Human Familial Adenomatous Polyposis Research Articles

The effects of antioxidant administration in the early stages of radiation-induced tumorigenesis.

ApcMin/+ mouse was a model mouse for human familial adenomatous polyposis, and irradiation at an early age increases tumors in the small and large intestine. To study the effects of antioxidant administration on tumor incidence after continuous whole-body exposure to gamma rays, ApcMin/+ mice were exposed to a medium-dose-rate, 200mGy/d, from postnatal Day 0 to 21 of age or a high-dose-rate of 0.65Gy/min (total dose 4.2Gy) on postnatal Day 7. The dams and pups were supplied with the N-acetylcysteine (NAC) in drinking water (7g/L), from gestation Day 15 until weaning (21days-old). A significant increase in the number of intestinal tumors were observed in ApcMin/+ mice irradiated with high dose-rate gamma rays as compared with the non-irradiated controls, but there was no significant difference in tumor counts between the non-irradiated controls and the medium-dose rate irradiation groups. NAC administration did not have any significant effect at least at this dose. These results suggest that the supplementation of anti-oxidant at the early stage of tumorigenesis does not suppress the formation of irradiation-induced small intestinal tumors.

Yogurt Prevents Colorectal Tumorigenesis in ApcMin/+ Mice.

Yogurt consumption is related to a decreased risk of colorectal cancer (CRC), but whether such association is causal remains unclear. Patients with familial adenomatous polyposis (FAP) are at increased risk of CRC development. Here, the study investigates the efficacy of yogurt for intestinal polyposis chemoprevention in ApcMin/+ mice, a preclinical model for human FAP. A 10-week yogurt supplementation (15gkg-1) in ApcMin/+ mice significantly reduces the intestinal polyp number (6.50±0.97 versus 1.80±0.49; p<0.001) compared to controls. 16S rRNA gene-based microbiota analysis suggests that yogurt supplementation may greatly modulate the gut microbiome composition, especially in the relative abundance of Lactobacillus and Bifidobacterium. Importantly, the fecal concentration of d-lactate (d-Lac, 0.39±0.04µmolg-1 versus 8.14±0.62µmolg-1; p < 0.001) is boosted by yogurt, while oral administration with d-Lac (125 or 250mgkg-1) reduces the polyp number by 71.43% or 77.14% (p<0.001), respectively. The study also observes that d-Lac does not affect cell viability and anchorage-independence in CRC cells, but it greatly suppresses epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation in preneoplastic cells. Mechanistically, it demonstrates that d-Lac may attenuate epithelial cell transformation by targeting PI3K/AKT/β-catenin axis. Yogurt protects against intestinal polyposis in ApcMin/+ mice, and d-Lac may partially account for the chemopreventive effects above.

Transition state analogue of MTAP extends lifespan of APCMin/+ mice

A mouse model of human Familial Adenomatous Polyposis responds favorably to pharmacological inhibition of 5′-methylthioadenosine phosphorylase (MTAP). Methylthio-DADMe-Immucillin-A (MTDIA) is an orally available, transition state analogue inhibitor of MTAP. 5′-Methylthioadenosine (MTA), the substrate for MTAP, is formed in polyamine synthesis and is recycled by MTAP to S-adenosyl-l-methionine (SAM) via salvage pathways. MTDIA treatment causes accumulation of MTA, which inhibits growth of human head and neck (FaDu) and lung (H359, A549) cancers in immunocompromised mouse models. We investigated the efficacy of oral MTDIA as an anti-cancer therapeutic for intestinal adenomas in immunocompetent APCMin/+ mice, a murine model of human Familial Adenomatous Polyposis. Tumors in APCMin/+ mice were decreased in size by MTDIA treatment, resulting in markedly improved anemia and doubling of mouse lifespan. Metabolomic analysis of treated mice showed no changes in polyamine, methionine, SAM or ATP levels when compared with control mice but indicated an increase in MTA, the MTAP substrate. Generation of an MTDIA-resistant cell line in culture showed a four-fold amplification of the methionine adenosyl transferase (MAT2A) locus and expression of this enzyme. MAT2A is downstream of MTAP action and catalyzes synthesis of the SAM necessary for methylation reactions. Immunohistochemical analysis of treated mouse intestinal tissue demonstrated a decrease in symmetric dimethylarginine, a PRMT5-catalyzed modification. The anti-cancer effects of MTDIA indicate that increased cellular MTA inhibits PRMT5-mediated methylations resulting in attenuated tumor growth. Oral dosing of MTDIA as monotherapy has potential for delaying the onset and progression of colorectal cancers in Familial Adenomatous Polyposis (FAP) as well as residual duodenal tumors in FAP patients following colectomy. MTDIA causes a physiologic inactivation of MTAP and may also have efficacy in combination with inhibitors of MAT2A or PRMT5, known synthetic-lethal interactions in MTAP−/− cancer cell lines.

