteome level, we used mass spectrometry analysis and identified deregulated proteins after ADNP silencing. Consistent with our transcriptome data, we found that upregulated proteins were significantly more abundant than downregulated proteins (2.8% vs. 1.43%, P < 0.0001, Fig. 2E). Furthermore strong correlation between our transcriptome and proteome results among 58 factors that were significantly deregulated in both datasets indicated consistency within these analyses (Pearson r² = 0.52, P = 0.001, data not shown). Among proteins that showed most significant upregulation were the WNT target DNMT1, a known driver of cell proliferation and interaction partner of β-Catenin, as well as TALIN-1, a recently identified key node of WNT signaling with roles in cell migration, invasion, and angiogenesis of human cancers (Fig. 2F; Supplementary Table S5; refs.17, 18).

We further addressed the effects of ADNP on WNT signaling in HCT116 and SW1222 colon cancer cells in vitro. In both cell lines, ADNP silencing increased expression of active-β-Catenin and the WNT targets DNMT1, AXIN2, and LEF-1 (Fig. 3A). Immunofluorescence of HCT116 cells confirmed these results with cytoplasmic and membranous, or nuclear increase of AXIN2 and DNMT1, respectively (Fig. 3B). In addition, upon ADNP silencing, both cell lines showed elevated WNT activity when assessed by TOPflash luciferase reporter assays (Fig. 3C). In contrast, overexpression of ADNP reduced β-Catenin and LEF-1 (Fig. 3D), and repressed WNT signaling in TOPflash assays (Fig. 3E). These results further corroborated the hypothesis that ADNP negatively regulates WNT signaling in colorectal cancer with repression of factors related to cell proliferation and other malignant traits of colorectal cancer cells.

Because WNT and its associated factors DNMT1 and TALIN-1 are known to regulate proliferation, migration, and invasion of colorectal cancer (17, 18), we tested the functional relevance of ADNP loss for these malignant traits in HCT116 and SW1222 colon cancer cells. We depleted ADNP by two different shRNAs and also generated ADNP knockout cells by CRISPR/Cas9 genome editing (Fig. 4A; Supplementary Fig. S2A). Importantly, ADNP depletion or knockout caused dramatic increases in Transwell cell migration and invasion of both colon cancer cell lines as determined by Boyden chamber assays (Fig. 4B and C; Supplementary Fig. S2C). In addition, ADNP depletion or knockout significantly increased cell proliferation of both cell lines as determined by impedance measurements (Fig. 4D; Supplementary Fig. S2D). Of note, these effects could be counteracted in ADNP knockout cells by concomitant depletion of β-Catenin, DNMT1, or TALIN-1 (Supplementary Fig. S2B–S2D). Moreover, ADNP overexpression showed opposite effects on invasion and migration (Supplementary Fig. S3). To further assess effects in vivo, we then injected 8 × 10⁶ SW1222 cells with and without stable ADNP knockdown subcutaneously into flanks of NOD/SCID mice and measured tumor growth over time. In line with our in vitro data, tumor xenografts derived from SW1222 cells with ADNP knockdown grew significantly faster, yielding larger tumors, with darker color due to increased intratumoral hemorrhage, when compared with tumor xenografts with normal ADNP levels (Fig. 4E). Immunohistochemical staining of these tumors for Ki67 revealed that proliferation was increased upon ADNP knockdown in vivo, explaining these observed differences in tumor growth and size (Fig. 4F). Taken together, these data showed strong effects of ADNP depletion on migration, invasion, and proliferation of colon cancer cells in vitro as well as tumor growth in vivo, and suggested a tumor suppressor function of ADNP through repression of WNT signaling in colorectal cancer.

Previous studies indicated that ADNP expression can be pharmacologically induced by subnarcotic doses of ketamine in cortical neurons (19, 20). Exploiting this potential, we treated colon cancer cell lines and primary colon cancer cells, which had high endogenous WNT activity (Supplementary Fig. S4), with low-dose ketamine and observed slight but steady increases of ADNP protein levels by immunoblotting (Fig. 5A). To determine the effects of ketamine-induced ADNP induction on WNT signaling, we then subjected these cell lines and primary colon cancer cells to TOPflash luciferase assays and indeed observed significant reductions in WNT activity under ketamine treatment (Fig. 5B). Importantly, these relative repressive effects of ketamine on WNT activity were decreased in ADNP-knockout cell lines, while the effects of a direct WNT inhibitor (XAV939) remained unchanged suggesting that WNT repression by ketamine in part depended on ADNP induction (Supplementary Fig. S5A and S5B). In addition, similar to the effects of ADNP overexpression, ketamine reduced migration and invasion of colon cancer cells (Supplementary Fig. S5C). Next, we treated NOD/SCID mice bearing colon cancer cell line or primary colon cancer xenografts with subnarcotic doses of ketamine and observed tumor growth over time. Ketamine treatment significantly slowed tumor growth (Fig. 5C) and prolonged tumor survival (Fig. 5D). These findings implicate that through induction of ADNP and WNT repression, subnarcotic doses of ketamine can inhibit colorectal cancer growth and tumor progression in vivo.

