Apc Mutant Mice Research Articles

Telomere dysfunction alters intestinal stem cell dynamics to promote cancer

Telomere dynamics are linked to aging hallmarks, and age-associated telomere loss fuels the development of epithelial cancers. In Apc-mutant mice, the onset of DNA damage associated with telomere dysfunction has been shown to accelerate adenoma initiation via unknown mechanisms. Here, we observed that Apc-mutant mice engineered to experience telomere dysfunction show accelerated adenoma formation resulting from augmented cell competition and clonal expansion. Mechanistically, telomere dysfunction induces the repression of EZH2, resulting in the derepression of Wnt antagonists, which causes the differentiation of adjacent stem cells and a relative growth advantage to Apc-deficient telomere dysfunctional cells. Correspondingly, in this mouse model, GSK3β inhibition countered the actions of Wnt antagonists on intestinal stem cells, resulting in impaired adenoma formation of telomere dysfunctional Apc-mutant cells. Thus, telomere dysfunction contributes to cancer initiation through altered stem cell dynamics, identifying an interception strategy for human APC-mutant cancers with shortened telomeres.

Abstract 3006: Morphogen-driven regulation of nuclear shape and its implication for colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide despite improved rates of early detection and development of targeted treatments. Dysregulation of nuclear shape and the emergence of aberrant chromatin architecture correlate with cancer onset and progression, including CRC. Both extrinsic and intrinsic factors, most critically cytoskeletal and nucleoskeletal organization, chromatin architecture, and metabolic signaling, impact nuclear shape. Yet, what precisely determines nuclear shape in health and disease and how it is linked to cell fate and function remains a fundamental open question. In the case of over 75% of CRC cases, the first transformative genetic event is the loss of function in the tumor suppressor gene, adenomatous polyposis coli (APC). Mechanistically, perturbed APC signaling leads to aberrant activation of Wnt/beta-catenin signaling. This pathway is essential for intestinal homeostasis, as is the regulation of proper nuclear shape and function. Here, we aim to decipher the crosstalk between Wnt gradients and nuclear shape regulation in non-transformed and APC-mutated epithelial cells in vitro, as well as in WT and APC-mutant mouse colon tissues. The long-term goal of this work is to test whether a mechanistic link exists between Wnt-driven cellular transformation and the emergence of aberrant nuclear shape and function. Citation Format: Mona A. El Gendi, Solène Hervé, Sudeep Gautam, Yekaterina A. Miroshnikova. Morphogen-driven regulation of nuclear shape and its implication for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3006.

Amine Oxidase, Copper Containing 3 (Aoc3) Knockout Mice Are More Prone to DSS-induced Colitis and Colonic Tumorigenesis.

Inflammatory bowel diseases and colorectal cancer are a major cause of morbidity and mortality. Amine oxidase, copper-containing 3 (AOC3) is a critical enzyme in the physiological trafficking of leukocytes and the regulation of inflammation. This study aimed to examine the effects of Aoc3 deficiency in mice models of colitis and colorectal tumorigenesis. C57BL/6 and Aoc3 knockout mice were used for Dextran Sodium Sulfate (DSS) induced acute colitis and the Azoxymethane (AOM)/DSS model of inflammation-related colon cancer. We also evaluated the effect of Aoc3 in an Apc mutant mice model of intestinal and colonic tumorigenesis. We observed that Aoc3 deficient mice were more prone to colitis induced by DSS in early phases and their survival was shorter. We also showed that Aoc3 deficient mice developed more tumors both in AOM/DSS and Apc mutant mice models. Furthermore, colonic tumors in the AOM/DSS groups in Aoc3 mutant mice were generally invasive type adenocarcinomas. Aoc3 deficiency promotes colitis and colonic tumorigenesis in mouse models.

