Dakota N Jackson

Kibrom M Alula

Yaritza Delgado-Deida

Redouane Tabti

Kevin Turner

Xuan Wang

K Venuprasad

Rhonda F Souza

Laurent Désaubry

Arianne L Theiss
email: arianne.theiss@cuanschutz.edu

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Keywords: colitis-associated cancer, colon cancer, natural compounds, -catenin degradation complex, adenomatous polyposis coli APC, adenomatous polyposis coli, AOM, azoxymethane, CA-CRC, colitisassociated cancer, CRC, colorectal cancer, DSS, dextran sodium sulfate, IBD, inflammatory bowel disease, PHB1, Prohibitin 1, WT, wild-type

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The Synthetic Small Molecule FL3 Combats Intestinal

Tumorigenesis via Axin1-Mediated Inhibition of Wnt/β-Catenin Signaling

INTRODUCTION

Patients with chronic inflammatory bowel disease (IBD) of the colon have increased risk for colorectal cancer (CRC), accounting for nearly 10-15% of deaths in IBD patients [START_REF] Mattar | Current management of inflammatory bowel disease and colorectal cancer[END_REF]. In these cases, CRC develops as a consequence of complications brought on by chronic colonic inflammation and is referred to as colitis-associated colorectal cancer (CA-CRC). Due to the well-established association of chronic inflammation with CRC development and malignant progression, use of anti-inflammatory agents to impede carcinogenesis is considered to be a highly effective strategy for CRC chemoprevention [START_REF] Fajardo | Chemoprevention in gastrointestinal physiology and disease. Anti-inflammatory approaches for colorectal cancer chemoprevention[END_REF].

Flavaglines are a relatively new class of molecules derived from medicinal Aglaia (family Meliaceae) plants [START_REF] Thuaud | Prohibitin ligands in cell death and survival: mode of action and therapeutic potential[END_REF]. Flavaglines have shown anti-cancer, anti-inflammatory, and cytoprotective activities in vitro [START_REF] Li-Weber | Molecular mechanisms and anti-cancer aspects of the medicinal phytochemicals rocaglamides (=flavaglines)[END_REF]. FL3 is a synthetic of the flavagline rocaglaol lacking the structural components linked to causing multidrug resistance and suboptimal pharmacokinetics, making it an ideal candidate for further investigation [START_REF] Thuaud | Prohibitin ligands in cell death and survival: mode of action and therapeutic potential[END_REF][START_REF] Liu | Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors[END_REF]. Previous studies demonstrated that FL3 at nanomolar concentrations exhibits cytoprotective effects in neurons [START_REF] Fahrig | A synthetic derivative of the natural product rocaglaol is a potent inhibitor of cytokine-mediated signaling and shows neuroprotective activity in vitro and in animal models of Parkinson's disease and traumatic brain injury[END_REF], cardiomyocytes [START_REF] Bernard | Flavaglines alleviate doxorubicin cardiotoxicity: implication of Hsp27[END_REF], and intestinal epithelial cells [START_REF] Han | Flavaglines Ameliorate Experimental Colitis and Protect Against Intestinal Epithelial Cell Apoptosis and Mitochondrial Dysfunction[END_REF] and presents little toxicity to healthy cells [START_REF] Thuaud | Prohibitin ligands in cell death and survival: mode of action and therapeutic potential[END_REF][START_REF] Qureshi | FL3, a Synthetic Flavagline and Ligand of Prohibitins, Protects Cardiomyocytes via STAT3 from Doxorubicin Toxicity[END_REF][START_REF] Yuan | Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45alpha pathway[END_REF]. Our previous data suggest that FL3 combats inflammation using a mouse model of colitis, although we did not elucidate the mechanism of FL3 action [START_REF] Han | Flavaglines Ameliorate Experimental Colitis and Protect Against Intestinal Epithelial Cell Apoptosis and Mitochondrial Dysfunction[END_REF]. In vitro studies suggest that flavaglines target Prohibitins as ligands, but this has not been established in the intestine [START_REF] Thuaud | Prohibitin ligands in cell death and survival: mode of action and therapeutic potential[END_REF]. Prohibitin 1 (PHB1) is a highly conserved protein with diverse functions including regulation of cell cycle progression, apoptosis, and transcription depending on its post-translational modifications and subcellular localization [START_REF] Basmadjian | Flavaglines: potent anticancer drugs that target prohibitins and the helicase eIF4A[END_REF]. In intestinal epithelial cells, PHB1 is predominantly localized in the mitochondria, where it has been shown to be required for optimal activity of the electron transport chain [START_REF] Bourges | Structural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin[END_REF][START_REF] Hsieh | Comparative proteomic studies on the pathogenesis of human ulcerative colitis[END_REF][START_REF] Theiss | Prohibitin protects against oxidative stress in intestinal epithelial cells[END_REF][START_REF] Tsutsumi | Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein[END_REF].

