Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis.

The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.

Assessment of circulating microRNA specific for patients with familial adenomatous polyposis.

Circulating microRNAs (miRNAs) are considered promising biomarkers for diagnosis, prognosis, and treatment efficacy of diseases. However, usefulness of circulating miRNAs as biomarkers for hereditary gastrointestinal diseases have not been confirmed yet. We explored circulating miRNAs specific for patients with familial adenomatous polyposis (FAP) as a representative hereditary gastrointestinal disease. Next-generation sequencing (NGS) indicated that plasma miR-143-3p, miR-183-5p, and miR-885-5p were candidate biomarkers for five FAP patients compared to three healthy donors due to moderate copy number and significant difference. MiR-16-5p was considered as an internal control due to minimum difference in expression across FAP patients and healthy donors. Validation studies by real-time PCR showed that mean ratios of maximum expression and minimum expression were 2.2 for miR-143-3p/miR-16-5p, 3.4 for miR-143-3p/miR-103a-3p, 5.1 for miR-183-5p/miR-16-5p, and 4.9 for miR-885-5p/miR-16-5p by using the samples collected at different time points of eight FAP patients. MiR-143-3p/16-5p was further assessed using specimens from 16 FAP patients and 7 healthy donors. MiR-143-3p was upregulated in FAP patients compared to healthy donors (P = 0.04), but not significantly influenced by clinicopathological features. However, miR-143-3p expression in colonic tumors was rare for upregulation, although there was a significant difference by existence of desmoid tumors. MiR-143-3p transfection significantly inhibited colorectal cancer cell proliferation compared to control microRNA transfection. Our data suggested regulation of miR-143-3p expression differed by samples (plasma or colonic tumors) in most FAP patients. Upregulation of plasma miR-143-3p expression may be helpful for diagnosis of FAP, although suppressive effect on tumorigenesis seemed insufficient in FAP patients.

Free Fatty Acids Signature in Human Intestinal Disorders: Significant Association between Butyric Acid and Celiac Disease.

Altered circulating levels of free fatty acids (FFAs), namely short chain fatty acids (SCFAs), medium chain fatty acids (MCFAs), and long chain fatty acids (LCFAs), are associated with metabolic, gastrointestinal, and malignant diseases. Hence, we compared the serum FFA profile of patients with celiac disease (CD), adenomatous polyposis (AP), and colorectal cancer (CRC) to healthy controls (HC). We enrolled 44 patients (19 CRC, 9 AP, 16 CD) and 16 HC. We performed a quantitative FFA evaluation with the gas chromatography-mass spectrometry method (GC-MS), and we performed Dirichlet-multinomial regression in order to highlight disease-specific FFA signature. HC showed a different composition of FFAs than CRC, AP, and CD patients. Furthermore, the partial least squares discriminant analysis (PLS-DA) confirmed perfect overlap between the CRC and AP patients and separation of HC from the diseased groups. The Dirichlet-multinomial regression identified only strong positive association between CD and butyric acid. Moreover, CD patients showed significant interactions with age, BMI, and gender. In addition, among patients with the same age and BMI, being male compared to being female implies a decrease of the CD effect on the (log) prevalence of butyric acid in FFA composition. Our data support GC-MS as a suitable method for the concurrent analysis of circulating SCFAs, MCFAs, and LCFAs in different gastrointestinal diseases. Furthermore, and notably, we suggest for the first time that butyric acid could represent a potential biomarker for CD screening.

Next-generation sequencing with comprehensive bioinformatics analysis facilitates somatic mosaic APC gene mutation detection in patients with familial adenomatous polyposis.

