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Constitutively active WNT signaling is a hallmark of colorectal cancers and a driver of malignant tumor progression. Therapeutic targeting of WNT signaling is difficult due to high pathway complexity and its role in tissue homeostasis. Here, we identify the transcription factor ADNP as a pharmacologically inducible repressor of WNT signaling in colon cancer. We used transcriptomic, proteomic, and analyses to identify ADNP expression in colorectal cancer and cell biology approaches to determine its function. We induced ADNP expression in colon cancer xenografts by low-dose ketamine Clinical associations were determined in a cohort of 221 human colorectal cancer cases. ADNP was overexpressed in colon cancer cells with high WNT activity, where it acted as a WNT repressor. Silencing ADNP expression increased migration, invasion, and proliferation of colon cancer cells and accelerated tumor growth in xenografts Treatment with subnarcotic doses of ketamine induced ADNP expression, significantly inhibited tumor growth, and prolonged survival of tumor-bearing animals. In human patients with colon cancer, high ADNP expression was linked to good prognosis. Our findings indicate that ADNP is a tumor suppressor and promising prognostic marker, and that ketamine treatment with ADNP induction is a potential therapeutic approach that may add benefit to current treatment protocols for patients with colorectal cancer. .

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<div>Abstract<p><b>Purpose:</b> Constitutively active WNT signaling is a hallmark of colorectal cancers and a driver of malignant tumor progression. Therapeutic targeting of WNT signaling is difficult due to high pathway complexity and its role in tissue homeostasis. Here, we identify the transcription factor ADNP as a pharmacologically inducible repressor of WNT signaling in colon cancer.</p><p><b>Experimental Design:</b> We used transcriptomic, proteomic, and <i>in situ</i> analyses to identify ADNP expression in colorectal cancer and cell biology approaches to determine its function. We induced ADNP expression in colon cancer xenografts by low-dose ketamine <i>in vivo</i>. Clinical associations were determined in a cohort of 221 human colorectal cancer cases.</p><p><b>Results:</b> ADNP was overexpressed in colon cancer cells with high WNT activity, where it acted as a WNT repressor. Silencing ADNP expression increased migration, invasion, and proliferation of colon cancer cells and accelerated tumor growth in xenografts <i>in vivo</i>. Treatment with subnarcotic doses of ketamine induced ADNP expression, significantly inhibited tumor growth, and prolonged survival of tumor-bearing animals. In human patients with colon cancer, high ADNP expression was linked to good prognosis.</p><p><b>Conclusions:</b> Our findings indicate that ADNP is a tumor suppressor and promising prognostic marker, and that ketamine treatment with ADNP induction is a potential therapeutic approach that may add benefit to current treatment protocols for patients with colorectal cancer. <i>Clin Cancer Res; 23(11); 2769–80. ©2016 AACR</i>.</p></div>

<p>Suppl. Figure 1. (A) Immunostaining for ADNP and β-Catenin on serial sections of two colon cancers. (B) Staining intensities of ADNP in colon cancer cells (n = 500, 5 different tumors) with high or low nuclear β- Catenin expression. P value is t test result. (C) Immunostaining for ADNP in normal mucosa. Right panels aremagnifications of areas boxed in left panel.Scale bars, 100 μm. Suppl. Figure 2. (A-B) Immunoblotting of indicated proteins on whole cell lysates in ADNP wild-type (WT) or knockout (KO) cell lines. Numbers below immunoblots indicate fold change by densitometry. (C) Representative micrographs (left panels) and quantification (right panels) of migrated or invaded tumor cells in transwell assays for indicated ADNP WT and KO cell lines, treated with indicated siRNAs or control siRNA (siCtrl). (D) Representative proliferation kinetics based on cell quantification by impedance measurements. Data are mean, n {greater than or equal to} 3. β-Cat = β-Catenin. Suppl. Figure 3. Representative micrographs (left panels) and quantification (right panels) of migrated or invaded HCT116 or SW1222 tumor cells with transient ADNP overexpression by transfection with p4.2-hADNP compared to empty p4.2 vector for 24 h in transwell assays. Suppl. Figure 4. (A) Immunoblotting of indicated proteins on whole cell lysates of SW1222, HCT116 and primary (P-Tu) colon cancer cells. Numbers below immunoblots indicate fold change by densitometry. (B) Immunostaining of SW1222, HCT116 and primary colon cancer (P-Tu) xenografts for β-Catenin. Scale bars, 50 μm. Suppl. Figure 5. (A-B) Dual-luciferase assays for indicatedADNPwild-type (WT) or knockout (KO) colon cancer cells transfected with TOPflash reporter constructs. Treatments with PBS, ketamine or the WNT inhibitor XAV939, as indicated. (C) Representativemicrographs (left panels) and quantification (right panels) ofmigrated or invaded HCT116 or SW1222 tumor cells in transwell assays, treated with PBS or 100 μM ketamine for 24 h, as indicated.Data aremean {plus minus}SD, Pvalues are t test results, n {greater than or equal to} 3. Suppl. Figure 6. Assessment of nuclear β-catenin staining in primary human colorectal cancers. Tumors were assigned scores from 0 (no nuclear β-catenin) to 3 (most tumor cells with strong nuclear β-catenin) and accordingly categorized as β-catenin low (score 0-1) and β-catenin high (score 2-3). Scale bars, 100 μm.</p>