Gut virome profiling identifies an association between temperate phages and colorectal cancer promoted by Helicobacter pylori infection

ABSTRACT Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. While a close correlation between chronic Helicobacter pylori infection and CRC has been reported, the role of the virome has been overlooked. Here, we infected Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive metagenomics analysis of H. pylori-induced changes in lower gastrointestinal tract bacterial and viral communities. We observed an expansion of temperate phages in H. pylori infected Apc +/1638N mice at the early stage of carcinogenesis. Some of the temperate phages were predicted to infect bacteria associated with CRC, including Enterococcus faecalis. We also observed a high prevalence of virulent genes, such as flgJ, cwlJ, and sleB, encoded by temperate phages. In addition, we identified phages associated with pre-onset and onset of H. pylori-promoted carcinogenesis. Through co-occurrence network analysis, we found strong associations between the viral and bacterial communities in infected mice before the onset of carcinogenesis. These findings suggest that the expansion of temperate phages, possibly caused by prophage induction triggered by H. pylori infection, may have contributed to the development of CRC in mice by interacting with the bacterial community.

The long non-coding RNA MALAT1 regulates intestine host-microbe interactions and polyposis.

The long non-coding RNA (lncRNA) Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) maintains the integrity of the intestinal epithelial barrier and regulates local inflammation. However, its influences on intestinal microbial communities and tissue susceptibility to cancer development remain unexplored. Here, we report that MALAT1 regulates host anti-microbial response gene expression and the composition of mucosal-associated microbial communities in a region-specific manner. In the APC mutant mouse model of intestine tumorigenesis, knocking out MALAT1 results in higher polyp counts in the small intestine and colon. Interestingly, intestine polyps that developed in the absence of MALAT1 were smaller in size. These findings highlight the unexpected bivalent role of MALAT1 in restricting and promoting cancer progression at different disease stages. Among the 30 MALAT1-targets shared by both the small intestine and colon, ZNF638 and SENP8 levels are predictive of colon adenoma patient overall survival and disease-free survival. Genomic assays further revealed that MALAT1 modulates intestinal target expression and splicing through both direct and indirect mechanisms. This study expands the role of lncRNAs in regulating intestine homeostasis, microbial communities, and cancer pathogenesis.

Helicobacter pylori promotes colorectal carcinogenesis by deregulating intestinal immunity and inducing a mucus-degrading microbiota signature

ObjectiveHelicobacter pylori infection is the most prevalent bacterial infection worldwide. Besides being the most important risk factor for gastric cancer development, epidemiological data show that infected individuals harbour a nearly...

Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium

BackgroundMethods for the long-term in situ transduction of the unperturbed murine intestinal epithelium have not been developed in past research. Such a method could speed up functional studies and screens to identify genetic factors influencing intestinal epithelium biology. Here, we developed an efficient method achieving this long-sought goal.ResultsWe used ultrasound-guided microinjections to transduce the embryonic endoderm at day 8 (E8.0) in utero. The injection procedure can be completed in 20 min and had a 100% survival rate. By injecting a small volume (0.1–0.2 μl) of concentrated virus, single shRNA constructs as well as lentiviral libraries can successfully be transduced. The new method stably and reproducibly targets adult intestinal epithelium, as well as other endoderm-derived organs such as the lungs, pancreas, liver, stomach, and bladder. Postnatal analysis of young adult mice indicates that single transduced cells at E8.0 gave rise to crypt fields that were comprised of 20–30 neighbouring crypts per crypt-field at 90 days after birth. Lentiviral targeting of ApcMin/+ mutant and wildtype mice revealed that heterozygous loss of Apc function suppresses the developmental normal growth pattern of intestinal crypt fields. This suppression of crypt field sizes did not involve a reduction of the crypt number per field, indicating that heterozygous Apc loss impaired the growth of individual crypts within the fields. Lentiviral-mediated shRNA knockdown of p53 led to an approximately 20% increase of individual crypts per field in both Apc+/+ and ApcMin/+ mice, associating with an increase in crypt size in ApcMin/+ mice but a slight reduction in crypt size in Apc+/+ mice. Overall, p53 knockdown rescued the reduction in crypt field size in Apc-mutant mice but had no effect on crypt field size in wildtype mice.ConclusionsThis study develops a novel technique enabling robust and reproducible in vivo targeting of intestinal stem cells in situ in the unperturbed intestinal epithelium across different regions of the intestine. In vivo somatic gene editing and genetic screening of lentiviral libraries has the potential to speed up discoveries and mechanistic understanding of genetic pathways controlling the biology of the intestinal epithelium during development and postnatal life. The here developed method enables such approaches.

