Intestinal Tumorigenesis In ApcMin Research Articles

MLKL inhibits intestinal tumorigenesis by suppressing STAT3 signaling pathway.

Mixed lineage kinase domain-like protein (MLKL) plays an important role in necroptosis, but the role and mechanism of MLKL in intestinal tumorigenesis remain unclear. Here, we found that hematopoietic- and nonhematopoietic-derived MLKL affected intestinal inflammation, but nonhematopoietic-derived MLKL primarily inhibited intestinal tumorigenesis. Loss of MLKL enhanced intestinal regeneration and the susceptibility to intestinal tumorigenesis in Apcmin/+ mice by hyperactivating the Janus kinase 2 (JAK2)/ signal transducer and activator of transcription 3 (STAT3) axis. Furthermore, MLKL deficiency increased interleukin-6 (IL-6) production in dendritic cells. Administration of anti-IL-6R antibody therapy reduced intestinal tumorigenesis in Apcmin/+Mlkl-/- mice. Notably, low MLKL expression in human colorectal tumors, which enhanced STAT3 activation, was associated with decreased overall survival. Together, our results reveal that MLKL exhibits a suppressive effect during intestinal tumorigenesis by suppressing the IL-6/JAK2/STAT3 signals.

High-throughput metabolomics for discovering metabolic biomarkers from intestinal tumorigenesis in APC min/+ mice based on liquid chromatography/mass spectrometry

As a major public health concern, colon cancer is one of the most common cancer types, which is also the second cause of cancer death in developed countries and the third most common cancer in other parts of the world. It was reported that patients diagnosed at early stage have a chance to obtain 5-year survival rates at least compared to patients with late stage. Facing the multistep process in intestinal tumorigenesis, there is an urgent need to develop more effective early detection strategies for ameliorating the patient clinical outcome. Metabolomics open up a novel avenue of seeking valuable potential biomarkers for assessing disease severity and prognosticating course by dynamic snapshot of small molecule metabolites. The study aims to provide deeper insights into the discovery, identification and functional pathways analysis of differentially expressed metabolites in intestinal tumorigenesis in APC min/+ mice used by the serum metabolomics, and bring about useful information for further effective prevention and treatment of the disease. 17 marker metabolites and related metabolism pathway were identified using non-targeted metabolomics based on liquid chromatography/mass spectrometry (LC/MS) associated with multivariate statistical analysis. The ingenuity pathway analysis platform involved multiple-pathways was applied to metabolic network analysis for further understanding the relationship between functional metabolic pathways and disease.

MACROD2 Haploinsufficiency Impairs Catalytic Activity of PARP1 and Promotes Chromosome Instability and Growth of Intestinal Tumors.

ADP-ribosylation is an important posttranslational protein modification that regulates diverse biological processes, controlled by dedicated transferases and hydrolases. Here, we show that frequent deletions (∼30%) of the MACROD2 mono-ADP-ribosylhydrolase locus in human colorectal cancer cause impaired PARP1 transferase activity in a gene dosage-dependent manner. MACROD2 haploinsufficiency alters DNA repair and sensitivity to DNA damage and results in chromosome instability. Heterozygous and homozygous depletion of Macrod2 enhances intestinal tumorigenesis in ApcMin/+ mice and the growth of human colorectal cancer xenografts. MACROD2 deletion in sporadic colorectal cancer is associated with the extent of chromosome instability, independent of clinical parameters and other known genetic drivers. We conclude that MACROD2 acts as a haploinsufficient tumor suppressor, with loss of function promoting chromosome instability, thereby driving cancer evolution.Significance: Chromosome instability (CIN) is a hallmark of cancer. We identify MACROD2 deletion as a cause of CIN in human colorectal cancer. MACROD2 loss causes repression of PARP1 activity, impairing DNA repair. MACROD2 haploinsufficiency promotes CIN and intestinal tumor growth. Our results reveal MACROD2 as a major caretaker tumor suppressor gene. Cancer Discov; 8(8); 988-1005. ©2018 AACR.See related commentary by Jin and Burkard, p. 921This article is highlighted in the In This Issue feature, p. 899.

Abstract 4158: Differential intestinal tumorigenesis and DNA repair response in APCMin/+ mice after acute and fractionated proton radiation