Fucoxanthin Prevents Colorectal Cancer Development in Dextran Sodium Sulfate-treated Apc Min/+ Mice.

A xanthophyll of fucoxanthin (Fx) is a potential chemopreventive agent. Familial adenomatous polyposis (FAP) is an inherited disease that is associated with a high risk of developing colorectal cancer. However, it remains unclear whether Fx can modify colorectal tumorigenesis in ApcMin/+ mice, a model mouse for human FAP. We investigated the chemopreventive effect of Fx in dextran sodium sulfate (DSS)-treated ApcMin/+ mice. Administration of Fx in the diet for 5 weeks significantly suppressed the number of colorectal adenocarcinomas in DSS-treated male ApcMin/+ mice, although the treatment did not affect the occurrence of colorectal dysplastic crypts and adenoma in the mice. In addition, Fx down-regulated cyclin D1 expression (0.6-fold) in colorectal mucosa of ApcMin/+ mice when compared with that of the control mice. Fx possesses chemopreventive potential against progression of colorectal carcinogenesis in ApcMin/+ mice that receive inflammatory stimuli.

Blockade of EIF5A hypusination limits colorectal cancer growth by inhibiting MYC elongation

ABSTRACTEukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusination impairs the expression of transcripts regulated by MYC, suggesting the involvement of this oncogene in the observed effect. Indeed, we demonstrate that EIF5A regulates MYC elongation without affecting its mRNA content or protein stability, by alleviating ribosome stalling at five distinct pausing motifs in MYC CDS. Of note, we show that blockade of the hypusination axis elicits a remarkable growth inhibitory effect in preclinical models of CRC and significantly reduces the size of polyps in APCMin/+ mice, a model of human familial adenomatous polyposis (FAP). Together, these data illustrate an unprecedented mechanism, whereby the tumor-promoting properties of hypusinated EIF5A are linked to its ability to regulate MYC elongation and provide a rationale for the use of DHPS/EIF5A inhibitors in CRC therapy.

Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1.

Background & AimsAspirin reduces colorectal cancer (CRC) incidence and mortality. Understanding the biology responsible for this protective effect is key to developing biomarker-led approaches for rational clinical use. Wnt signaling drives CRC development from initiation to progression through regulation of epithelial-mesenchymal transition (EMT) and cancer stem cell populations. Here, we investigated whether aspirin can rescue these proinvasive phenotypes associated with CRC progression in Wnt-driven human and mouse intestinal organoids.MethodsWe evaluated aspirin-mediated effects on phenotype and stem cell markers in intestinal organoids derived from mouse (ApcMin/+ and Apcflox/flox) and human familial adenomatous polyposis patients. CRC cell lines (HCT116 and Colo205) were used to study effects on motility, invasion, Wnt signaling, and EMT.ResultsAspirin rescues the Wnt-driven cystic organoid phenotype by promoting budding in mouse and human Apc deficient organoids, which is paralleled by decreased stem cell marker expression. Aspirin-mediated Wnt inhibition in ApcMin/+ mice is associated with EMT inhibition and decreased cell migration, invasion, and motility in CRC cell lines. Chemical Wnt activation induces EMT and stem-like alterations in CRC cells, which are rescued by aspirin. Aspirin increases expression of the Wnt antagonist Dickkopf-1 in CRC cells and organoids derived from familial adenomatous polyposis patients, which contributes to EMT and cancer stem cell inhibition.ConclusionsWe provide evidence of phenotypic biomarkers of response to aspirin with an increased epithelial and reduced stem-like state mediated by an increase in Dickkopf-1. This highlights a novel mechanism of aspirin-mediated Wnt inhibition and potential phenotypic and molecular biomarkers for trials.