Role of Reduced Bdnf Expression in Novel Apc Mutant Allele-induced Intestinal and Colonic Tumorigenesis in Mice.

Brain-derived neurotrophic factor (BDNF) is a growth factor of the neurotrophin family. Recent studies indicate that its expression is regulated by Wnt/β-catenin signaling. In this study, we aimed to examine the effects of reduced Bdnf levels in an Apc mutant intestinal/colonic tumor mouse model. We crossed Apc+/- and Bdnf+/- C57BL/6 mice. After genotyping the litters, Apc+/+ Bdnf+/+ (wild-type, wt), Apc+/- Bdnf+/+ (Apc mutant), Apc+/+ Bdnf+/- (Bdnf mutant), and Apc+/- Bdnf+/- (Apc/Bdnf double mutant) mice cohorts were generated. All mice were followed daily for 36 weeks and weighed once a week, and mice that died or reached a terminal stage before this period were also recorded and dissected. At the end of this period, all surviving mice were sacrificed, and tissue samples were collected. Polyp numbers in the small intestine and colon were counted. Microscopic slides were prepared for histopathological examination. Protein extraction was performed both for tumor and normal tissue analysis. A significant weight gain was observed in the Bdnf mutant and Apc/Bdnf double mutant cohorts compared to wt and Apc mutant controls. In Apc/Bdnf double mutant mice, the small intestinal polyp count was slightly decreased, and the colon polyp count increased significantly, and developed the disease phenotype significantly later than Apc mutant mice. Bdnf level has an important role in the Apc mutant intestinal and colonic tumorigenesis model. Modulation of Bdnf levels can be a potential therapeutic target in colorectal cancer.

Abstract 3721: MTORC1/2 and HDAC1/2 inhibition as therapy for colorectal cancer with PIK3CA mutation

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related deaths. PIK3CA mutations are found in 18% of CRCs which lead to the constitutive activation of the PI3K/MTOR pathway and can promote tumorigenesis. Here, we examine the efficacy of the FDA approved inhibitors copanlisib, a PI3K/MTOR inhibitor, and romidepsin, a HDAC1/2 inhibitor, in CRC with a PIK3CA mutation. Methods: CRC mouse derived cancer organoids (MDCOs) were derived from Apc and Pik3ca mutant mice (Fc1Apcfl/+Pik3caH1047R/+). Brightfield images of the MDCOs were taken prior to treatment with copanlisib, romidepsin, or the combination and 48 hours post-treatment. The change in diameter of each organoid over the 48-hour treatment was determined. Additionally, immunoblotting was performed to confirm known targets of copanlisib and romidepsin were altered in response to drug treatment. MTORC1/2 and HDAC1/2 inhibition were also investigated in vivo. SW48 and SW48PIK3CA-H1047R (SW48PK) xenograft mice were treated with a vehicle control, copanlisib, romidepsin, or the combination therapy. Results: In the diameter analysis study, the combination therapy had the largest effect size compared to control (Glass’s Δ 2.82). Single agents copanlisib and romidepsin had smaller effect sizes (Glass’s Δ 1.75 and 2.04, respectively). Immunoblotting results indicated a decrease in phosphoAKT (Ser473) and pRPS6 (Ser235/236) in the copanlisib treated samples and an increase in H3K27 acetylation was seen in the romidepsin treated samples. There was also increased cleaved PARP, an indicator of apoptosis, in the romidepsin and combination therapy treatment groups. In vivo, SW48 xenografts showed a greater response in the combination therapy compared to either single agent therapy (median relative change in tumor volume: control=339%; combination therapy=107%(p-value<0.05), copanlisib=225%(p-value=0.17), romidepsin=200%). A similar trend was seen in the SW48PK xenograft mice (median relative change in tumor volume: control=241%; combination therapy=70% (p-value<0.05), copanlisib=132%(p-value<0.05), romidepsin=177%). Conclusion: MDCOs with Apc and Pik3ca mutations had an increased response to treatment with the combination therapy as compared to the single agents alone. Additionally, in vivo studies with human CRC xenografts showed enhanced inhibition of tumor growth with both MTORC1/2 and HDAC1/2 inhibition. These data demonstrate potential for this combination treatment strategy for the treatment of PIK3CA mutant CRC and this combination warrants further investigation in other models and clinically. Citation Format: Autumn M. Olson, Rebecca A. DeStefanis, Alyssa K. DeZeeuw, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. MTORC1/2 and HDAC1/2 inhibition as therapy for colorectal cancer with PIK3CA mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3721.