Abstract Ionizing radiation (IR) exposure is a risk factor for colorectal cancer (CRC) development. With increasing interest in exploring outer space, astronauts on prolonged space missions such as a mission to Mars are expected to receive IR doses that could heighten the risk of CRC. It is estimated that about 90% of the IR in outer space is energetic protons, and the radiation dose during solar particle events (SPEs) could reach up to 2 Gy behind shielding. However, the risk of CRC after energetic proton radiation is not yet fully defined due to lack of human or animal model data. The purpose of the current study was to quantitatively and qualitatively compare and characterize differences in intestinal tumorigenesis between acute and fractionated proton irradiation in APCMin/+ mice, a well-established mouse model for human CRC. Mice were exposed to 1.88 Gy of proton radiation delivered in a single fraction or in 4 equal daily fractions (0.47 Gy x 4). Since proton dose to astronauts are variable based on space environment, in this initial study we chose a high single dose and a lower daily dose pattern to assess intestinal tumorigenesis. Intestinal tumor frequency and grade were noted, and tumor samples were collected from irradiated and control mice euthanized 100 to 110 days after radiation exposure. Molecular analysis was focused on DNA damage and repair, and cellular proliferative signaling pathways relevant to CRC using immunoblots, immunohistochemistry, and qPCR. Relative to control and fractionated-protons, there was significantly higher intestinal tumor frequency and grade after acute (single dose) proton irradiation. Decreased cellular differentiation and increased oxidative damage to DNA was observed after acute proton radiation. At the molecular level, our data showed increased DNA double strand breaks associated with decreased levels of DNA repair proteins, and upregulation of proliferative β-catenin and Akt signaling after acute proton exposure. Since increased DNA damage was not associated with concomitant increased apoptosis, our data suggest continued proliferation of cells bearing sub-lethal damage to promote tumorigenesis. In the fractionated proton group, tumor frequency and grade as well as molecular changes were comparable to sham-irradiated control group. When considered along with decreased tumor frequency and upregulation of DNA repair pathways after fractionation, our data are suggestive of marked differences in carcinogenic effects of acute vs. fractionated proton radiation. In summary, our data suggest that acute exposure to proton radiation is associated with higher risk of intestinal tumorigenesis in APCMin/+ mice with implications for gastrointestinal cancer risk in astronauts undertaking long duration space missions and in patients undergoing proton radiotherapy. Citation Format: Kamal Datta, Shubhankar Suman, Santosh Kumar, Albert J. Fornace. Differential intestinal tumorigenesis and DNA repair response in APCMin/+ mice after acute and fractionated proton radiation [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 4158.

Abstract 4984: Celastrol inhibits high fat diet-induced obesity and intestinal tumorigenesis in APCMin/+ mice by modulating gut microbes and inflammation

Abstract Obesity and inflammation play a vital role in colorectal cancer (CRC). Antiobesity agents may be beneficial for CRC prevention. Celastrol is a triterpene bioactive compound derived from Tripterygium wilfordii (TW) plant, which possesses antiobesity and anti-inflammatory properties. In the present study, we tested celastrol for its intestinal tumor inhibitory efficacy, modulation of intestinal microbiome, induction of UCP-1 in inguinal fat, and inflammation under obese conditions using APCMin/+ mouse model. For efficacy study, six-week-old male and female C57BL/6J-APCMin/+ mice (10 mice/group/gender) were fed high-fat diets (HFD; 60% Kcal fat) containing 0 and 150 ppm celastrol and a group of mice with a low-fat diet (LFD; 10% Kca fat) for 11 weeks. At termination, intestinal tumors were evaluated histologically and serum was assayed for fasting glucose, uric acid, liver enzymes (ALT, AST), triglycerides and cholesterol levels. Untreated and treated intestinal tumors were assayed for apoptosis and inflammatory markers by real-time PCR method. Results suggest that administration of LFD showed lower intestinal tumor formation by 52% (p<0.02) in males; 74% (p<0.0009) in females compared to HFD fed animals. Importantly, administration of HFD containing celastrol suppressed the intestinal polyp formation by 92% (p<0.0001) in males and 83.6% (p<0.0002) in females compared to control HFD fed mice. Also, significant inhibition of colonic tumor suppression was observed in (34%) male and (100%) female mice fed with celastrol. HFD fed animals showed 55% high-grade adenomas whereas HFD containing celastrol treatment showed 25% high-grade adenomas in colon. HFD containing celastrol treatment resulted in significantly reduced body weight gain, p<0.002, compared to HFD alone in both genders with upregulation of UCP1 protein in inguinal fat indicating increased thermogenesis. HFD celastrol treatment significantly reduced fasting glucose and triglycerides levels with an increase in uric acid with no effect on cholesterol, ALT and AST levels compared to control HFD fed mice. A sequence-based analysis of fecal microbiota of mice fed with HFD celastrol showed significantly decreased number of inflammation-causing microbes belonging to genus Prevotella (84%), Dorea (77%), Allobaculum (74%), and Aneroplasma (100%) and increased number of beneficial microbes belonging to genus Bifidobacterium (100%), Akkermansia (99.9%), Mucispirillum (94%) and Coprococcus (97%) compared to control HFD fecal samples. Celastrol treatment altered mRNA expression of IFN-γ, Ccl6, TGFβ1, and Aimp1. This is first study to report the chemopreventive properties of celastrol against the small intestinal and colonic neoplasia, modulation of gut microbiome and inflammation in APCMin/+ mice. Stephenson Cancer Center Grant. Citation Format: Naveena B. Janakiram, Venkateshwar Madka, Yuting Zhang, Nicole Stratton, Hima Bezawada, Beth Griesel, Altaf Mohammed, Ann L. Olson, Chinthalapally V. Rao. Celastrol inhibits high fat diet-induced obesity and intestinal tumorigenesis in APCMin/+ mice by modulating gut microbes and inflammation [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 4984.