Accumulation of select DNA mutations in tumor suppressors and oncogenes in colonic epithelial cells drives CRC, including CA-CRC, initiation and progression [START_REF] Van Der Kraak | Colitis-associated colon cancer: Is it in your genes?[END_REF]. The majority of CRC, including sporadic (up to 80%) and inflammation-induced (~50%), carry a genetic mutation in either Adenomatous polyposis coli (APC) or CTNNB1 (encoding β-catenin), resulting in aberrant Wnt activation [START_REF] Conlin | The prognostic significance of K-ras, p53, and APC mutations in colorectal carcinoma[END_REF][START_REF] Yamauchi | Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum[END_REF]. Although Wnt/-catenin pathway mutations tend to occur late in CA-CRC development, studies suggest that activation of the Wnt/-catenin pathway by numerous inflammatory signaling networks contribute to the onset and progression of CA-CRC [START_REF] Koch | Extrinsic control of Wnt signaling in the intestine[END_REF]. APC is a tumor suppressor due to its scaffolding role in the '-catenin destruction complex', which is a key node in regulating Wnt signaling [START_REF] Wang | Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase[END_REF]. Axin1 and Axin2 are pivotal scaffold proteins that coordinate the assembly of APC, kinases CK1 and GSK3, and their substrate -catenin to facilitate the phosphorylation and subsequent proteasomal degradation of -catenin under basal conditions. During Wnt stimulation, the destruction complex is inhibited, resulting in accumulation and nuclear translocation of -catenin and transcriptional activation by binding to TCF/LEF sites on Wnt target genes that control proliferation, survival, and migration [START_REF] Daugherty | Phospho-regulation of Beta-catenin adhesion and signaling functions[END_REF]. Alterations in the APC gene found in CRC generate truncated mutants lacking all binding sites for Axin and abolish the formation of the -catenin destruction complex. Current therapeutics cannot directly target APC. CRC mutations in CTNNB1 render -catenin resistant to phosphorylation and proteasomal degradation, leading to overexpression of -catenin and Wnt target genes [START_REF] Tortelote | Complexity of the Wnt/betacatenin pathway: Searching for an activation model[END_REF].

Many inhibitors of the Wnt/-catenin pathway have been characterized, with some reaching early clinical trials [START_REF] Krishnamurthy | Targeting the Wnt/beta-catenin pathway in cancer: Update on effectors and inhibitors[END_REF]. However, current Wnt-targeting compounds exhibit adverse reactions since Wnt signaling regulates homeostasis of many adult tissues, with the intestine being extremely vulnerable. For this reason, advancement of these compounds in clinical trials is impeded [START_REF] Shang | The regulation of beta-catenin activity and function in cancer: therapeutic opportunities[END_REF]. Here, using human CRC cell lines with or without mutations in the CTNNB1

MATERIALS AND METHODS

Description of histological dysplasia/adenoma scoring, SDS-PAGE and western immunoblot analysis, cell viability assay, MTT proliferation assay, cell migration assay, RNA isolation and quantitative real-time PCR analysis, TCF/LEF luciferase reporter, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, immunofluorescence and immunohistochemistry, chromatin immunoprecipitation, and RNAseq analysis is provided in the Supplementary Methods.

FL3 synthesis

FL3 was synthesized in our laboratory as previously described [START_REF] Thuaud | Synthetic analogue of rocaglaol displays a potent and selective cytotoxicity in cancer cells: involvement of apoptosis inducing factor and caspase-12[END_REF].

Animal models

All mice were grouped-housed in standard cages under a controlled temperature (25˚C) and photoperiod (12- AOM-DSS model: Age-matched male and female WT C57Bl/6 mice (Jackson Labs), Phb1 fl/fl mice (C57Bl/6 genetic background), and Phb1 i∆IEC mice (C57Bl/6 background and previously described ( 26)) at 8-weeks old were intraperitoneally (i.p.) injected with 7.6 mg/kg azoxymethane (AOM; Sigma-Aldrich) followed by 2 cycles of 3.0% (wt/vol) dextran sodium sulfate (DSS; molecular weight, 50,000; MP Biomedicals) in drinking water for 7 days followed by 14 or 21 days of recovery (regular drinking water). One day before and each day of DSS administration, mice were i.p. injected daily with 0. sacrifice, colon was excised from the ileocecal junction to anus, cut open longitudinally, neoplasia number and size were quantitated using a dissecting microscope, and prepared for histological evaluation or biochemical analyses.

Apc Min/+ model: Age-matched male and female Apc Min/+ mice (Jackson Labs) and WT C57BL/6 littermates were i.p. injected with 0.1 mg/kg FL3 or vehicle (100 l volume) twice per week starting at 15 weeks to 20 weeks. Upon sacrifice, the entire small intestine and colon were excised, cut open longitudinally, neoplasia number and size were quantitated using a dissecting microscope and prepared for histological evaluation or biochemical analyzes.

Human CRC and matched-normal specimens

Fresh, de-identified specimens of CRC, stage II and III, and matched-normal colon tissue were obtained from Baylor University Medical Center Pathology Department, which obtained written informed consent from patients undergoing resection surgery. The studies were conducted in accordance with the Belmont Report ethical principles and under Institutional IRB approval. Tissue specimens were stored in Advanced DMEM/F12 supplemented with penicillin/streptomycin until culturing which averaged 2-8 h later.