Olfactomedin 4 Is Not a Precise Marker for Human Intestinal Stem Cells, But Is Involved in Intestinal Carcinogenesis

Olfactomedin 4 Is Not a Precise Marker for Human Intestinal Stem Cells, But Is Involved in Intestinal Carcinogenesis

Fatty acid metabolism and colon cancer protection by dietary methyl donor restriction

IntroductionA methyl donor depleted (MDD) diet dramatically suppresses intestinal tumor development in Apc-mutant mice, but the mechanism of this prevention is not entirely clear.ObjectivesWe sought to gain insight into the mechanisms of cancer suppression by the MDD diet and to identify biomarkers of cancer risk reduction.MethodsA plasma metabolomic analysis was performed on ApcΔ14/+ mice maintained on either a methyl donor sufficient (MDS) diet or the protective MDD diet. A group of MDS animals was also pair-fed with the MDD mice to normalize caloric intake, and another group was shifted from an MDD to MDS diet to determine the durability of the metabolic changes.ResultsIn addition to the anticipated changes in folate one-carbon metabolites, plasma metabolites related to fatty acid metabolism were generally decreased by the MDD diet, including carnitine, acylcarnitines, and fatty acids. Some fatty acid selectivity was observed; the levels of cancer-promoting arachidonic acid and 2-hydroxyglutarate were decreased by the MDD diet, whereas eicosapentaenoic acid (EPA) levels were increased. Machine-learning elastic net analysis revealed a positive association between the fatty acid-related compounds azelate and 7-hydroxycholesterol and tumor development, and a negative correlation with succinate and β-sitosterol.ConclusionMethyl donor restriction causes dramatic changes in systemic fatty acid metabolism. Regulating fatty acid metabolism through methyl donor restriction favorably effects fatty acid profiles to achieve cancer protection.

Abstract 2969: Impact of baseline culture condition on the growth and treatment response of mouse derived cancer organoids

Abstract Impact of baseline culture condition on the growth and treatment response of mouse derived cancer organoidsAuthors: Autumn Olson, Rebecca A. DeStefanis, Alyssa DeZeeuw, Samantha Anderson, Gioia Sha, Jeremy Kratz, Cheri Pasch, Linda Clipson, Dustin A. DemingIntroduction: Colorectal cancer (CRC) is a leading cause of cancer related deaths and there has been an increased focus on developing therapies targeted to the mutational profile of individual patients. Mouse derived cancer organoids (MDCOs) are used as a model for therapeutic drug testing. How the culture conditions can impact the determination of treatment response from MDCOs has been understudied and is the focus of these investigations. Here, we examine how baseline size, passage number, plating density, location within the Matrigel droplet, and lineage impact MDCO growth and treatment response.Methods: CRC MDCOs were derived from multiple tumors across several Apc and Pik3ca mutant mice (Fc1 Apcfl/+ Pik3caH1047R/+) totaling 3,152 individual MDCOs. Untreated MDCO growth was analyzed across 902 MDCOs. Additionally, the PI3K pathway inhibitors, AZD2014, Everolimus, BEZ235, MLN0128, Palbociclib, and Copanlisib were analyzed across 2,250 MDCOs. In each study, baseline brightfield images of the MDCOs were taken and following 48 hours. Individual MDCOs were evaluated for the impact of baseline size, passage number (passage 1-15), plating density, location within the Matrigel droplet, and the generation of MDCOs across different mice on MDCO growth and treatment response. A change point analysis was used to assess the impact of baseline size. The percent relative change in diameter over 48 hours was compared to each other culture condition by calculating an R2 value.Results: A change in the organoid baseline growth rate was observed if the MDCOs were greater than 0.373 mm in baseline was identified. There were no significant differences in growth due to changes in the other culture conditions for the untreated MDCOs. The R2 values comparing the percent relative change in diameter over 48 hours to the culture conditions of passage number, plating density, location within the Matrigel droplet, and from which mouse the MDCOs were derived from were 0.0103, 0.0243, 0.0001, and 0.0295, respectively. There were no significant differences in the response to PI3K pathway inhibition of the MDCOs related to passage number, plating density, location within the Matrigel droplet, and from which mouse the MDCOs were derived from (R2 values: 0.1174, 0.0979, 0.0221, and 0.1267, respectively).Conclusions: Mouse derived cancer organoids are a useful tool in modeling drug response of cancers with specific mutational profiles. While the baseline size of MDCOs can change the growth rate, the growth rate and treatment response of MDCOs is not impacted by the passage number, plating density, location within the Matrigel, and from which mouse the MDCOs were derived. Citation Format: Autumn Olson, Rebecca A. DeStefanis, Alyssa DeZeeuw, Samantha Anderson, Gioia Sha, Jeremy Kratz, Cheri Pasch, Linda Clipson, Dustin A. Deming. Impact of baseline culture condition on the growth and treatment response of mouse derived cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2969.