Abstract A25: Intestinal tumorigenesis in APCMin/+ mice was higher after acute relative to fractionated proton radiation: Implications for space radiation-induced colorectal carcinogenesis

Abstract Ionizing radiation (IR) is a risk factor for colorectal cancer (CRC) based on epidemiologic studies. With increasing interest in exploring outer space, astronauts on prolonged space missions such as a mission to Mars will be exposed to IR doses that are expected to heighten the risk of CRC. Sources of space radiation beyond Earth's magnetosphere are galactic cosmic radiation (GCR) and solar particle event (SPE) and about 90% of the IR in outer space is energetic protons, which are more damaging than photon radiations such as γ-rays. While astronauts on deep-space missions are estimated to receive ~250 μGy/day of proton, radiation dose during SPE could reach up to ~2 Gy behind shielding. However, the risk of CRC after energetic proton radiation is not yet fully defined due to lack of human or animal model data. Limitations in obtaining adequate human data mandate that in vivo mechanistic data for CRC risk estimates be obtained using animal models and surrogate biologic and molecular end points relevant to human disease process. The purpose of the current study was to quantitatively and qualitatively compare and characterize differences in intestinal tumorigenesis between acute and fractionated proton irradiation in APCMin/+ mice, a well-established mouse model for human CRC. Mice were exposed to 1.88 Gy of proton radiation delivered in a single fraction or in 4 equal daily fractions (0.47 Gy x 4). Since proton dose to astronauts is variable based on space environment, in this initial study we chose a high single dose and a low daily dose pattern to assess intestinal tumorigenesis. Intestinal tumor frequency and grade were noted, and tumor and normal tissue samples were collected from irradiated and control mice euthanized 100 to 110 days after radiation exposure. Molecular analysis was focused on DNA damage, genomic instability, specific epigenetic changes and mutation analysis, and cellular proliferative signaling pathways relevant to CRC using immunoblots, immunohistochemistry, and PCR. Relative to control and fractionated protons, there was significantly higher intestinal tumor frequency and grade after acute proton irradiation. At the molecular level our data showed increased DNA damage and DNA double-strand breaks, decreased genomic stability, and upregulation of proliferative β-catenin and Akt signaling after acute proton exposure. Since increased DNA damage was not associated with concomitant increased apoptosis and there was increased microsatellite instability, our data are suggestive of continued proliferation of cells bearing sublethal damages. Mutational analysis showed significantly more tumors had higher overall mutation frequency and increased p53 hot spot mutations after acute relative to fractionated proton exposure. In fractionated proton groups, tumor frequency and grade as well as molecular changes were statistically comparable to control groups. When considered along with higher mutational frequency and altered promoter methylation of key CRC-related genes in tumor adjacent normal tissues, our data are suggestive of marked differences in carcinogenesis of acute vs. fractionated proton radiation with higher tumorigenic effects associated with acute exposures. Citation Format: Shubhankar Suman, Bo-Hyun Moon, Albert J. Fornace, Jr., Kamal Datta. Intestinal tumorigenesis in APCMin/+ mice was higher after acute relative to fractionated proton radiation: Implications for space radiation-induced colorectal carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A25.

Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis in Mice via a Toll-Like Receptor 4/p21-Activated Kinase 1 Cascade.

The underlying pathogenic mechanism of Fusobacterium nucleatum in the carcinogenesis of colorectal cancer has been poorly understood. Using C57BL/6-ApcMin/+ mice, we investigated gut microbial structures with F. nucleatum, antibiotics, and Toll-like receptor 4 (TLR4) antagonist TAK-242 treatment. In addition, we measured intestinal tumor formation and the expression of TLR4, p21-activated kinase 1 (PAK1), phosphorylated-PAK1 (p-PAK1), phosphorylated-β-catenin S675 (p-β-catenin S675), and cyclin D1 in mice with different treatments. Fusobacterium nucleatum and antibiotics treatment altered gut microbial structures in mice. In addition, F. nucleatum invaded into the intestinal mucosa in large amounts but were less abundant in the feces of F. nucleatum-fed mice. The average number and size of intestinal tumors in F. nucleatum groups was significantly increased compared to control groups in ApcMin/+ mice (P < 0.05). The expression of TLR4, PAK1, p-PAK1, p-β-catenin S675, and cyclin D1 was significantly increased in F. nucleatum groups compared to the control groups (P < 0.05). Moreover, TAK-242 significantly decreased the average number and size of intestinal tumors compared to F. nucleatum groups (P < 0.05). The expression of p-PAK1, p-β-catenin S675, and cyclin D1 was also significantly decreased in the TAK-242-treated group compared to F. nucleatum groups (P < 0.05). Fusobacterium nucleatum potentiates intestinal tumorigenesis in ApcMin/+ mice via a TLR4/p-PAK1/p-β-catenin S675 cascade. Fusobacterium nucleatum-induced intestinal tumorigenesis can be inhibited by TAK-242, implicating TLR4 as a potential target for the prevention and therapy of F. nucleatum-related colorectal cancer.