Finally, we tested for ADNP expression and disease outcome of patients with colorectal cancer. Using IHC, we scored overall ADNP expression in a collection of 221 stage I and II human colorectal cancers with recorded clinical follow-up data. ADNP expression varied substantially between cases, ranging from negative or barely detectable (score 0) to strong expression (score 3; Fig. 6A; Supplementary Table S6). Using Kaplan–Meier statistics, we found that differential ADNP expression was strongly linked to cancer-specific survival. All patients with strong ADNP expression completely survived their follow-up period (score 3, 5-year survival rate 100%). In contrast, moderate (score 2, 5-year survival rate 88.7%), weak (score 1, 5-year survival rate 78.2%), and barely detectable or negative ADNP expression (score 0, 5-year survival rate 60.4%) were significantly linked to cancer-specific deaths at increasing frequencies (Fig. 6B). Testing for disease-free survival yielded comparable, yet slightly less stark results (Fig. 6B). We then categorized ADNP expression into low (scores 0 and 1) and high (scores 2 and 3), and tested for an overall association with β-Catenin expression levels (Supplementary Fig. S6). Although not significant, high ADNP expression tended to be more frequent in cases with high nuclear β-Catenin (Supplementary Table S6). Interestingly however, high and low ADNP expression separated survival probabilities particularly well in the subset of colorectal cancer cases with high nuclear β-Catenin expression (Fig. 6C). We then evaluated co-occurrences of ADNP expression and other clinical/pathologic variables and found that T-stages were significantly associated with different ADNP expression scores, with a tendency of lower T-stages linked to higher ADNP expression. Also, low tumor grade tended to associate with low ADNP expression, while the other core clinical variables age and sex were not linked to ADNP (Supplementary Table S6). Including these variables and β-Catenin expression levels into proportional hazards regression analyses revealed that ADNP expression was an independent predictor of favorable outcome (Supplementary Table S7). Collectively, high ADNP expression was a marker of good prognosis in patients with colorectal cancer, which is in agreement with its tumor-suppressive function in this malignancy.

Constitutive activation of WNT signaling by inactivating mutations in APC is hallmark of most colorectal cancers and drives tumor progression via target genes that promote cell proliferation, invasion, and spawn putative cancer stem cell traits (2, 4, 5). Therefore, identifying effectors or regulators of WNT signaling, which are involved in governing these malignant traits, may hold keys for a deeper understanding of colon cancer biology, and the development of more effective therapies (21). In this context, we here identified upregulation of the transcription factor ADNP in colon cancer cells with high WNT signaling activity. Although differential expression of ADNP in colon cancer cell subpopulations with high and low WNT activity was relatively small, we demonstrate high consistency of this finding on mRNA and protein levels. However, a direct regulation of ADNP through WNT remained unclear, as modulation of WNT signaling in colon cancer and other cells yielded no measurable effects on ADNP expression. How ADNP expression itself is regulated in colon cancer cells, therefore, still needs to be determined, keeping in mind that ADNP is known to engage in autoregulatory feedback loops, as others report (22).

Consistent upregulation of ADNP in colon cancer cells with high WNT activity prompted us to further investigate its functional relevance. Unexpectedly, ADNP depletion caused significant upregulation of WNT target genes, as assessed on multiple levels of the transcriptome, proteome, for individual factors, and in reporter assays, while, in line with these findings, overexpression of ADNP in colon cancer cells showed opposite effects. ADNP therefore exhibited a previously unknown function in suppressing WNT activity in colon cancer. Specifically, ADNP knockdown caused overexpression of WNT targets such as DNMT1 and the recently identified WNT signaling node TALIN-1, which are reported drivers of tumor cell proliferation, invasion, and migration, respectively (17, 18). Indeed, we found that these attributes of malignancy were strongly unleashed in colorectal cancer cells under ADNP knockdown or knockout in vitro, and that ADNP depletion strongly enhanced tumor growth in vivo. ADNP therefore acted as a tumor suppressor gene in colorectal cancer and our data suggest that this may mainly be transduced through WNT repression. Because of the strongest ADNP expression in tumor cells with high WNT activity, its function may thus partially mirror that of WNT feedback inhibitors such as AXIN2 (23). However, although these effects may be mediated through chromatin remodeling, since ADNP has been shown to interact with SWI/SNF complexes that also are known to impact on WNT signaling (13, 24), the exact mechanism of ADNP function, and the detailed dynamics of its WNT and tumor-suppressive effects in colon cancer yet remain to be determined. Moreover, since ADNP knockdown also upregulated genes of other pathways driving colon cancer progression, such as angiogenesis and EGFR signaling (25, 26), we hypothesize that tumor suppressor effects of ADNP may additionally transduce through other pathways than WNT.