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal tumor characterized by numerous adenomatous colonic polyps that often lead to colon cancer. Although most patients with FAP harbored germline mutations in APC gene, it was recently recognized that patients with clinical FAP, but without detectable pathogenic mutations, could be associated with somatic mosaic APC mutation. METHODS: We reanalyzed the nest-generation sequencing (NGS) gene panel testing results of patients who were diagnosed with FAP, but did not have APC mutations, at Yonsei Cancer Prevention Center between July 2016 and March 2018. We tested several variant calling algorithms to identify low level mosaic variants. In one patient with a low frequency APC mutation, NGS analysis was performed together with endoscopic biopsy. Variant calling tools HaplotypeCaller, MuTect2, VarScan2, and Pindel were used. We also used 3'-Modified Oligonucleotides (MEMO)-PCR or conventional PCR for confirmation. RESULTS: Among 28 patients with clinical suspicion of FAP but no detectable pathogenic variants of colonic polyposis associated genes, somatic mosaic pathogenic variants were identified in seven patients. The variant allele frequency ranged from 0.3 to 7.7%. These variants were mostly detected through variant caller MuTect2 and Pindel, and were further confirmed using mutant enrichment with MEMO-PCR. CONCLUSIONS: The NGS with an adequate combination of bioinformatics tools is effective to detect low level somatic variants in a single assay. Because mosaic APC mutations are more frequent than previously thought, the presence of mosaic mutations must be considered when analyzing genetic tests of patients with FAP.

Mean platelet volume/platelet count ratio in colorectal cancer: a retrospective clinical study.

BACKGROUND: Mean platelet volume (MPV) is a marker of platelet activation. MPV and platelet count (PC) are negatively correlated, and their ratio (MPV/PC) is informative for the diagnosis of malignant tumors. However, the relationship between MPV/PC and colorectal cancer is unclear. This retrospective clinical study aimed to evaluate the diagnostic value of MPV/PC in colorectal cancer. METHODS: Hematological examinations were performed at initial diagnosis in patients with colorectal cancer (n = 186) or adenomatous polyp (n = 132) and healthy controls (n = 108). Hematological parameters evaluated included white blood cells, red blood cells, hemoglobin, neutrophils, lymphocytes, monocytes, PC, and MPV. Statistical analyses included Student's t-test, one-way ANOVA or Kruskal-Wallis H test, chi-square tests, Spearman's correlation test and receiver operating characteristic (ROC). ROC curve was used to evaluate the diagnostic values of MPV and MPV/PC in colorectal cancer. RESULTS: Among these groups, MPV was significantly lower in colorectal cancer than in adenomatous polyp (p = 0.002) and healthy controls (p < 0.001) but did not significantly differ between adenomatous polyp and healthy controls (p = 0.210). MPV/PC was lower in colorectal cancer compared with adenomatous polyp and healthy controls (p < 0.001) and in adenomatous polyp compared with healthy controls (p = 0.010). MPV did not significantly differ among colorectal cancer subgroups, while MPV/PC significantly differed between TNM stages and the presence/absence of lymph node metastasis. MPV/PC was negatively correlated with the neutrophil to lymphocyte ratio(NLR) (p = 0.002) and platelet to lymphocyte ratio(PLR) concentration (p < 0.001). In the differential diagnosis between colorectal cancer and adenomatous polyp, MPV/PC produced a larger ROC curve than MPV, NLR or PLR alone. Using MPV/PC to distinguish between colorectal cancer and controls produced a larger AUC than using MPV or NLR alone. CONCLUSIONS: MPV/PC may be useful for the diagnosis of colorectal cancer. However, further studies are warranted to include additional regions and more data, to assess the utility of MPV/PC as a novel diagnostic screening tool for colorectal cancer.

Serum metabolite profiling of familial adenomatous polyposis using ultra performance liquid chromatography and tandem mass spectrometry.