Human or mouse CRC tumoroid and colonoid culture

CRC tumoroids were derived from tumors digested in 125 g/ml dispase type II, 75 U/ml collagenase type IX, and 0.2% Primocen. Colonoids were derived from isolated colonic crypts from mice or human specimens and cultured in Matrigel Basement Membrane Matrix (Corning) as previously described [START_REF] Sato | Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium[END_REF]. Colonoids were grown in WENR media (Advanced DMEM/F12 supplemented with penicillin/streptomycin, 10 mM Hepes, 1× L-glutamine, 1× N2, 1× B27 (all from Life Technologies)), containing 100 ng/mL Wnt3, 50 ng/mL recombinant epidermal growth factor (EGF), 500 ng/mL R-spondin1, and 100 ng/mL Noggin (all from R&D Systems).

Tumoroids were grown in WNR medium (containing Wnt3, Noggin, and R-spondin1) since CRC cells require no addition of growth factors such as EGF [START_REF] Sato | Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium[END_REF]. Tumoroids and colonoids were grown for 7 d and treated with 10 or 50 nM FL3. For collection of protein lysates for Western blot, tumoroids were collected using Cultrex Organoid Harvesting Solution (Fisher Scientific) to remove Matrigel.

To determine whether human tumoroids exhibited deletion at S45 of -catenin, genomic DNA was isolated from cultured tumoroids using the DNeasy Kit (Qiagen), 100 ng genomic DNA was amplified by PCR using the following primers: sense: 5′-CAATGGGTCATATCACAGATTCTT-3′; antisense: 5′-TCTCTTTTCTTCACCACAACATTT-3′, and the PCR products were gel-purified and sequenced.

For transfection of sgPHB1, human tumoroids grown in matrigel were trypsinized into single cells and Nucleofected with human Prohibitin 1 CRISPR/Cas9 KO Plasmid (sc-416271, Santa Cruz) and human Prohibitin 1 HDR Plasmid (sc-416271-HDR, Santa Cruz) using Hepatocyte Nucleofector Kit (VPL-1004, Lonza) for 72 h.

Cell culture

RKO (CRL-2577), HCT-116 (CCL-247), SW48, and IEC-6 cells were acquired from the American Type Culture Collection (ATCC) in 2017. Cells were cultured in 1X Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 40 mg/L penicillin and 90 mg/L streptomycin. Cells were maintained in an incubator with 5% CO 2 at 37°C. All experiments were performed on RKO cells between passages 5 and 19, HCT-116 between passages 7 and 12, SW48 between passages 5 and 9, and IEC-6 between passages 19 and 26. All cell lines were verified to be mycoplasma-free using Genlantis MycoScope PCR Detection kit (Fisher Scientific) as recently as March 15, 2020 (RKO, HCT116, IEC-6) or April 7, 2020 (SW48). To generate RKO cells stably expressing -catenin harboring deletion of S45, RKO cells were transfected with Pcl-neo--catenin-∆45 (Addgene) using Nucleofector T kit (Lonza). 48 h following transfection, transfected cells were selected with 1000 ug/mL of G418 (Geneticin; Gibco) for 3 to 5 weeks. Single clones were then isolated, maintained under 200 ug/mL G418 selection, and DNA sequenced for validation of exogenous -catenin-∆45 expression using the following primers: sense: 5′-ATGGCCATGGAACCAGACAG-3′; antisense: 5′-CTGAGAAAATCCCTGTTCCCAC-3′. Since these cell lines still express wild-type endogenous -catenin, sequencing confirmed heterozygous S45 deletion. To knockdown Axin1 expression, RKO cells were Nucleofected with 20 M of three pooled unique 27mer siRNA duplexes against Axin1 (SR305433; Origene) or 20 μM Stealth RNAi non-specific Negative Control Med GC (siNC; Invitrogen).

Statistical analysis

Data are presented as individual data points ± SEM. An unpaired two-tailed Student's t test was used for single comparisons, one-way ANOVA with Bonferroni post hoc test for multiple comparisons, and two-way ANOVA with Bonferroni post hoc test for assessing the combination of Axin1 knockdown and FL3 treatment (GraphPad Prism 8.0). For RNAseq analysis, genes were filtered for CPM ≥ 1 and differential gene expression was determined using R package DESeq2 (Bioconductor), P < 0.05, FDR ≤ 0.05, log2FC > 0.32. Ingenuity Pathway Analysis (Qiagen) identified significantly altered pathways.

RESULTS

FL3 decreases AOM-DSS-induced colonic tumorigenesis

To induce CA-CRC in mice, wild-type (WT) C57BL/6 mice were i.p. injected with AOM, followed by 2 cycles of DSS, consisting of 3.0% (wt/vol) DSS given in their drinking water for 7 days and 14 or 21 days recovery (regular drinking water). One day prior to and during each cycle of DSS, mice were i.p. injected daily with 0.1 mg/kg FL3 or vehicle. FL3 decreased the number and size of AOM-DSS-induced colonic neoplasia (Figure 1A and1B) and decreased the histological occurrence of adenocarcinoma (Figure 1C and 1D), suggesting that FL3 prevents the progression of colitis to cancer. Importantly, the mice displayed no overt signs of toxicity or abnormal behavior from FL3 treatment. Colon tumoroids cultured from three independent AOM-DSS-treated mice and treated with FL3 at a low nanomolar concentration (10 nM) exhibited decreased viability and increased apoptosis (Figure 1E, 1F and Supplemental Figure S1A).