Abstract 3059: Heavy ion radiation exposure perturbs intestinal stem cell homeostasis and induces accelerated tumorigenesis in apc mutant mouse intestine

Abstract In a previous study, we demonstrated long-term decreased intestinal epithelial cell migration after low-dose heavy ion iron radiation, and this was associated with increased senescence-associated secretory phenotype (SASP) signaling. However, we know very little about the long-term effects of low dose space radiation on intestinal stem cell (ISC) senescence and SASP that have implications for intestinal homeostasis. Male Lgr5+ (Lgr5-EGFP-IRES-creERT) mice or Lgr5+/APC1638N/+ were exposed to 0.5 Gy of sham, γ-rays, or 28Si (69 keV/μm) radiation and were euthanized 60 or 150 days after irradiation. ISCs were FACS sorted for oxidative stress analysis and assessed for senescence and SASP signaling in tissue sections by immunostaining. Greater cellROX or mitoSOX staining was observed after 28Si indicating higher oxidative stress compared to γ-rays or control Lgr5 positive cells. β-gal co-staining with Lgr5, lysozyme, IL1R, or ILβ showed highest staining in 28Si irradiated compared to controls ISCs samples. γ-H2AX staining showed greater foci numbers in 28Si relative to γ-rays or control group. Higher expression of secretory cytokines IL8 and IL15 was observed in 28Si irradiated ISCs relative to γ-rays or control group at both time points. Elevated expression of DKK2 and activation of NF-κB and p38 MAPK were observed in senescent stem cells of 28Si irradiated relative to γ-rays or control group at these time points. A higher GI tumor count was observed in 28Si compared to γ-irradiated or control Lgr5+/APC1638N/+ mice. In summary, our results demonstrate low dose heavy ion exposure induces ongoing DNA damage, persistent oxidative stress, senescence and acquisition of the SASP in ISCs. Higher levels of oxidative stress, senescence and SASP were observed at 150 compared to the 60-day timepoint. Overall, space radiation-induced oxidative stress, senescence, SASP, and activated p38MAPK/NF-κB pathway may very well have implications in GI carcinogenesis, accelerated aging, and pathological conditions, such as intestinal dysfunction, in astronauts during deep space flights. Since similar effects have been seen in our studies with other particle beams including carbon ions, this has potential relevance to particle radiotherapy as well. This work was supported by NASA NNX15AI21G Citation Format: Santosh Kumar, Shubhankar Suman, Kamal Datta, Albert J. Fornace. Heavy ion radiation exposure perturbs intestinal stem cell homeostasis and induces accelerated tumorigenesis in apc mutant mouse intestine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3059.