Abstract 6364: LFA-9, a dual mPGES-1 and 5-LOX inhibitor, suppresses colon cancer stemness and inflammogen-induced inflammatory response

Abstract Chronic inflammation triggers tumorigenesis of the colorectum; hence several non-steroidal anti-inflammatory drugs (NSAIDs) have been explored for prevention and treatment of inflammatory diseases, including colorectal cancer (CRC). Although traditional NSAIDs and COX-2 inhibitors are effective chemopreventive agents for CRC, unwanted side effects (gastrointestinal and renal) hinder their use. Mechanistic studies suggest that the side effects are in part due to a COX-2 inhibition-associated increase in 5-lipoxygenase (5-LOX) metabolites and reduced PGI2 levels. Hence, agents targeting microsomal prostaglandin synthase-1 (mPGES-1) and 5-LOX inhibitors may prove to be more effective and safer agents for chemoprevention. LFA-9 was designed based on the pharmacophore of licofelone by adding 2-aminoethanoic acid (Glycine) at its carboxylic end to enhance renal clearance and selectivity towards dual mPGES-1 and 5-LOX inhibition. Here we characterized LFA-9 in detail for its mPGES1/5-LOX selectivity over COX-1/2 using in silico molecular modelling, in vitro enzyme activity assays, and ex vivo inhibition studies with human/mouse/rat mPGES-1 and 5-LOX enzymes to facilitate pre-clinical experimentation and clinical translation. In silico, LFA-9 showed strong binding affinity against 5-LOX (binding energies -199.85 and -215.3 Kcal/mol) and mPGES-1 (binding energies -238.27 and -257.76 Kcal/mol), but not against COX-1/2. LFA-9 substantially inhibited mPGES-1 (IC50 0.52 - 1.40 µM) and 5-LOX (IC50 0.89 - 2.75 µM) but not COX-1/2 (IC50&amp;gt;1mM) in cell-free assays. Ex-vivo, LFA-9 inhibited PGE2 (IC50 0.47 - 0.78 µM) and LTB4 (IC50 0.66 - 1.24 µM) production and spared PGI2 (IC50&amp;gt;1 mM) and TXB2 (IC50&amp;gt;1 mM) production in LPS-stimulated human/mouse/rat macrophages and whole blood (cell-based system). Circular dichroism and isothermal calorimetric-based studies demonstrated that LFA-9 strongly binds and induces changes in the secondary structure of human mPGES-1 and 5-LOX enzymes, thereby inhibiting their activities. Since PGE2 from mPGES1 activity promotes colon tumor stemness, LFA-9 was evaluated on colonic cancer organoids (spheroids) derived from PIRC (Polyposis in Rat Colon) rats that replicate human familial adenomatous polyposis. LFA-9 significantly inhibited the growth of colonic spheroids at 25 µM as compared to that of untreated as well as licofelone-treated spheroids. Anti-inflammatory properties of LFA-9 were evaluated in vivo using an inflammogen (carrageenan)-induced rat paw edema model. Dietary LFA-9 inhibited edema formation after carrageenan injection. In summary, LFA-9 showed anti-inflammatory and anti-tumor properties through mPGES1/5-LOX inhibition. Our data supports LFA-9, a novel mPGES-1 and 5-LOX inhibitor, as a safer drug candidate for prevention and therapy of colorectal cancer. (Supported by R01CA 213987, NIH/NCI HHSN261201500024I and VA merit). Citation Format: Nagendra Sastri Yarla, Gopal Pathuri, Hariprasad Gali, Venkateshwar Madka, Janani Panneerselvam, Parthasarathy Chandrakesan, Courtney W. Houchen, Elizabeth R. Glaze, Jennifer Fox, David McCormick, Chinthalapally V. Rao. LFA-9, a dual mPGES-1 and 5-LOX inhibitor, suppresses colon cancer stemness and inflammogen-induced inflammatory response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6364.

Suppression of tensin 2 promotes intestinal tumorigenesis by liberating integrin-linked kinase-induced nuclear translocation of β-catenin

Tensin 2 (TNS2) is a focal adhesion-localized multidomain protein expressed in various tissues. TNS2 expression significantly decreases in many tumor cell lines, and low TNS2 expression is associated with a poorer relapse-free survival in some cancers, suggesting that the loss of TNS2 may be related to tumor progression. Deregulation of Wnt/β-catenin signaling is frequently observed in colorectal cancer. In the present study, we found that TNS2 negatively regulated Wnt signaling by suppressing the nuclear translocation of β-catenin by reducing integrin-linked kinase (ILK) activity in colon cancer cell lines. To investigate the role of TNS2 in intestinal tumorigenesis in vivo, we introduced Tns2 mutation into ApcMin/+ mouse, a model of human familial adenomatous polyposis. The compound mutant mice showed a significant increase in tumor number and size in the small intestine and colon. Thus, this study may contribute to the discovery of novel mechanisms underlying cancer malignancy, and pave the way for the development of treatment strategies for intestinal cancers.