Sitagliptin, a dipeptidyl peptidase-4 inhibitor, suppresses CXCL5 and SDF-1 and does not accelerate intestinal neoplasia formation in ApcMin/+ mice fed a high-fat diet.

The relationship between type 2 diabetes mellitus and intestinal neoplasia has been shown epidemiologically. A high-fat diet (HFD) is also known to promote insulin resistance, which is a risk factor for intestinal neoplasia. Dipeptidyl peptidase-4 (DPP-4) inhibitors are used in the clinic for the treatment of type 2 diabetes and also to prolong the effects of glucagon-like peptide-1 (GLP-1). However, since the intestinotrophic hormone GLP-2 and chemokines, such as CXCL5 and stromal cell-derived factor-1 (SDF-1), are also substrates of DPP-4, DPP-4 inhibitors may increase the risk of intestinal carcinogenesis. In this study, we evaluated the impact of a DPP-4 inhibitor on intestinal tumorigenesis in ApcMin/+ mice fed a HFD. Six-week-old male ApcMin/+ mice were randomized to either a normal diet (10 kcal% fat) group, a HFD (60 kcal% fat) group, or a HFD group treated with sitagliptin (STG). The mice were euthanized nine weeks after the start of treatment. Daily treatment with STG did not increase number of intestinal tumors in the HFD group; however, this increase was not statistically significant. The mucosal concentration of total GLP-2 was significantly increased in the HFD group. The chemokine protein array showed elevated plasma concentrations of CXCL5 and SDF-1 in the HFD group. The administration of STG significantly suppressed the levels of plasma CXCL5 and SDF-1 in mice fed a HFD. Since CXCL5 expression is increased in patients with type 2 diabetes, and GLP-2, CXCL5 and SDF-1 are associated with tumor progression, DPP-4 inhibition may have potential as an agent for decreasing the risk of cancer in obese or diabetic patients.

Epithelial-derived IL-33 promotes intestinal tumorigenesis in ApcMin/+ mice

Increased expression of Interleukin (IL)-33 has been detected in intestinal samples of patients with ulcerative colitis, a condition associated with increased risk for colon cancer, but its role in the development of colorectal cancer has yet to be fully examined. Here, we investigated the role of epithelial expressed IL-33 during development of intestinal tumors. IL-33 expression was detected in epithelial cells in colorectal cancer specimens and in the ApcMin/+ mice. To better understand the role of epithelial-derived IL-33 in the intestinal tumorigenesis, we generated transgenic mice expressing IL-33 in intestinal epithelial cells (V33 mice). V33 ApcMin/+ mice, resulting from the cross of V33 with ApcMin/+ mice, had increased intestinal tumor burden compared with littermate ApcMin/+ mice. Consistently, ApcMin/+ mice deficient for IL-33 receptor (ST2), had reduced polyp burden. Mechanistically, overexpression of IL-33 promoted expansion of ST2+ regulatory T cells, increased Th2 cytokine milieu, and induced alternatively activated macrophages in the gut. IL-33 promoted marked changes in the expression of antimicrobial peptides, and antibiotic treatment of V33 ApcMin/+ mice abrogated the tumor promoting-effects of IL-33 in the colon. In conclusion, elevated IL-33 signaling increases tumor development in the ApcMin/+ mice.

CCR2 promotes intestinal tumorigenesis through enhancing inflammation via IL-17 and IL-23 pathway