Abstract 440: Dual role of epithelial MyD88 inApc-mutant intestinal tumors

Abstract Mutational activation of the Wnt/β-catenin signaling plays essential roles in initiating the adenoma-carcinoma sequence for the majority of colorectal cancers (CRCs) as well as in maintaining the transformed phenotypes of progressed CRCs. However, the Wnt/β-catenin signaling itself is not easy to target for therapy. Here we addressed the roles of MyD88, a critical adaptor protein in the interleukin (IL)-1β and Toll-like receptor (TLR) pathways, in intestinal epithelial cells (IECs) during intestinal tumor formation in Apc+/Δ716 mice, a mouse model of familial adenomatous polyposis as well as the early-stage spontaneous colorectal cancer. Conditional knockout of MyD88 in IECs significantly reduced the number of intestinal tumors in Apc+/Δ716 mice. Although suppression of tumor formation in Apc mutant mice by IEC-specific knockout of MyD88 was previously reported by others, our mechanistic studies uncovered the downstream molecular pathways of MyD88 involved in tumor formation. (1) IEC-specific MyD88 loss induced inactivation of the JNK/mTORC1 signaling, a pathway we previously demonstrated to be essential in the growth of Apc+/Δ716 tumors, leading to decreased proliferation of tumor cells in vivo. (2) IEC-specific MyD88 loss reduced the level of NF-κB p65 and attenuated the Wnt/β-catenin signaling, leading to apoptosis of intestinal tumor cells in vivo. (3) Induction of MyD88 deletion in the intestinal tumor-derived organoids, but not in the normal IEC-derived organoids, caused apoptotic cell death and suppressed their growth in vitro. (4) Knockdown of MYD88 in colorectal cancer cell lines harboring mutations in the Wnt/β-catenin signaling reduced their proliferation and induced apoptosis. These results demonstrate a novel mechanism by which epithelial MyD88 contributes to intestinal tumorigenesis, and also suggest synthetic lethality between MyD88 deficiency and mutations in the Wnt/β-catenin signaling in CRCs. Citation Format: Rie Kajino-Sakamoto, Teruaki Fujishita, Makoto Mark Taketo, Masahiro Aoki. Dual role of epithelial MyD88 inApc-mutant intestinal tumors [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 440.

Integration of genomics, metagenomics, and metabolomics to identify interplay between susceptibility alleles and microbiota in adenoma initiation

BackgroundColorectal cancer (CRC) is a multifactorial disease resulting from both genetic predisposition and environmental factors including the gut microbiota (GM), but deciphering the influence of genetic variants, environmental variables, and interactions with the GM is exceedingly difficult. We previously observed significant differences in intestinal adenoma multiplicity between C57BL/6 J-ApcMin (B6-Min/J) from The Jackson Laboratory (JAX), and original founder strain C57BL/6JD-ApcMin (B6-Min/D) from the University of Wisconsin.MethodsTo resolve genetic and environmental interactions and determine their contributions we utilized two genetically inbred, independently isolated ApcMin mouse colonies that have been separated for over 20 generations. Whole genome sequencing was used to identify genetic variants unique to the two substrains. To determine the influence of genetic variants and the impact of differences in the GM on phenotypic variability, we used complex microbiota targeted rederivation to generate two Apc mutant mouse colonies harboring complex GMs from two different sources (GMJAX originally from JAX or GMHSD originally from Envigo), creating four ApcMin groups. Untargeted metabolomics were used to characterize shifts in the fecal metabolite profile based on genetic variation and differences in the GM.ResultsWGS revealed several thousand high quality variants unique to the two substrains. No homozygous variants were present in coding regions, with the vast majority of variants residing in noncoding regions. Host genetic divergence between Min/J and Min/D and the complex GM additively determined differential adenoma susceptibility. Untargeted metabolomics revealed that both genetic lineage and the GM collectively determined the fecal metabolite profile, and that each differentially regulates bile acid (BA) metabolism. Metabolomics pathway analysis facilitated identification of a functionally relevant private noncoding variant associated with the bile acid transporter Fatty acid binding protein 6 (Fabp6). Expression studies demonstrated differential expression of Fabp6 between Min/J and Min/D, and the variant correlates with adenoma multiplicity in backcrossed mice.ConclusionsWe found that both genetic variation and differences in microbiota influences the quantitiative adenoma phenotype in ApcMin mice. These findings demonstrate how the use of metabolomics datasets can aid as a functional genomic tool, and furthermore illustrate the power of a multi-omics approach to dissect complex disease susceptibility of noncoding variants.