Abstract 5074: Optimizing erlotinib plus sulindac dosing regimens in a preclinical model of FAP

Abstract Introduction Colorectal cancer (CRC) involves sporadic cases and hereditary syndromes, such as familial adenomatous polyposis (FAP). In FAP patients, surgical intervention often is coupled to prevention strategies using nonsteroidal anti-inflammatory drugs. However, none of the current therapeutic options is fully effective. A recent trial in FAP patients combined standard of care Sulindac (SUL) treatment with daily Tarceva/erlotinib (ERL), and had good efficacy in reducing adenomatous polyp burden. Toxicity concerns, however, raised questions over the combination strategy for long-term prevention (Samadder et al. 2016). Using the Apc-mutant polyposis in rat colon (Pirc) model as a mimic of human FAP, we sought to optimize the dosing regimens for SUL+ERL for an improved safety profile, while retaining efficacy in the GI tract. Methods In a short-term pharmacodynamic biomarker study, rats (n=7) were fed AIN control diet or AIN diet containing 250 ppm SUL, either alone or in combination with ERL, given by oral gavage at 6 or 12 mg/kg (daily), 21 mg/kg (twice weekly), or 42 mg/kg (once weekly). Colon and small intestine (SI) polyps were resected at 0.5, 1, 2, 3, 7, 10, and 14 days after dosing, and assessed by immunoblotting (IB) or RT-qPCR for changes in pErk/Erk, pAkt/Akt, and other biomarkers. In a follow-up efficacy study, rats (n=10) were given SUL, ERL, or ERL+SUL using dosing regimens that were dictated by recovery times for pErk in the biomarker study. Colon and SI polyps were recorded at necropsy for location, incidence, multiplicity, and volume. Tumors and adjacent normal tissues were taken for histopathology and molecular analyses. Results The biomarker study revealed that pErk was inhibited in Pirc colon polyps for up to 10 days after discontinuing ERL treatment, with full recovery on or around day 14. Nuclear β-catenin, c-Myc, Mmp-7 and Cyclin D1 also were attenuated by ERL+SUL in colon polyps. In the efficacy study, results accrued via endoscopy at 3, 4 and 5 months were remarkably consistent with data obtained during final necropsy, at 6 months. Compared to AIN controls, multiple ERL+SUL groups exhibited significant suppression of polyps in the colon (78-98% inhibition, p&amp;lt;0.001) and SI (91-100% inhibition, p&amp;lt;0.001). Molecular analyses revealed that pErk was inhibited in adenomatous polyps, along with downregulation of β-catenin targets (c-Myc, Mmp-7). No overt toxicity was detected, other than mild skin changes in groups given ERL. Conclusions These studies have the potential to define safe and effective dosing strategies for SUL+ERL, improving efficacy against colon and SI polyps, while circumventing toxicity and resistance. Outcomes from the current work, plus an ongoing one-year toxicity/resistance trial in Pirc, should be directly translatable to the clinical management of FAP patients exhibiting similar pathology and phenotype. Supported by NCI Contract Number HHSN261201500018I, Task Order HHSN26100004. Citation Format: Ahmetmursel Ulusan, Praveen Rajendran, Wan-Mohaiza Dashwood, Altaf Mohammed, Shizuko Sei, Powel H. Brown, Eduardo Vilar-Sanchez, Roderick H. Dashwood. Optimizing erlotinib plus sulindac dosing regimens in a preclinical model of FAP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5074.

Role of tropomyosin 4 gene misregulation in colon epithelial cell tumorigenesis.