Abstract Chemokines and their receptors have been implicated in several cancers and metastasis including colon cancer. CCL2, a classic chemokine mediates biological functions via CCR2 receptor. In this study, we investigated the role of CCR2 in progression of intestinal tumors using spontaneous intestinal tumorigenesis mouse model, ApcMin/+ mice. Ablation of CCR2 in ApcMin/+ mice significantly increased the survival, reduced tumor burden and corrected immune abnormalities caused by ApcMin/+ mutation. Immune cell analysis in spleen and mesenteric lymph nodes (mLNs) suggested that CCR2−/−ApcMin/+ mice exhibited significant reduction in myeloid cell population and increase in IFNγ producing T-cell populations compared to ApcMin/+ mice. CCR2−/−ApcMin/+ mice also displayed decreased tumor infiltrating F4/80+ and increased CD8+ cells. Polyps in CCR2−/−ApcMin/+ displayed significant reduction in IL-17 and IL-23 cytokines compared to polyps of ApcMin/+. Adoptive transfer of splenic CD4+ T cells from ApcMin/+ mice into Rag2−/− mice develops severe colitis. However, transfer of CD4+ T cells from CCR2−/− ApcMin/+ failed to develop colitis in Rag2−/− mice. The levels of IL-17 in these mice significantly reduced suggesting importance of CCR2 expression on CD4+cells to promote IL 17 mediated colonic inflammation. Microbiota analysis revealed that ablation of CCR2 in ApcMin/+ lead to decrease in tumor promoting Bacteroides genus compared to ApcMin/+. In conclusion, CCR2 plays a critical role in enhancing tumor-promoting inflammation via IL-17 and IL-23 leading to increased intestinal tumorigenesis in ApcMin/+ mice. Blocking CCR2-CCL2 axis potentially offers an alternative therapeutic option to control intestinal tumorigenesis.

Evaluation of Rint1 as a modifier of intestinal tumorigenesis and cancer risk.

The Rad50 Interacting Protein 1 (Rint1) influences cellular homeostasis through maintenance of endoplasmic reticulum, Golgi and centrosome integrity and regulation of vesicle transport, autophagy and the G2/M checkpoint. Rint1 has been postulated to function as a tumor suppressor as well as an oncogene, with its role depending perhaps upon the precise cellular and/or experimental context. In humans, heterozygosity for germline missense variants in RINT1 have, in some studies, been associated with increased risk of both breast and Lynch syndrome type cancers. However, it is not known if these germline variants represent loss of function alleles or gain of function alleles. Based upon these findings, as well as our initial consideration of Rint1 as a potential candidate for Mom5, a genetic modifier of intestinal tumorigenesis in ApcMin/+ mice, we sought to explicitly examine the impact of Rint1 on tumorigenesis in ApcMin/+ mice. However, heterozygosity for a knockout of Rint1 had no impact on tumorigenesis in Rint1+/-; ApcMin/+ mice. Likewise, we found no evidence to suggest that the remaining Rint1 allele was lost somatically in intestinal tumors in ApcMin/+ mice. Interestingly, in contrast to what has been observed in Rint1+/- mice on a mixed genetic background, Rint1+/- mice on a pure C57BL/6J background did not show spontaneous tumor development. We also evaluated colorectal cancer data available in the COSMIC and ONCOMINE databases and found that RINT1 overexpression, as well as the presence of somatic missense mutations in RINT1 were associated with colorectal cancer development. In vitro evaluation of two missense variants in RINT1 suggested that such variants do have the potential to impact RINT1 function.

TNIK inhibition abrogates colorectal cancer stemness.

Canonical Wnt/β-catenin signalling is essential for maintaining intestinal stem cells, and its constitutive activation has been implicated in colorectal carcinogenesis. We and others have previously identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulatory component of the T-cell factor-4 and β-catenin transcriptional complex. Consistent with this, Tnik-deficient mice are resistant to azoxymethane-induced colon tumorigenesis, and Tnik−/−/Apcmin/+ mutant mice develop significantly fewer intestinal tumours. Here we report the first orally available small-molecule TNIK inhibitor, NCB-0846, having anti-Wnt activity. X-ray co-crystal structure analysis reveals that NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apcmin/+ mice and the sphere- and tumour-forming activities of colorectal cancer cells. TNIK is required for the tumour-initiating function of colorectal cancer stem cells. Its inhibition is a promising therapeutic approach.

Lack of interferon-γ receptor results in a microenvironment favorable for intestinal tumorigenesis

IFN-γ plays an important role in innate and adaptive immunity. IFN-γ signaling is also involved in tumorigenesis, with both pro- and antitumor activities documented. We here report the characterization of intestinal tumorigenesis in ApcMin/+ mice that lack IFN-γ receptor. We observed that Ifngr1−/−ApcMin/+ mice are shorter-lived than Ifngr1+/+ApcMin/+ mice. The tumors in Ifngr1−/−ApcMin/+ mice are more likely to progress into invasive adenocarcinomas. Gene expression profiling by RNA sequencing revealed a significant upregulation of genes involved in inflammation and tissue remodeling in tumors of Ifngr1−/−ApcMin/+ mice when compared to those in Ifngr1+/+ApcMin/+ mice. In particular, five genes encoding matrix metallopeptidases (MMPs) were among the upregulated. On the other hand, genes that promote or maintain intestinal differentiation, such as Cdx2, Cdhr2 and Cdhr5, were downregulated. Tumor-associated macrophages were more abundant and were more favored toward M2 polarization in Ifngr1−/−ApcMin/+ mice than in Ifngr1+/+ApcMin/+ mice. Furthermore, the Ifngr1 was significantly downregulated in intestinal tumors when compared to mucosa. A similar trend was noted for human colorectal carcinomas. Together, our results indicate that adequate IFN-γ signaling is critical for maintaining a tumor-prohibitive microenvironment.