Theracurmin inhibits intestinal polyp development in Apc-mutant mice by inhibiting inflammation-related factors.

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Therefore, it is important to establish useful methods for preventing CRC. One prevention strategy involves the use of cancer chemopreventive agents, including functional foods. We focused on the well‐known cancer chemopreventive agent curcumin, which is derived from turmeric. However, curcumin has the disadvantage of being poorly soluble in water due to its high hydrophobicity. To overcome this problem, the formation of submicron particles with surface controlled technology has been applied to curcumin to give it remarkably improved water solubility, and this derived compound is named Theracurmin. To date, the preventive effects of Theracurmin on hereditary intestinal carcinogenesis have not been elucidated. Thus, we used Apc‐mutant mice, a model of familial adenomatous polyposis, to evaluate the effects of Theracurmin. First, we showed that treatment with 10‐20 µM Theracurmin for 24 hours reduced nuclear factor‐κB (NF‐κB) transcriptional activity in human colon cancer DLD‐1 and HCT116 cells. However, treatment with curcumin mixed in water did not change the NF‐κB promoter transcriptional activity. As NF‐κB is a regulator of inflammation‐related factors, we next investigated the downstream targets of NF‐κB: monocyte chemoattractant protein‐1 (MCP‐1) and interleukin (IL)‐6. We found that treatment with 500 ppm Theracurmin for 8 weeks inhibited intestinal polyp development and suppressed MCP‐1 and IL‐6 mRNA expression levels in the parts of the intestine with polyps. This report provides a proof of concept for the ongoing Theracurmin human trial (J‐CAP‐C study).

Antiangiogenic immunotherapy suppresses desmoplastic and chemoresistant intestinal tumors in mice.

Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.

Methyl Donor Deficiency Blocks Colorectal Cancer Development by Affecting Key Metabolic Pathways.

Our understanding of the role of folate one-carbon metabolism in colon carcinogenesis remains incomplete. Previous studies indicate that a methyl donor-deficient (MDD) diet lacking folic acid, choline, methionine, and vitamin B12 is associated with long-lasting changes to the intestinal epithelium and sustained tumor protection in Apc-mutant mice. However, the metabolic pathways by which the MDD diet affects these changes are unknown. Colon samples harvested from ApcΔ14/+ mice fed the MDD diet for 18 weeks were profiled using a GC-MS and LC-MS/MS metabolomics platform. Random forest and pathway analyses were used to identify altered metabolic pathways, and associated gene expression changes were analyzed by RT-PCR. Approximately 100 metabolites affected by the MDD diet were identified. As expected, metabolites within the methionine cycle, including methionine (-2.9-fold, P < 0.001) and betaine (-3.3-fold, P < 0.001), were reduced. Elevated homocysteine (110-fold, P < 0.001) was associated with increased flux through the transsulfuration pathway. Unexpectedly, levels of deoxycholic acid (-4.5-fold, P < 0.05) and several other secondary bile acids were reduced. There were also unexpected reductions in the levels of carnitine (-2.0-fold, P < 0.01) and a panel of acylcarnitines involved in fatty acid β-oxidation. Finally, metabolites involved in redox balance, including ascorbate and hypotaurine, were found to be persistently elevated. These findings provide clues to the molecular changes underlying MDD-mediated tumor protection and identify regulatable metabolic pathways that may provide new targets for colon cancer prevention and treatment. IMPLICATIONS: Metabolomic profiling reveals molecular changes underlying MDD-induced tumor protection and may provide new targets for colorectal cancer prevention and treatment.