e15184 Background: Ordered migration of colon epithelial cells from the bottom to the top of the crypt is required for their differentiation. Because aberrant colon cell migration may contribute to early stages of colon tumorigenesis, elucidation of how this migration is regulated provides insight into how colon cancer can be prevented and treated before malignancy. Certain actin-binding tropomyosins, including tropomyosin 4 (TPM4), regulate cytoskeletal contractility and migration in many different cell types. Methods: To determine whether aberrant TPM4 regulation may be an early event in colon cell tumorigenesis, we measured TPM4 RNA expression in uninvolved, grossly normal colon tissue from 10 human familial adenomatous polyposis (FAP) patients genetically predisposed to colon cancer versus 7 patients not genetically predisposed (normal). We also examined TPM4 expression in a colon adenocarcinoma cell line (Caco2). These are highly proliferative, undifferentiated cells but can be induced to growth arrest and differentiate, thus acquiring many characteristics of normal absorptive colon epithelial cells. Reporter assays were used to identify sequences that regulate TPM4 gene expression in Caco2 cells. Results: Average TPM4 RNA expression is 2.59 fold higher in the FAP versus normal tissue. TPM4 RNA and protein are highly expressed in proliferating, undifferentiated Caco2 cells but are severely reduced in growth arrested, differentiated cells. Reporter assays have identified genetic regions, near the TPM4 transcriptional start site, that may regulate TPM4 gene expression in proliferating Caco2 cells. Conclusions: This data suggests that aberrantly upregulated TPM4 may be an early event in colon tumorigenesis and that normal TPM4 expression, by fostering ordered colon epithelial cell migration, may promote normal cell differentiation and mitigate adoption of a premalignant state.

The expression of TAP1 candidate gene, but not its polymorphism and methylation, is associated with colonic polyp formation in a porcine model of human familial adenomatous polyposis

In humans, the dysfunction of the adenomatous polyposis coli (APC) gene causes hereditary familial adenomatous polyposis (FAP) and increased risk of colorectal cancer (CRC). The severity of polyposis varies between individuals, but genetic basis for this is in large part unknown. This variability also occurs in our porcine model of FAP, based on an APC1311 mutation (orthologous to human APC1309). Since loss of TAP1 function can lead to CRC in humans, we searched for germline polymorphisms in APC1311/+ pigs with low (LP) and high (HP) levels of polyposis, as well as in wild-type pigs representing six breeds and a commercial line. The distribution of 40 identified polymorphic variants was similar in the LP and HP pigs. In contrast, the TAP1 transcript level was significantly higher in normal colon mucosa of HP pigs than in LP pigs. Moreover, six SNPs showed significant effects on TAP1 promoter activity, but no correlation with severity of polyposis was observed. Analysis of DNA methylation in the promoter region showed that one CpG site differed significantly between LP and HP pigs. We conclude that TAP1 genotype may not itself be associated with polyposis, but our findings concerning its expression suggest a role in the development of polyps.

The application of ApcMin/+ mouse model in colorectal tumor researches.

ApcMin/+ mouse is an excellent animal model bearing multiple intestinal neoplasia, used to simulate human familial adenomatous polyposis and colorectal tumors. The key point of this model is the mutation of Apc gene, which is a significant tumor-suppressor gene in the Wnt signaling pathway. There are also some other possible mechanisms responsible for the development of colorectal tumors in the ApcMin/+ mouse model, such as tumor-associated signaling pathways activation, the changes of tumor-related genes, and the involvement of some related proteins or molecules. The relevant literatures about ApcMin/+ mouse model from PUBMED databases are reviewed in this study. In recent years, increasing studies have focused on the application of ApcMin/+ mouse model in colorectal tumor, trying to find effective therapeutic targets for further use. This article will give a brief review on the related molecular mechanisms of the ApcMin/+ mouse model and its application in colorectal tumor researches.

Suppression of polyps formation by saffron extract in Adenomatous polyposis coliMin/+mice

Saffron (Crocus sativus L.) has been used both as a food additive for flavoring and coloring and in traditional medicine. Saffron extract and its main component crocin decrease the growth of several types of human cancer, including colorectal cancer in vitro. Numerous polyps develop in the small intestine in the Adenomatous polyposis coli (Apc) deficiency mice. ApcMin/+ mice are models for human familial adenomatous polyposis and human colon cancer patients. In this study, we examined the efficacy of saffron extract added to diet on reducing the polyp density in ApcMin/+ mice. ApcMin/+ mice were either given a placebo or saffron extract (0.1% and 0.5%) diet for 4 weeks. At 12 weeks of age, intestines were analyzed for polyp number in the small intestine. Our analysis confirmed that crocin (1), crocin-2 (2), and crocin-4 (4) are the major compounds in the saffron extract and the content of 1 in the tested saffron extract was 29.2%. Saffron extract decreased the number of intestinal polyps in a concentration-dependent manner in ApcMin/+ mice. Notably, the number of polyps in the distal small intestine of the mice fed with 0.5% saffron extract was significantly decreased compared with the placebo. These results indicate that saffron extract can reduce the polyp number in the ApcMin/+ mice. Abbreviations Used: FAP: Familial adenomatous polyposis; Apc: Adenomatous polyposis coli.