RNA sequencing (RNAseq) analysis of AOM-DSS colon tumoroids treated with FL3 followed by Ingenuity Pathway Analysis identified Wnt/-catenin signaling as the top canonical pathway altered by FL3 (P = 4.3E-04; Figure 1G). Of the genes identified by RNAseq, the expression of Axin1 was most significantly altered by FL3 (P = 1.6E-08), followed by Smek1 (P = 4.6E-07), Tle4 (P = 2.8E-07), and Klf4 (P = 2.3E-05). Western blotting confirmed increased Axin1 protein expression by FL3 as compared to negligible increases in Tle4, Smek1, or Klf4 (Supplemental Figure S1B). Significant increases in Jun (P = 8.9E-07), ep300 (P = 4.6E-07) were also demonstrated by RNAseq, but this was not evident at the protein level (Supplemental Figure S1B). These results identify a potential role of Wnt/-catenin inhibition in mediating antitumorigenic properties of FL3. To determine whether FL3 inhibits the Wnt/-catenin pathway, we utilized various human CRC cell lines with known APC and CTNNB1 gene mutation status. 10 nM FL3 significantly decreased viability of RKO and SW48 cells but was not effective against HCT-116 cells or the non-transformed intestinal epithelial cell line IEC-6 (Supplemental Figure S2A). HCT-116 cells harbor a heterozygous deletion of S45 of -catenin coupled with loss of heterozygosity (LOH), which is the site of phosphorylation by CK1 and the priming step necessary for phosphorylation of -catenin at other sites that drive it to proteasomal degradation [START_REF] Tortelote | Complexity of the Wnt/betacatenin pathway: Searching for an activation model[END_REF]. Phosphorylation at S33 of -catenin (mutant in SW48 cells) is dispensable if S45, S37, T41 are phosphorylated [START_REF] Tortelote | Complexity of the Wnt/betacatenin pathway: Searching for an activation model[END_REF]. FL3 induced cell death as measured by TUNEL staining and cleaved Caspase 3 expression in RKO and SW48 cells but not in HCT-116 or IEC6 cells (Supplemental Figure S2B-D). FL3 did not alter cell proliferation in any cell line tested and decreased migration of RKO and SW48 cells, but not HCT-116 cells (Supplemental Figure S3). These results suggest that FL3 therapeutic response to decrease cell viability and migration is dependent on -catenin mutation status.

FL3 decreases transcriptional activation by -catenin and protein expression in RKO and

SW48 CRC cells

We next measured -catenin activation and expression during FL3 treatment. Western blotting and immunofluorescent staining confirmed that 10 nM FL3 decreased -catenin protein expression in RKO and SW48 cells, but not in HCT-116 or IEC6 cells (Figure 2A and2B).

Human CRC cell lines were transfected with a TCF/LEF luciferase reporter plasmid to measure transcriptional activation by -catenin and treated with FL3. 10 nM FL3 abolished relative luciferase expression to levels similar to negative control in RKO and SW48 cells, but in HCT-116 cells relative luciferase remained 6-fold higher than negative control (Figure 2C). Indeed, FL3 decreased expression of Wnt target genes in RKO cells (Figure 2D) and increased phosphorylation of -catenin at S45 that inversely correlated with -catenin degradation (Supplemental Figure S4A). Since HCT-116 cells harbor heterozygous G13D mutation of K-Ras in addition to heterozygous deletion of S45 of -catenin, we stably-transfected RKO cells with a on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216 plasmid carrying the same HCT-116 S45--catenin deletion (RKO mut). RKO mut cells were no longer susceptible to FL3-induced -catenin degradation (Supplemental Figure S4B andS4C) and cell death (Supplemental Figure S4D-F), suggesting that S45 of -catenin is crucial for FL3 action. A cell viability comparison of FL3 to common CRC chemotherapeutic drugs 5-fluorouracil (5-FU), irinotecan, and oxaliplatin in a normal intestinal epithelial cell line (IEC6), a FL3responsive CRC cell line (RKO), and a FL3-nonreponsive CRC cell line (HCT-116) demonstrates that FL3 did not decrease viability in IEC6 cells compared to 5-FU, irinotecan, or oxaliplatin (Supplemental Figure S5). 5-FU or oxaliplatin decreased cell viability in IEC-6 cells similarly to that in RKO or HCT-116 cells, suggesting equal toxicity by these drugs in normal intestinal epithelial cells and CRC cells (Supplemental Figure S5). Irinotecan did not alter viability compared to vehicle in any cell line tested.