ADNP can be pharmacologically induced in neurons by ketamine (19, 20) and our data demonstrate how this aspect may translate into a therapeutic approach for colorectal cancer. Treatment with subnarcotic ketamine induced ADNP, suppressed WNT signaling, inhibited migration and invasion of colon cancer cells, and significantly slowed tumor growth of colon cancer xenografts in vivo. Although the mechanism of ADNP induction by ketamine is currently unknown (19), we demonstrate that the effects of ketamine treatment were similar to those of ADNP overexpression, and decreased in ADNP knockout cells, suggesting that the tumor suppressive effects of ketamine in part depend on ADNP-mediated WNT repression. These findings are of specific interest when considering that transcription factors are usually difficult drug targets due to their binding promiscuity and the intrinsically disordered nature of their binding sites (27). Moreover, direct targeting of WNT signaling poses substantial challenges due to complexity of its signaling cascade and cross talk from various other signaling pathways (8, 28, 29). The benefit for patients with colorectal cancer with advanced disease by repressing WNT through ADNP induction with low-dose ketamine may quite easily be tested as an add-on to existing treatment regimens, as this substance and its pharmacologic characteristics are well studied (30). Of course, side effects of this treatment are to be carefully evaluated and strictly balanced with potential therapeutic benefits, especially since others reported adverse effects of ketamine for patients with other malignancies, such as breast cancer (31).

Although ADNP was consistently linked to nuclear β-Catenin expression within individual colorectal cancers, we were surprised to find only a weak and nonsignificant overall association of ADNP and nuclear β-Catenin expression in our tissue collection. We hypothesize that this may be due to different genetic backgrounds of colorectal cancers with differential influence of other signaling pathways on WNT activity that may not completely reflect in nuclear β-Catenin expression levels (2, 32). However, in support of the idea that ADNP functions as a tumor suppressor, we found that high ADNP expression predicted better outcomes for cancer and disease-free survival in human colorectal cancer, while this was independent of other core clinical variables. Because ADNP levels separated different outcomes particularly well in cases with high nuclear β-Catenin expression that have been associated with more aggressive behavior in some studies (33), ADNP may predominantly exhibit its protective role in this subset of colorectal cancer cases. As more than 50% of colorectal cancers progress and/or develop metastases during the course of the disease, markers predicting prognosis and individual risk may guide personalized therapy regimens (34). In this context, patients with low-stage colorectal cancer but loss of ADNP expression may benefit from increased clinical attention and intensified or adjuvant treatment protocols. In regard to ketamine treatment, since individual colon cancers showed significantly different expression levels of ADNP, it may be a useful biomarker in predicting therapy response, a hypothesis to be addressed in further preclinical and eventually clinical trials.

In conclusion, we here identified ADNP as a transcription factor that is overexpressed in colon cancer cells with high WNT signaling activity, counteracts WNT activity in these tumor cells, and exhibits tumor suppressor functions. These characteristics may be therapeutically exploited, since ADNP is inducible by low-dose ketamine treatment which reduces tumor growth in preclinical xenograft models in vivo. Moreover, in human colorectal cancer patients, ADNP predicts superior clinical outcome. We propose that these potentials of ADNP as a prognostic marker and therapeutic target may be considered in further trials to improve management and treatment options for patients with colorectal cancer.

T. Kirchner reports receiving speakers bureau honoraria from AstraZeneca and Merck. No potential conflicts of interest were disclosed by the other authors.

Conception and design: C. Blaj, D. Horst

Development of methodology: C. Blaj, D. Horst

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Blaj, A. Bringmann, E.M. Schmidt, M. Urbischek, S. Lamprecht, T. Fröhlich, G.J. Arnold, S. Krebs, H. Blum, H. Hermeking, A. Jung, T. Kirchner, D. Horst

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Blaj, E.M. Schmidt, M. Urbischek, S. Lamprecht, T. Fröhlich, G.J. Arnold, S. Krebs, A. Jung, T. Kirchner, D. Horst

Writing, review, and/or revision of the manuscript: C. Blaj, E.M. Schmidt, S. Lamprecht, T. Fröhlich, S. Krebs, H. Hermeking, A. Jung, T. Kirchner, D. Horst

Study supervision: D. Horst

We are grateful to Anne Küchler and Sabine Sagebiel-Kohler for experimental assistance, Jutta Engel for clinical follow-up data, Vivien Bubb for ADNP overexpression vectors, and the HTCR for primary colon cancer cells.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (HO4325/4-1 to D. Horst).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.