Elevated expression of p53 in early colon polyps in a pig model of human familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is a hereditary predisposition to formation of colon polyps that can progress to colorectal cancer (CRC). The severity of polyposis varies substantially within families bearing the same germline mutation in the adenomatous polyposis coli (APC) tumour suppressor gene. The progressive step-wise accumulation of genetic events in tumour suppressor genes and oncogenes leads to oncogenic transformation, with driver alterations in the tumour protein p53 (TP53) gene playing a key role in advanced stage CRC. We analysed groups of pigs carrying a truncating mutation in APC (APC1311/+; orthologous to human APC1309/+) to study the influence of TP53 polymorphisms and expression on the frequency of polyp formation and polyp progression in early-stage FAP. Five generations of APC1311/+ pigs were examined by colonoscopy for polyposis severity and development. A total of 19 polymorphisms were found in 5′-flanking, coding, and 3′ untranslated regions of TP53. The distribution of TP53 genotypes did not differ between APC1311/+ pigs with low (LP) and high (HP) number of colon polyps. p53 mRNA expression was analysed in distally located normal mucosa samples of wild-type pigs, APC1311/+ LP and HP pigs, and also in distally located polyp samples histologically classified as low-grade (LG-IEN) and high-grade intraepithelial dysplastic (HG-IEN) from APC1311/+ pigs. p53 mRNA expression was found to be significantly elevated in HG-IEN compared to LG-IEN samples (p = 0.012), suggesting a role for p53 in the early precancerous stages of polyp development.

An intestinal stem cell niche in Apc mutated neoplasia targetable by CtBP inhibition.

C-terminal binding protein 2 (CtBP2) drives intestinal polyposis in the Apc min mouse model of human Familial Adenomatous Polyposis. As CtBP2 is targetable by an inhibitor of its dehydrogenase domain, understanding CtBP2’s role in adenoma formation is necessary to optimize CtBP-targeted therapies in Apc mutated human neoplasia. Tumor initiating cell (TIC) populations were substantially decreased in Apc min Ctbp2+/- intestinal epithelia. Moreover, normally nuclear Ctbp2 was mislocalized to the cytoplasm of intestinal crypt stem cells in Ctbp2+/- mice, both Apc min and wildtype, correlating with low/absent CD133 expression in those cells, and possibly explaining the lower burden of polyps in Apc min Ctbp2+/- mice. The CtBP inhibitor 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) also robustly downregulated TIC populations and significantly decreased intestinal polyposis in Apc min mice. We have therefore demonstrated a critical link between polyposis, intestinal TIC’s and Ctbp2 gene dosage or activity, supporting continued efforts targeting CtBP in the treatment or prevention of Apc mutated neoplasia.

Abstract 68: Targeting macrophage that forms a chemotherapy-induced niche significantly reduces cancer stem cells and tumors

Abstract Failure in cancer therapy is often caused by relapse resulting largely from chemo-resistant cancer stem cells (CSCs). Most colorectal cancer patients, after receiving chemoradiotherapy, still harbor treatment-resistant residual tumor cells that are akin to colorectal adenoma. We therefore exploited the APCMin adenoma model that allowed us to study the natural and dynamic interaction between CSCs and their microenvironment in vivo following therapeutic stress. We observed that macrophages were directly recruited from blood vessel to the CSC niche in response to chemotherapy, playing a role in protecting and further promoting activation of CSCs. Reduction in the Ly6c-CD11bintCX3CR1+ MHCII+ population of tumor associated macrophages (TAMs) by celecoxib or clodrosome was correlated with a decline in slow-cycling drug-resistant CSCs and tumorigenesis. Molecularly, we found that COX-2 inhibitor celecoxib suppressed Akt and Wnt signaling, downstream of macrophage-dependent PGE2-EP pathway, thus reducing CSC survival and activation. Taken together, in the naturally formed adenoma reflecting human familial adenomatous polyposis, Chemotherapy-induced recruitment of TAMs to the CSC niche protected and promoted activation of surviving CSCs via PGE2-EP mediated Akt-Wnt signaling, leading to tumor regrowth. Targeting the TAMs will benefit clinical treatment of colorectal cancer. Citation Format: Xi He, Paloma I. Giles, Edward H. Lin, Linheng Li. Targeting macrophage that forms a chemotherapy-induced niche significantly reduces cancer stem cells and tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 68.