FL3 inhibition of Wnt/-catenin and induction of cell death in RKO cells is dependent on

Axin1

Axin1 and Axin2 are pivotal scaffold proteins crucial for the formation of the -catenin destruction complex. Axin is the least abundant component of the destruction complex due to proteasomal degradation by tankyrase and even small increases in Axin protein alters -catenin [START_REF] Wang | Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase[END_REF]. Unlike Axin2, which is a transcriptional target of -catenin-dependent Wnt signaling, Axin1 is ubiquitously expressed and is classically thought of as a tumor suppressor protein [START_REF] Mazzoni | AXIN1 and AXIN2 variants in gastrointestinal cancers[END_REF]. RKO cells with knockdown of Axin1 by 3 pooled siRNAs were resistant to FL3-induced -catenin degradation (Figure 3A and 3B), -catenin transcriptional activation (Figure 3C), and cell death (Figure 3D-F). These results suggest that FL3 anti-cancer mechanism in CRC is dependent on Axin1 degradation of -catenin. To determine whether FL3 is able to ameliorate colon tumorigenesis in a model of robust Wnt/-catenin overexpression, Apc Min/+ mice were administered 0.1 mg/kg FL3 or vehicle twice weekly from 15 to 20 weeks of age. FL3 significantly decreased the number and size of adenomas and severity of dysplasia in Apc Min/+ mice (Figure 4A-C). This was associated with decreased -catenin expression (Figure 4D) and increased cell death in adenomas (Figure 4E-G) but no change in cell proliferation (Supplemental Figure S6). Quantitation of TUNEL + cells in non-tumor regions demonstrates that FL3 did not alter the number of apoptotic cells in normal crypts or villi in vivo (Figure 4F). These studies demonstrate that FL3 is sufficient to impact tumor growth in the absence of normal APC activity. WT mice injected with FL3 alongside Apc Min/+ mice demonstrated normal gastrointestinal histology including small intestinal villus length and small intestinal and colonic crypt length (Supplemental Figure S7).

FL3 decreases intestinal tumorigenesis in the Apc

FL3 decreases viability of human CRC tumoroids but not matched-normal colonoids

Historically, preclinical gastrointestinal translational research has relied on cell cultures that are of limited relevance to human physiology [START_REF] Sato | Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium[END_REF]. These models often fail to predict human toxicity and/or efficacy [START_REF] Howell | Generating intestinal tissue from stem cells: potential for research and therapy[END_REF]. The establishment of 3D matrigel-based intestinal enteroid cultures in recent years provides a rapid and simple means to test toxicity and efficacy of pharmaceutical compounds in human specimens [START_REF] Sato | Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium[END_REF]. To assess translational utility of FL3, tumoroids were cultured from human CRC specimens and colonoids from matched-normal tissue and treated with FL3. FL3 decreased viability of CRC tumoroids derived from 5 out of 6 patients, but did not affect cell viability in normal colonoids (Figure 5A and5B). Sequencing analysis confirmed that tumoroids derived from Patient 3, which were resistant to FL3 treatment, harbored heterozygous S45 deletion of -catenin, corroborating our results in HCT-116 cells. All other patients were wild-type at S45 of -catenin.

on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

PHB1 expression is required for FL3 anti-tumorigenic action in the colon

Mice with Villin-CreER T2 tamoxifen inducible deletion of Phb1 from the intestinal epithelium (Phb1 iIEC ) were recently described [START_REF] Jackson | Mitochondrial dysfunction during loss of prohibitin 1 triggers Paneth cell defects and ileitis[END_REF]. We utilized this model to determine whether PHB1 is necessary for FL3 to combat colonic tumorigenesis. Since we have not observed spontaneous colonic neoplasia in Phb1 iIEC mice up to 12 weeks following deletion of Phb1, we induced CA-CRC in Phb1 iIEC and Phb1 fl/fl control mice using AOM-DSS and cultured tumoroids.

FL3 induced death of tumoroids derived from Phb1 fl/fl mice, whereas FL3 had no effect on tumoroids derived from Phb1 iIEC mice (Supplemental Figure S8A andS8B). Additionally, FL3 induction of Axin1 was absent in Phb1 deficient tumoroids (Supplemental Figure S8C). We next selected human CRC tumoroids from a patient responsive to FL3 (Patient 1; Figure 5B) and targeted PHB1 using CRISPR-Cas9 sgRNAs (Figure 5C). FL3 was ineffective to increase Axin1 expression (Figure 5C) and to decrease viability in PHB1-targeted human CRC tumoroids (Figure 5D). These data suggest that PHB1 is required for FL3 anti-tumor action in the colon. To demonstrate aberrant glycosylation in our tumoroid model compared to colonoids, which is an important feature of cancer cells [START_REF] Kang | Cancer Cell Glycocalyx and Its Significance in Cancer Progression[END_REF], CRC tumoroids exhibited increased sialylated-Tn (a shortened glycan formed from incomplete synthesis of O-glycans common in cancer) and increased N-acetylglucosamine transferase (GnT-V) that is commonly upregulated in cancer (Figure 5E). These findings are important for the translational relevance of FL3 using CRC tumoroid models and are in agreement with previous reports demonstrating glycocalyx expression in intestinal and colonic organoids [START_REF] Fujii | Modeling Human Digestive Diseases With CRISPR-Cas9-Modified Organoids[END_REF][START_REF] Rouhanifard | Engineered Glycocalyx Regulates Stem Cell Proliferation in Murine Crypt Organoids[END_REF].