Genetic Manipulation of Homologous Recombination In Vivo Attenuates Intestinal Tumorigenesis.

Although disruption of DNA repair capacity is unquestionably associated with cancer susceptibility in humans and model organisms, it remains unclear if the inherent tumor phenotypes of DNA repair deficiency syndromes can be regulated by manipulating DNA repair pathways. Loss-of-function mutations in BLM, a member of the RecQ helicase family, cause Bloom's syndrome (BS), a rare, recessive genetic disorder that predisposes to many types of cancer. BLM functions in many aspects of DNA homeostasis, including the suppression of homologous recombination (HR) in somatic cells. We investigated whether BLM overexpression, in contrast with loss-of-function mutations, attenuated the intestinal tumor phenotypes of Apc(Min/+) and Apc(Min/+);Msh2(-/-) mice, animal models of familial adenomatous polyposis coli (FAP). We constructed a transgenic mouse line expressing human BLM (BLM-Tg) and crossed it onto both backgrounds. BLM-Tg decreased adenoma incidence in a dose-dependent manner in our Apc(Min/) (+) model of FAP, although levels of GIN were unaffected and concomitantly increased animal survival over 50%. It did not reduce intestinal tumorigenesis in Apc(Min/) (+);Msh2(-/-) mice. We used the pink-eyed unstable (p(un)) mouse model to demonstrate that increasing BLM dosage in vivo lowered endogenous levels of HR by 2-fold. Our data suggest that attenuation of the Min phenotype is achieved through a direct effect of BLM-Tg on the HR repair pathway. These findings demonstrate that HR can be manipulated in vivo to modulate tumor formation at the organismal level. Our data suggest that lowering HR frequencies may have positive therapeutic outcomes in the context of specific hereditary cancer predisposition syndromes, exemplified by FAP.

Targeting phospholipase D1 attenuates intestinal tumorigenesis by controlling β-catenin signaling in cancer-initiating cells.

Expression of the Wnt target gene phospholipase D1 (PLD1) is up-regulated in various carcinomas, including colorectal cancer (CRC). However, the mechanistic significance of its elevated expression in intestinal tumorigenesis remains unknown. In this study, we show that genetic and pharmacological targeting of PLD1 disrupts spontaneous and colitis-associated intestinal tumorigenesis in Apc(Min/+) and azoxymethane/dextran sodium sulfate mice models. Intestinal epithelial cell-specific PLD1 overexpression in Apc(Min/+) mice accelerated tumorigenesis with increased proliferation and nuclear β-catenin levels compared with Apc(Min/+) mice. Moreover, PLD1 inactivation suppressed the self-renewal capacity of colon cancer-initiating cells (CC-ICs) by decreasing expression of β-catenin via E2F1-induced microRNA (miR)-4496 up-regulation. Ultimately, low expression of PLD1 coupled with a low level of CC-IC markers was predictive of a good prognosis in CRC patients, suggesting in vivo relevance. Collectively, our data reveal that PLD1 has a crucial role in intestinal tumorigenesis via its modulation of the E2F1-miR-4496-β-catenin signaling pathway. Modulation of PLD1 expression and activity represents a promising therapeutic strategy for the treatment of intestinal tumorigenesis.

Modulation of colon cancer by nutmeg.

Colon cancer is the most common cancer and the third leading cause of cancer mortality in humans. Using mass spectrometry-based metabolomics, the current study revealed the accumulation of four uremic toxins (cresol sulfate, cresol glucuronide, indoxyl sulfate, and phenyl sulfate) in the serum of mice harboring adenomatous polyposis coli (APC) gene mutation-induced colon cancer. These uremic toxins, likely generated from the gut microbiota, were associated with an increase in the expression of the proinflammatory cytokine IL-6 and a disorder of lipid metabolism. Nutmeg, which exhibits antimicrobial activity, attenuated the levels of uremic toxins and decreased intestinal tumorigenesis in Apc(min/+) mice. Nutmeg-treated Apc(min/+) mice had decreased IL-6 levels and normalized dysregulated lipid metabolism, suggesting that uremic toxins are responsible, in part, for the metabolic disorders that occur during tumorigenesis. These studies demonstrate a potential biochemical link among gut microbial metabolism, inflammation, and metabolic disorders and suggest that modulation of gut microbiota and lipid metabolism using dietary intervention or drugs may be effective in colon cancer chemoprevention strategies.

Obesity-Related Colorectal Cancer: The Role of Leptin.