Collaborating genomic, transcriptomic and microbiomic alterations lead to canine extreme intestinal polyposis

Extreme intestinal polyposis in pet dogs has not yet been reported in literature. We identified a dog patient who developed numerous intestinal polyps, with the severity resembling human classic familial adenomatous polyposis (FAP), except the jejunum-ileum junction being the most polyp-dense. We investigated this dog, in comparison with 22 other dogs with spontaneous intestinal tumors but no severe polyposis, and with numerous published human cancers. We found, not APC mutation, but three other alteration pathways as likely reasons of this canine extreme polyposis. First, somatic truncation mutation W411X of FBXW7, a component of an E3 ubiquitin ligase, over-activates MYC and cell cycle-promoting network, accelerating crypt cell proliferation. Second, genes of protein trafficking and localization are downregulated, likely associated with germline mutation G406D of STAMBPL1, a K63-deubiquitinase, and MYC network activation. This inhibits epithelial apical-basolateral polarity establishment, preventing crypt cell differentiation. Third, Bacteroides uniformis, a commensal gut anaerobe, thrives and expresses abundantly thioredoxin and nitroreductase. These bacterial products could reduce oxidative stress linked to host germline mutation R51X of CYB5RL, a cytochrome b5 reductase homologue, decreasing cell death. Our work emphasizes the close collaboration of alterations across the genome, transcriptome and microbiome in promoting tumorigenesis.

Abstract 3901: Therapeutic targeting of C-terminal binding protein: a key dependency for polyposis and cancer stem cell activity in APC mutant neoplasia

Abstract The C terminal binding proteins (CtBP) 1 and 2 are a family of transcriptional co-repressors overexpressed in a variety of cancers, and are frequently associated with poor prognosis and chemoresistance. CtBP has also been characterized in cell culture models as drivers of migration/invasion and epithelial-mesenchymal transition. CtBP mediates its transcriptional corepressor activity via its dehydrogenase domain, and inhibition of this domain interferes with CtBP oncogenic functions. The role of CtBP in APC mutant neoplasia remains obscure even though APC is responsible for degradation of both β-catenin and CtBP in suppressing colorectal tumorigenesis. Our prior work demonstrates that CtBP proteins can be effectively therapeutically targeted with substrate analogues of their intrinsic dehydrogenase domains. We now observe that pharmacologic inhibition of CtBP using either the 1st generation inhibitor 2-keto-4-methylthiobutyrate (MTOB) or the 2nd generation inhibitor hydroxyimino-3-phenylpropanoic acid (HIPP) significantly reduces the burden of intestinal polyps in the Apcmin mouse model of the human cancer predisposition syndrome Familial Adenomatous Polyposis. The number of intestinal polyps in mice with CtBP2 inhibitors were significantly lower (21 polyps/mouse for MTOB, 15 polyps/mouse for HIPP) as compared with vehicle treated mice (45 polyps/mouse) with a P value &amp;lt;0.0001. Pointing to the anti-neoplastic mechanism of MTOB and HIPP, polyps that did grow in MTOB or HIPP treated mice demonstrated robust downregulation of β catenin and Ctbp2 proteins. This observation points to the role of Ctbp2 as a putative driver oncogene that can be therapeutically targeted in vivo. In addition, CtBP2 has been reported to play an important role in human colon cancer stem cells via its interaction with the transcription factor TCF4 on chromatin. We now show that inhibiting CtBP with HIPP or HIPP analogues attenuates CtBP2 protein levels in colon cancer stem cells with resultant decreased expression of CtBP transcriptional target genes, such as the motility/invasion regulator TIAM1, and c-MYC, a known driver of the cancer stem cell phenotype. Moreover, colonospheres treated with CtBP inhibitor showed downregulation of cancer stem cell markers CD133 and c-MYC, as compared with vehicle treatment. Flow cytometry analysis of intestinal epithelium from Apcmin mice treated with CtBP inhibitor vs. vehicle indicated a significant reduction in CD133+/CXCR4+ cells that are associated with a tumor initiating cell phenotype. Our data demonstrate that CtBP inhibition may reduce colon neoplasia via downregulation of c-MYC and the cancer stem cell/tumor initiating cell compartment, and that CtBP is a promising and novel target for therapy in colon cancer. Citation Format: Ayesha T. Chawla, Agnes Cororaton, Rashmi Seth, Barbara Szomju, Evan T. Sumner, Steven R. Grossman. Therapeutic targeting of C-terminal binding protein: a key dependency for polyposis and cancer stem cell activity in APC mutant neoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3901. doi:10.1158/1538-7445.AM2017-3901