The role of PHB1 in cancer depends upon the tumor type, with expression of PHB1 being reported as increased in CRC [START_REF] Chen | Mitochondrial proteomics analysis of tumorigenic and metastatic breast cancer markers[END_REF][START_REF] Hammoudi | Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer (CRC)[END_REF]. Dynamic partitioning of PHB1 between nuclear and cytoplasmic compartments of tumor cells has been shown to be signal-dependent and necessary for the induction of apoptosis [START_REF] Rastogi | Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM-1-dependent mechanism[END_REF]. Compared to the non-transformed cell line IEC6, CRC cells exhibited increased phosphorylation of PHB1 at Thr258 (Figure 6A), a post- translational modification that induces mitochondrial localization of PHB1 in other types of cancer and correlates with enhanced proliferation, invasion, and metastasis [START_REF] Chiu | Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane[END_REF][START_REF] Jiang | Akt phosphorylates Prohibitin 1 to mediate its mitochondrial localization and promote proliferation of bladder cancer cells[END_REF]. FL3 inhibited phosphorylation of PHB1 at Thr258 in CRC cells (Figure 6A) with a concomitant nuclear translocation of PHB1 in CRC cells but not in normal IECs (Figure 6B). In the nucleus, PHB1 has been shown to act as a co-regulator of transcription by interacting with various transcription factors such as p53, Rb, and E2F [START_REF] Thuaud | Prohibitin ligands in cell death and survival: mode of action and therapeutic potential[END_REF]. A previous study identified PHB1 as a novel factor able to bind to the (TGYCC)n motif in the promoter of p53-inducible gene 3 (PIG3) [START_REF] Guan | Identification of prohibitin and prohibiton as novel factors binding to the p53 induced gene 3 (PIG3) promoter (TGYCC)(15) motif[END_REF]. This motif is similar to the underlined portion of the p53 binding site (RRRCWWGYYY; R = A or G, W = A or T, Y = C or T) which repeats this motif with a 0-21 base pair spacer in-between.

Using UCSC Genome Browser, the promoter regions of Axin1, Smek1, Tle4, and Klf4 genes (the genes most significantly increased by FL3 in RNAseq analysis shown in Figure 1G) were searched for WGYYY or RGYYY motifs with or without a spacer. None were found in Smek1, Tle4, or Klf4 promoters. However, the Axin1 promoter contains the sequence 5-GGCCTGGGCTTCGGCGCTCTGGCTCGGGCTCTGGCTC-3 located -5668 to -5631 from the transcriptional start site (TSS) in the 5UTR, which contains 5 RGYYY motifs (underlined), and we will refer to as putative PHB1 binding motif herein. ChIP assays demonstrated that PHB1 associates with this sequence in the Axin1 promoter and that PHB1 binding is enriched by FL3 treatment in RKO cells (Figure 6C translational potential of FL3 since mutation in the APC gene is present in 49.5% of all CRC patients, a much higher percentage than mutation in the CTNNB1 gene (4.99% of CRC patients) or Axin1 gene (2.72% of CRC patients) [START_REF]AACR Project GENIE: Powering Precision Medicine through an International Consortium[END_REF], suggesting that the majority of CRC patients will likely respond to FL3 treatment based on APC and CTNNB1 genetic mutation status. Heterogeneity of CA-CRC and sporadic CRC includes involvement of chromosomal instability (CIN), microsatellite instability (MSI), and promoter CpG island methylator phenotype (CIMP) [START_REF] Habermann | Pronounced chromosomal instability and multiple gene amplifications characterize ulcerative colitis-associated colorectal carcinomas[END_REF][START_REF] Scarpa | Aberrant gene methylation in non-neoplastic mucosa as a predictive marker of ulcerative colitisassociated CRC[END_REF][START_REF] Shivakumar | Copy number variations are progressively associated with the pathogenesis of colorectal cancer in ulcerative colitis[END_REF]. Future studies will further analyze these classifications in regards to FL3 response. Based on the CRC cell lines used in this study, HCT-116, RKO and SW48 are CIN  /MSI-High, and therefore does not alter response to FL3. CIMP  , which is the status of HCT-116 cells, could contribute to diminished FL3 responsiveness, whereas CIMP + , which is the status of RKO and SW48, could be an important indicator of FL3 effectiveness. Future studies using other CIN + /MSS/CIMP  cells such as Caco2 and HT29 will further elucidate FL3 response with these classifications.