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited intestinal polyposis syndrome accounting for about 1% of colorectal cancers (CRC). Despite increasing researches on the molecular pathogenesis of CRC, we are still unclear about metabolic pathways and alterations probably involved in the development of CRC. To obtain new insights into the mechanisms underlying APC mutation and to elucidate the mechanisms of CRC development, we performed to identify the potential metabolites in FAP based on metabolomic strategy. Serum metabolites from FAP patients (n = 30) and healthy individuals (n = 34) were detected and qualified using Ultra Performance Liquid Chromatography and Tandem Mass Spectrometry (UPLC- MS/MS). 118 metabolites were identified with statistical tests of orthogonal partial least-squares-discriminant analysis (OPLS-DA), with the conditions of variable importance in projection (VIP) >1, p < 0.05 using the Mann-Whitney U test, and fold change (FC) ≥2 or ≤0.5. OPLS-DA model was useful for distinguishing FAP patients from healthy controls. Unique metabolic signatures were pooled in FAP patients covering tricarboxylic acid (TCA) cycle, amino acids metabolism, vitamin D, fatty acids metabolism, and bile acids (BAs) metabolism. Our results demonstrated that metabolites alterations in FAP can be helpful for further analysis of metabonomics induced by APC mutation, and these alterations might be involved in the progress of intestinal carcinogenesis.

Burden and Profile of Somatic Mutation in Duodenal Adenomas from Patients with Familial Adenomatous- and MUTYH-associated Polyposis.

Purpose: Duodenal polyposis and cancer are important causes of morbidity and mortality in familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP). This study aimed to comprehensively characterize somatic genetic changes in FAP and MAP duodenal adenomas to better understand duodenal tumorigenesis in these disorders.Experimental Design: Sixty-nine adenomas were biopsied during endoscopy in 16 FAP and 10 MAP patients with duodenal polyposis. Ten FAP and 10 MAP adenomas and matched blood DNA samples were exome sequenced, 42 further adenomas underwent targeted sequencing, and 47 were studied by array comparative genomic hybridization. Findings in FAP and MAP duodenal adenomas were compared with each other and to the reported mutational landscape in FAP and MAP colorectal adenomas.Results: MAP duodenal adenomas had significantly more protein-changing somatic mutations (P = 0.018), truncating mutations (P = 0.006), and copy number variants (P = 0.005) than FAP duodenal adenomas, even though MAP patients had lower Spigelman stage duodenal polyposis. Fifteen genes were significantly recurrently mutated. Targeted sequencing of APC, KRAS, PTCHD2, and PLCL1 identified further mutations in each of these genes in additional duodenal adenomas. In contrast to MAP and FAP colorectal adenomas, neither exome nor targeted sequencing identified WTX mutations (P = 0.0017).Conclusions: The mutational landscapes in FAP and MAP duodenal adenomas overlapped with, but had significant differences to those reported in colorectal adenomas. The significantly higher burden of somatic mutations in MAP than FAP duodenal adenomas despite lower Spigelman stage disease could increase cancer risk in the context of apparently less severe benign disease. Clin Cancer Res; 23(21); 6721-32. ©2017 AACR.

Extensive metabolic disorders are present in APC(min) tumorigenesis mice.

Wnt signaling plays essential role in mesenchymal stem cell (MSC) differentiation. Activation of Wnt signaling suppresses adipogenesis, but promotes osteogenesis in MSC. Adenomatous polyposis coli (APC) is a negative regulator of ß-catenin and Wnt signaling activity. The mutation of APC gene leads to the activation of Wnt signaling and is responsible for tumorigenesis in APC(min) mouse; however, very few studies focused on its metabolic abnormalities. The present study reports a widespread metabolic disorder phenotype in APC(min) mice. The old APC(min) mice have decreased body weight and impaired adipogenesis, but severe hyperlipidemia, which mimic the phenotypes of Familial Adenomatous Polyposis (FAP), an inherited disease also caused by APC gene mutation in human. We found that the expression of lipid metabolism and free fat acids (FA) use genes in the white adipose tissue (WAT) of the APC(min) mice is much lower than those of control. The changed gene expression pattern may lead to the disability of circulatory lipid transportation and storage at WAT. Moreover, the APC(min) mice could not maintain the core body temperature in cold condition. PET-CT determination revealed that the BAT of APC(min) mice has significantly impaired ability to take up (18)FDG from the blood. Morphological studies identified that the brown adipocytes of APC(min) mice were filled with lipid droplets but fewer mitochondria. These results matched with the findings of impaired BAT function in APC(min) mice. Collectively, our study explores a new mechanism that explains abnormal metabolism in APC(min) mice and provides insights into studying the metabolic disorders of FAP patients.