Abstract 2226: Sildenafil suppresses tumorigenesis in ApcMin/+ mouse model

Abstract The cGMP signaling pathway regulates homeostasis in the colon epithelium and has been implicated in the suppression of colorectal cancer. Exisulind is a weak phosphodiesterase-5 (PDE5) inhibitor that was previously shown to reduce polyp formation in familial adenomatous polyposis (FAP) patients. Despite this validation of PDE5 as a therapeutic target, the failure of Exisulind due to toxicity halted further trials of cGMP elevating agents. The present study tested the effect of the PDE5 inhibitor sildenafil, and the receptor guanylyl-cyclase C (GCC) agonist linaclotide on tumorigenesis in the ApcMin/+ mouse model of colon cancer. Administration of either sildenafil or linaclotide to both wild type and ApcMin/+ mice caused dramatic effects on homeostasis that included reduced proliferation and increased goblet cell differentiation. With treatment beginning at 4 weeks, sildenafil and linaclotide caused a 50% and 67% (respectively) reduction in the number of polyps per mouse. Furthermore, the polyps in sildenafil and linaclotide treated animals exhibited a reduced proliferative index and increased mucus density compared to polyps from untreated mice. The mean polyp size was not changed by treatment with either drug. In support of a tumor-suppressive role for cGMP signaling in the intestine, the ApcMin/+ mice showed reduced levels of endogenous GCC agonists in the intestinal mucosa compared to wild type animals. This suggests that part of the tumor preventative effects observed here could be due to compensation for this deficit by augmenting cGMP signaling with PDE5 inhibitors or GCC agonists. While the tumor suppressive mechanism is not fully understood, the results presented here demonstrate in a preclinical model, that increasing intestinal cGMP levels by targeting PDE5 or GCC may be a viable chemoprevention strategy for human FAP patients. Citation Format: Sarah K. Sharman, Bianca N. Islam, Yali Hou, Allison Bridges, Nagendra Singh, Subbaramiah Sridhar, Frankin G. Berger, Darren D. Browning. Sildenafil suppresses tumorigenesis in ApcMin/+ mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2226. doi:10.1158/1538-7445.AM2017-2226

Abstract PR03: The pig as a model for colorectal cancer

Abstract Colorectal cancer is the fourth most common cancer worldwide with a mortality of more than 50%. Contrary to the increasing advances in cancer research colorectal cancer deaths are increasing in number. Most animal models also for colorectal cancer are in rodents. However, mice differ significantly in size, lifespan, physiology, anatomy and diet. Furthermore, genetically engineered mouse models for colorectal cancer show polyp development in the small intestine, contrary to the human phenotype that develops polyps in the large intestine. Therefore, a more suitable animal model is necessary. Pigs are increasingly recognized as a valuable model facilitating diagnostic and therapeutic strategies at human scale, longitudinal studies of disease initiation, progression and treatment response in preclinical trials. We generated gene targeted cloned pigs carrying the nonsense mutation, APC1311 that is orthologous to a human mutation associated with a very severe phenotype of colorectal cancer and the inherited condition familial adenomatous polyposis (FAP). In pigs the APC1311 mutation results in multiple adenomatous polyps in the colon and rectum that, so far, have progressed to carcinoma in situ. Histological and molecular analysis show that the porcine model recapitulates all major features of early stage human FAP. Breeding and analysis of the mutant pig herd has revealed segregation of traits that affect polyposis severity in animals carrying the same APC1311 mutation. We are currently investigating the genetic basis for this effect by comparing normal mucosa samples from low and high polyp animals and have discovered some genes that are differentially expressed. A progression study comparing low-grade with high-grade adenomas also identified differentially expressed genes and miRNAs. Further improvements of this model are targeted at generating organ/tissue specific Cre pigs to activate latent mutations such as KRAS and TP53 mutations in the colon. Additionally to encourage cancer progression we are working on the introduction of ubiquitous Cas9 expression in our APC1311 pigs to allow the activation of oncogenic pathways locally in vivo. Our porcine model provides a powerful new resource for preclinical cancer research, including identifying and investigating novel biomarkers for early detection. This abstract is also being presented as Poster A20. Citation Format: Carolin Wander, Tatiana Flisikowska, Krzysztof Flisikowski, Érica Schulze, Hans-Rudolf Fries, Stefan Bauersachs, Alexander Kind, Angelika Schnieke. The pig as a model for colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR03.