Obesity is linked to increased risk of colon cancer. Consequently, rising levels of obesity worldwide are likely to have a significant impact on obesity-related colon cancers in the decades to come. Understanding the molecular mechanisms whereby obesity increases the risk of colon cancer is, thus, a focus for research on strategies to prevent the increasing trend in obesity-related cancers. The epidemiological evidence clearly indicates common factors linking obesity and colon cancer. Both are associated with consumption of high-energy diets, a sedentary lifestyle, increased age and reduced consumption of fruit, vegetables and fiber [1-3]. All these factors influence adipose tissue, now firmly established as the body’s largest endocrine organ [4]. These factors have the potential to influence the production of adipose-derived hormones and cytokines from the adipose organ [5-8]. Leptin and adiponectin are two of the most abundant and most investigated adipose-derived hormones. Friedman and Halaas [9] positionally cloned the ob gene and demonstrated that it was encoded for a hormone that they called leptin (after the Greek ‘leptos’ for thin). The proposed role of leptin was as a growth factor in the colon, stimulating proliferation of the colon epithelium and inhibiting apoptosis. That leptin increased colon tumor growth in obese patients subsequent to the initiation of colon cancer. Leptin is important for colorectal cancer (CRC) growth in obese patients and acts as a growth factor for CRC at stages subsequent to tumor initiation in colorectal carcinogenesis [10]. However, leptin failed to promote the growth of colon cancer xenografts in nude mice and did not increase intestinal tumorigenesis in ApcMin/ + mice [11]. The present study also failed to demonstrate the impact of leptin for increasing tumorigenesis and improving the prognosis. Moreover, an inverse association of nodal stage with high leptin expression was found. Therefore, the authors concluded that a higher leptin expression level was a predictor of a better oncologic outcome and that high leptin expression in colorectal-cancer tissue might be a good prognostic factor. Assessment of leptin signaling in tumor tissue needs further investigation to determine its possible impact on prognosis [12]. The complexity of this issue is confounded by various aspects associated with leptin production and signaling in human coloncancer patients. Leptin receptors are expressed by normal colon epithelial cells in human subjects [13, 14]. High expression of the long-form signaling receptor, ObRb, has been associated with increased age, proximally-located tumors, and high levels of microsatellite instability and lymphocyte infiltration [13]. The impact of plasma leptin is confounded by inconsistent and conflicting data reported from studies on patients with colon cancer [15-20]. A more recent report failed to determine significant differences in serum leptin between patients with colon cancer and controls [15]. The conflicting reports may be due, in part, to cachexia and anorexia, which are common in patients with CRC [21]. Leptin expression in colon tissue may be positively correlated with tumor features that are associated with improved survival of patients with CRC [22]. At present, the significant relationship between leptin expression and clinicopathological details, such as tumor stage and prognosis, suggests a possible role for leptin as a novel growth factor driving the development and the progression of CRC. Large clinical studies are needed to determine the mechanisms underlying the effects of leptin on the development and the progression of CRC.

Wip1 abrogation decreases intestinal tumor frequency in APC(Min/+) mice irrespective of radiation quality.

Low-linear energy transfer (low-LET) γ-ray exposure is a risk factor for colorectal cancer (CRC). Due to their high-LET nature, energetic iron ions found in space are expected to pose greater CRC risks to astronauts undertaking long-duration space missions beyond low Earth orbit. Wild-type p53-induced phosphatase 1 (Wip1) is important for cellular DNA damage response and its abrogation has been shown to inhibit spontaneous intestinal tumorigenesis in APC(Min/+) mice, a well-studied mouse model of human CRC. However, the relationship of Wip1 to radiation-induced intestinal tumorigenesis, especially by energetic iron ions, has not been investigated in APC(Min/+) mice. We have previously reported that there is a greater intestinal tumorigenic potential of iron-ion radiation relative to (137)Cs γ rays, so the purpose of the current study was to investigate whether Wip1 abrogation could influence high-LET dependent intestinal tumorigenesis in APC(Min/+) mice. Intestinal tumor frequency and grade were assessed in APC(Min/+)/Wip1(-/-) mice and results were compared to those in APC(Min/+)/Wip1(+/+) mice after exposure to a mean absorbed dose of 2 Gy from (137)Cs γ rays or 1.6 Gy from 1 GeV/n iron ions. Cellular differentiation and proliferation were also assessed in the intestinal tumors of sham-irradiated and irradiated mice. Decreased tumor frequency and lower tumor grade were observed in APC(Min/+)/Wip1(-/-) relative to APC(Min/+)/Wip1(+/+) mice. Notably, a similar decrease (∼6-fold in both groups) in tumor number was observed in sham-irradiated and γ-irradiated APC(Min/+)/Wip1(-/-) relative to APC(Min/+)/Wip1(+/+) mice. However, tumorigenesis in the energetic iron-ion exposed group was reduced ∼8-fold in APC(Min/+)/Wip1(-/-) relative to APC(Min/+)/Wip1(+/+) mice. A significantly lower proliferation/differentiation index in tumors of iron-ion exposed APC(Min/+)/Wip1(-/-) relative to APC(Min/+)/Wip1(+/+) mice suggests that reduced proliferation and enhanced differentiation as a result of Wip1 abrogation maybe involved. In conclusion, the current study demonstrated that the absence of Wip1 blocked radiation-induced intestinal tumorigenesis irrespective of radiation quality and has implications for developing preventive strategies against the tumorigenic potential of radiation exposure on earth and in outer space.