Abstract 5073: mPGES-1 deficiency impairs self-renewal properties of colon cancer stem cells

Abstract Dysregulation of prostaglandin E2 (PGE2) signaling is a hallmark of many cancers, including colorectal cancer (CRC). Direct suppression of inducible PGE2 synthesis by genetic deletion of Ptges1 (mPGES-1) affords dramatic cancer protection to the colon. However, the precise mechanisms by which this effect occurs remain incompletely understood. Recent evidence points to a fundamental role of PGE2 signaling in the expansion of cancer stem cells. In the present study, using an organoid system established from Apc mutant mice, we have evaluated the influence of PGE2 and related prostanoids on the viability of cancer stem cells in the colon. To establish cancer organoids, colon tumors were harvested from 16 week-old ApcΔ14/+ mice with or without mPGES-1 (D14:WT and D14:KO). Colon tumor multiplicity in D14:KO mice is approximately half the number of tumors in D14:WT mice. Tumor tissues were digested with collagenase and dispase, and cultured in Matrigel with complete organoid media. Organoid growth was scored daily over a one-week time period, and passaged. Tumor organoids generated from D14:WT and D14:KO mice showed no obvious differences in their growth characteristics nor morphological features during the first week of ex-vivo growth. After the first passage, however, the growth rate of D14:KO organoids was significantly reduced (175% vs. 80%, WT and KO, respectively), and by the second passage, D14:KO organoids failed to maintain cell viability. Immunohistochemical analysis of the organoids showed strong nuclear localization of ß-catenin and loss of Apc expression regardless of mPGES-1 genotype, indicating intact Wnt-driven cancer growth mechanisms that were independent of inducible PGE2 signaling. These observations indicate that alternative mechanisms are contributing to the compromised stem cell expansion observed in the D14:KO organoids. Since mPGES-1 is expressed within the tumor stroma, we postulated that the inactivation of mPGES-1 would cause a marked microenvironmental change. To test this possibility, we used GC-MS/MS to measure a panel of prostanoids within the tumor tissue. Targeted lipidomic analysis showed significant metabolite redirection of tissue prostanoids, with a significant increase (2-fold) in PGD2in the D14:KO mice. Interestingly, administration of PGD2 was recently shown to reduce the growth and regeneration potential of stem cells within a hair follicle organ culture system. Given new evidence that PGD2 may have a direct influence on ‘stem cell-ness' in other organ systems, we propose that the colon tumor microenvironment, deficient in inducible PGE2, may undergo permanent molecular changes that directly influence the growth characteristics and viability of cancer stem cells as reflected in our organoid culture system. New data is presented to define the long-lasting influence of eicosanoid metabolic changes on cancer-derived stem cell viability and proliferative capacity. Citation Format: Masako Nakanishi, Daniel W. Rosenberg. mPGES-1 deficiency impairs self-renewal properties of colon cancer stem cells [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 5073.

SAT-LB057 High-Fructose Corn Syrup Enhances Intestinal Tumor Growth in Mice

An increasing number of studies have suggested a potential link between sugar consumption and cancer. However, a direct relationship between the two remains controversial. Here, we treated APC-mutant mice daily with a modest amount of high-fructose corn syrup (HFCS) via oral gavage to mimic sugar-sweetened beverage (SSB) consumption and evaluated the effects of SSBs on intestinal tumorigenesis. We observed a dramatic increase in tumor size and tumor grade in HFCS-treated mice despite no signs of obesity or metabolic syndrome. A bolus of HFCS spikes the level of fructose and glucose in the intestinal lumen and serum, respectively, allowing tumors to take up both sugars efficiently. Ketohexokinase (KHK) in tumors rapidly converts fructose to fructose-1-phosphate (F1P), lowering cytosolic ATP in the process. Consequently, this leads to the activation of glycolysis as ATP depletion relieves the allosteric inhibition of phosphofructokinase (PFK), the most critical regulatory enzyme in glycolysis. This accelerated glycolysis contributes to the increased de novo lipogenesis pathway that enhances tumor growth in HFCS-treated mice. Importantly, the deletion of KHK or fatty acid synthase (FASN) in APC-mutant mice abolishes the HFCS-induced metabolic changes and tumor growth. Overall, our results provide a mechanistic rationale for restricting SSB consumption to decrease the rate of growth of early stage colorectal cancers. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.