The role of PHB1 in cancer depends upon the tumor type, with expression of PHB1 being reported as increased in CRC [START_REF] Chen | Mitochondrial proteomics analysis of tumorigenic and metastatic breast cancer markers[END_REF][START_REF] Hammoudi | Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer (CRC)[END_REF]. Dynamic partitioning of PHB1 between nuclear and cytoplasmic compartments of tumor cells has been shown to be signal-dependent and necessary for the induction of apoptosis in tumor cells [START_REF] Rastogi | Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM-1-dependent mechanism[END_REF]. Although human PHB1 does not possess a mitochondrial targeting sequence, the N-terminal transmembrane domain of PHB1 (aa 2-24) targets it to the mitochondria where it is anchored to the inner mitochondrial membrane [START_REF] Peng | Multifaceted role of prohibitin in cell survival and apoptosis[END_REF]. At the C-terminus of PHB1, a leucine/isoleucine nuclear export sequence (NES) between aa 257-270 facilitates its export from the nucleus via CRM-1 export receptor to the cytoplasm [START_REF] Rastogi | Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM-1-dependent mechanism[END_REF]. This specific structure of PHB1 allows active shuttling between the organelles. Previous studies have shown that phosphorylation of PHB1 at Thr258 enhances its mitochondrial localization in bladder and cervical cancer cells and is crucial to increased proliferation, invasion, and metastasis in these cancer cells [START_REF] Yuan | Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45alpha pathway[END_REF][START_REF] Chiu | Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane[END_REF][START_REF] Jiang | Akt phosphorylates Prohibitin 1 to mediate its mitochondrial localization and promote proliferation of bladder cancer cells[END_REF]. Here, we demonstrate on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216 that RKO, HCT-116, and SW48 CRC cells exhibit increased phosphorylation of PHB1 at Thr258, which is inhibited by FL3. Nuclear localization of PHB1 is induced by FL3 treatment in RKO CRC cells but not in non-transformed IEC6 cells. We show by ChIP that PHB1 binding to the 5UTR of the Axin1 gene is increased by FL3 in RKO CRC cells and that PHB1 deficiency in mouse and human CRC tumoroids abolishes FL3-induced Axin1 expression and cell death.

These results suggest that, FL3 is an inhibitor of Wnt/-catenin via blockade of PHB1 phosphorylation at Thr258 and subsequent PHB1 nuclear translocation where it induces transcription of Axin1, the core molecule and limiting component of the -catenin degradation complex. PHB1 was shown to inhibit Wnt signaling in murine liver and human hepatocellular carcinoma cells via regulation of the transcription factor E2F1 [START_REF] Mavila | Prohibitin 1 Acts As a Negative Regulator of Wingless/Integrated-Beta-Catenin Signaling in Murine Liver and Human Liver Cancer Cells[END_REF]. Interestingly, similar to FL3, other Axin1 stabilizers that act via tankyrase inhibition have been shown to impact tumorigenesis in the absence of normal APC activity [START_REF] Lau | A novel tankyrase smallmolecule inhibitor suppresses APC mutation-driven colorectal tumor growth[END_REF]. The tight control Axin1 expression exerts on the -catenin destruction complex is a property currently being exploited in the development of novel therapeutics for Wnt-driven cancers [START_REF] Wang | Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase[END_REF] and our results suggest that FL3 at low nanomolar concentrations upregulates Axin1 expression. We admit that PHB1 regulates multiple signaling pathways dependent on cell type studied, post-translational modifications of PHB1, and subcellular localization of PHB1. As examples, PHB1 can interact with C-RAF, Akt, IKK, and Stat3 [START_REF] Wang | Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases[END_REF]. Given our unbiased RNAseq data implicating FL3 inhibition of Wnt/catenin signaling in CRC, we currently focused on this pathway. Future studies will elucidate the role of other known PHB1 signaling pathways in FL3 anti-tumorigenic properties in CRC.

Polymorphisms of the PHB1 gene have been linked to gastric, breast, ovarian, and skin cancers but it remains unknown whether these genetic alterations affect PHB1 post-translational modifications or function [START_REF] Chowdhury | PHB in Cardiovascular and Other Diseases: Present Knowledge and Implications[END_REF]. Our results elucidating FL3 mechanism via PHB1 action may provide an ability to stratify CRC patients by those who are most likely to respond to FL3 treatment (those with high p-Thr258-PHB1 expression and absence of S45 -catenin mutation).

  hour light /dark cycle) and were allowed standard chow and tap water ad libitum. All experiments were approved by the Baylor Research Institute and the University of Colorado School of Medicine Institutional Animal Care and Use Committees. Experiments were performed with age-and gender-matched littermate mice.

  and 6D). Collectively, these results suggest a model for the in vivo anti-cancer mechanism of FL3 involving PHB1-induced Axin1 expression and -catenin degradation. on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216DISCUSSIONFL3 is a novel pharmacologic agent, not previously tested in cell or pre-clinical models of intestinal tumorigenesis. Here, we provide proof-of-principle for fundamental mechanisms of FL3 to combat intestinal tumorigenesis without significant toxicity. Using an unbiased approach (RNAseq analysis), we identify inhibition of Wnt/-catenin as the mechanism of FL3 anti-cancer activity dependent on PHB1 signaling. Importantly, we demonstrate translational utility of FL3 using human CRC tumoroids and matched-normal colonoids. FL3 provides a novel small molecule to combat cancers dependent on the Wnt pathway such CRC.It is well established that genetic mutations in various genes are associated with different prognosis and response to CRC therapy. For instance, activating mutations in K-Ras render EGFR inhibition ineffective[START_REF] Shi | Molecular testing in colorectal cancer: diagnosis of Lynch syndrome and personalized cancer medicine[END_REF] and tp53 mutation diminishes response to 5-fluorouracil[START_REF] Iacopetta | TP53 mutation in colorectal cancer[END_REF]. Response to FL3 was diminished in CRC cell lines harboring deletion of S45 of CTNNB1 including HCT-116. HCT-116 cells harbor heterozygous G13D mutation of K-Ras in addition to heterozygous deletion of S45 of -catenin. To determine which mutation diminishes FL3 response, stably-transfected RKO cells with a plasmid carrying the same HCT-116 S45-catenin deletion (RKO mut cells) were no longer susceptible to FL3-induced -catenin degradation and cell death, suggesting that S45 of -catenin is crucial for FL3 action. DNA sequencing revealed that the human CRC tumoroids unresponsive to FL3 (Patient 3) carried mutation at S45 of -catenin. Collectively, these results suggest that FL3 anti-cancer action is dependent on degradation of -catenin, requiring phosphorylation at S45. FL3 decreased tumor number, size, and severity of dysplasia while increasing tumor cell death in two mouse models of intestinal tumorigenesis, one inflammation-induced model (AOM-DSS) and one sporadic model (Apc Min/+ mice). FL3 efficacy in Apc Min/+ mice demonstrates that FL3 is sufficient to combat tumorigenicity in a model driven by aberrant Wnt/-catenin overexpression and in the absence of normal APC activity. This is especially important for the on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216