Mo1913 Influence of a Dipeptidyl Peptidase-4 Inhibitor on Intestinal Neoplasia Formation in APC MIN/+ Mice Fed a High Fat Diet

Background: The association between type 2 diabetes mellitus and intestinal neoplasia has been shown epidemiologically. It is also well known that a high fat diet (HFD) promotes insulin resistance which is a risk factor for intestinal neoplasia. Dipeptidyl peptidase-4 (DPP4) inhibitors are used clinically in diabetes therapy to prolong the effects of glucagon-like peptide-1 (GLP-1). However, because another derivative from proglucagon, the intestinotrophic hormone GLP-2, is also a substrate for DPP-4, DPP-4 inhibitionmay enhance intestinal carcinogenesis by increasing endogenous active GLP-2. We therefore conducted this study to investigate the influence of a DPP-4 inhibitor on intestinal tumorigenesis in Apc Min/+ mice fed a HFD. Material and Methods: Segmental differences in DPP-4, 8, and 9 mRNA expression and DPP enzyme activity were determined in the mice. The DPP-4 inhibitor, sitagliptin (0.3, 1 or 3 mg/kg), was given orally. Mucosal DPP activity and GLP concentrations were measured 24 hr after administration. Six-week-old male Apc Min/+ mice were randomized to either a HFD (60 kcal fat) group or control diet (10 kcal fat) group for 9 weeks. Each group was divided into two groups, which were either treated or not treated with sitagliptin (3 mg/kg/day, i.g.). The mice were sacrificed 9 weeks after the initiation of treatment. The small intestine was coiled up into a Swiss roll, which was used to determine the number, size, and localization of tumors. Results: DPP-4 mRNA was expressed at high levels in the jejunum and ileum, with high DPP-4 activity also being detected. Oral sitagliptin caused a dose-dependent suppression in mucosal DPP-4 activity. Although the mucosal concentration of total GLP-2 was not affected, the concentration of GLP-1(7-36) was significantly elevated in the mucosa of the ileum. From seven to eight weeks after the initiation of sitagliptin treatment, bloody and inconsistent stools were observed mainly in the control diet and sitagliptin-treated mice. These mice also showed significantly lower body weight gains. In the control diet group, daily treatment with sitagliptin caused a significant increase in the number of intestinal tumors compared with mice not treated with sitagliptin. Daily treatment with sitagliptin suppressed the number of intestinal tumors in the HFD group, although this change was not statistically significant. Conclusion: This study demonstrated that chronic treatment with sitagliptin increased endogenous mucosal GLPs in the intestine and promoted intestinal tumorigenesis in lean mice, whereas sitagliptin treatment tended to suppress tumorigenesis in mice fed a HFD. Because a HFD promotes insulin resistance and intestinal neoplasia, DPP-4 inhibition may also be useful as an agent for preventing cancer in obese or diabetic patients.

Spermidine/spermine N1-acetyltransferase activity associates with white blood cell count in myeloid leukemias

The metabolism of polyamines, the cationic small molecules essential for cell proliferation and differentiation, is altered in cancer cells and can be exploited in cancer diagnosis and therapy. Spermidine/spermine N(1)-acetyltransferase (SSAT), which regulates intracellular levels of polyamines by catabolizing spermidine and spermine, has a controversial role in the development of cancers. In this study, the polyamine metabolism and function of SSAT were characterized in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia patient samples. Also, mice overexpressing SSAT and having a myeloproliferative phenotype were analyzed for their response to decitabine and histone deacetylase inhibitor trichostatin A. The presence of epigenetic factors in the bone marrow cells of SSAT mice was analyzed. Elevated levels of spermidine and spermine, as well as increased activity of SSAT, were detected in AML, CML, and acute lymphoid leukemia patients compared with the controls. However, we found SSAT activity to be associated with white blood cell count only in AML and CML patients. Decitabine treatment brought the peripheral blood and bone marrow cell counts of SSAT mice to the level of wild-type mice. Spermidine/spermine N(1)-acetyltransferase mice had increased histone methylation and an increased level of histone deacetylase 1 in their bone marrow cells. The study suggests that SSAT influences the development of myeloid malignancies, and epigenetic factors partly contribute to the SSAT overexpression-induced myeloproliferative disease in mice.