on

  July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216 FL3 treatment of non-transformed cells alongside CRC cells and CRC tumoroids, as well as no change by FL3 treatment in the number of TUNEL+ cells in normal crypts or villi in vivo suggests that FL3 lacks toxicity in normal intestinal epithelial cells. We propose that the targeting of PHB1 by FL3 provides a novel mechanism to inhibit Wnt signaling without intestinal toxicity since PHB1 nuclear shuttling response to FL3 is specific to CRC cells. Although many inhibitors of the Wnt/-catenin pathway have been characterized with some reaching early clinical trials (23), these compounds exhibit significant adverse reactions given the role of Wnt signaling in maintaining homeostasis of many adult tissues, particularly the intestine. As examples, Wnt pathway inhibitors XAV939 and LGK974 result in severe intestinal toxicity in mice and OMP18R5 (Vantictumab) induces abdominal pain and diarrhea in patients preventing advancement in clinical trials (23). FL3 provides a novel small molecule Wnt/-catenin inhibitor presenting little toxicity to healthy intestinal epithelial cells, as shown previously in other healthy cell types (3, 9, 10). Our study defines a link between FL3, PHB1, and Wnt/-catenin inhibition, which is a key pathway mediating intestinal carcinogenesis. These results provide important insight in the nuclear trafficking of PHB1 and its contribution to tumor death dependent on cellular localization and post-transcriptional modifications. We provide the first identification of in vivo molecular targets of FL3 and specifically identify a novel role of PHB1 in regulating Axin1 levels. FL3 provides a novel small molecule inhibitor of -catenin via upregulation of Axin1 expression as a potential therapeutic for Wnt pathway-driven cancers such as CRC. Future studies are poised to build the pre-clinical biosafety profile of FL3 and test the role of FL3 as a sensitizing agent in chemoresistant CRC cell lines. on July 15, 2020. © 2020 American Association for Cancer Research.

Figure legends Figure 1 .

 legends1 Figure legendsFigure 1. FL3 decreases AOM-DSS-induced colonic tumorigenesis. One day prior to and

Figure 2 .

 2 Figure 2. FL3 decreases transcriptional activation by -catenin and protein expression in

Figure 3 .

 3 Figure 3. FL3 inhibition of Wnt/-catenin and induction of cell death in RKO cells is

Figure 4 .

 4 Figure 4. FL3 decreases intestinal tumorigenesis in Apc Min/+ mice. Beginning at 15 weeks of age, Apc Min/+ mice were i.p. injected with 0.1 mg/kg FL3 of vehicle twice weekly for 5 weeks. (A)

Figure 5 .

 5 Figure 5. FL3 decreases viability of human CRC tumoroids but not matched-normal

Figure 6 .

 6 Figure 6. In CRC cells, FL3 blocks phosphorylation of T258-PHB1 and induces nuclear

  Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216

	gene, two mouse models of intestinal tumorigenesis, and mouse and human CRC tumoroids,
	we demonstrate that the targeting of PHB1 by FL3 provides a novel mechanism to inhibit
	aberrant Wnt signaling without intestinal toxicity.

on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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1 mg/kg FL3 or vehicle (100 l volume). Upon on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Min/+ mice on

  July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216

  Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216

on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216

Acknowledgments

We thank Dr. Xiaofang Huo and Dr. Qiuyang Zhang for providing key antibodies, Jie Han and Masha Sorouri for technical assistance, Beth Cook for histology processing, Cynthia Smitherman for RNAseq analysis, and Jinghua Gu for biostatistical support (Baylor Scott & White Research Institute). This work was supported by National Institutes of Health grants R01-DK117001 (ALT) and Litwin IBD Pioneers Crohn's Colitis Foundation 301869 (ALT). on July 15, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Financial support

This work was supported by National Institutes of Health grants R01-DK117001 (ALT) and Litwin IBD Pioneers Crohn's Colitis Foundation 301869 (ALT). and design: RFS, LD, ALT. Acquisition of data: DNJ, KMA, YDD, RT, KT, ALT. Analysis and interpretation of data: DNJ, KV, RFS, ALT.

Conflict of interest

The authors declare no potential conflicts of interest.

Author Manuscript Published OnlineFirst on July 14, 2020; DOI: 10.1158/0008-5472.CAN-20-0216