Peptide Patterns as Discriminating Biomarkers in Plasma of Patients With Familial Adenomatous Polyposis.

BACKGROUND: Familial adenomatous polyposis (FAP) is one of the most important clinical forms of inherited susceptibility to colorectal cancer. So far, no accepted prognostic markers are present to monitor patients with FAP. Consequently, the major problem in managing patients with FAP is the difficulty to predict when the switch between adenoma and malignant carcinoma occurs, leading to the necessity of preventive surgery. Proteomics is one of the most suitable approaches to identify biomarkers, and it is widely used in cancer research. In this investigation, we studied the circulating plasma peptides in samples collected from patients with FAP and compared the obtained results with adenoma, colorectal cancer, and control samples to discover peptides able to distinguish different phenotypes. MATERIALS AND METHODS: The peptide fingerprint was obtained by matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry. After statistical analysis, a subset of 45 ionic species was found differently expressed in the 4 groups considered, 12 of them peculiar to patients with FAP. Moreover, 4 ionic species were found significantly changed in the switch between adenoma and malignant carcinoma. RESULTS: Potentially prognostic peptides identified by this study derive mainly from circulating proteins, some of which are involved in the inflammatory response, such as complement C3 and C4 subjected to an exoprotease activity that seemed pathology related. CONCLUSIONS: In this study, we defined for the first time a specific panel of peptides for monitoring patients with FAP that could be profitably used to monitor and predict the pathologic evolution in adenocarcinoma malignancy.

Profiling of volatile compounds in APC(Min/+) mice blood by dynamic headspace extraction and gas chromatography/mass spectrometry.

Various volatile compounds as well as hydrophilic compounds exist in the blood. For example, 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes have been reported as oxidized lipid-derived volatiles in blood. These specific volatiles have been associated with diseases; however, multi-volatile analyses have not been performed. In this study, volatile profiling of APC(Min/+) mouse plasma by dynamic headspace extraction was performed for multi-volatile analysis. In total, 19 volatiles were detected in the plasma of mice, based on information regarding oxidized lipid-derived volatile compounds, and eight of these compounds differed significantly between normal and diseased mice. 2-Methyl-2-butanol and benzyl alcohol were previously unreported in blood samples. Furthermore, 3,5,5-trimethyl-2(5H)-furanone was only detected in normal mice. 5-Methyl-3-hexanone and benzaldehyde have been detected in subjects with gastrointestinal diseases and lung cancer, respectively. Therefore, volatile profiling can be used to detect differences between samples and to identify compounds associated with diseases.

Early detection of high oxidative activity in patients with adenomatous intestinal polyps and colorectal adenocarcinoma: myeloperoxidase and oxidized low-density lipoprotein in serum as new markers of oxidative stress in colorectal cancer.

OBJECTIVES: To detect whether signs of oxidative stress appear at early stages of colorectal adenocarcinoma (CRC), particularly in the polyp stage. We also aimed to evaluate the specific entities myeloperoxidase (MPO) and oxidized low-density lipoprotein (oxLDL) as novel markers of oxidation in the plasma of patients with CRC and to study the relationship between oxidative status in plasma and patient survival. METHODS: We assayed serum or plasma specimens from healthy control subjects (n = 14), from patients with intestinal polyps (n = 39), and from patients with CRC (n = 128) to calculate the modified oxidative balance score (MOBS) using several serum markers (ß-carotene, lycopene, vitamin A, vitamin E, MPO, and oxLDL). We also assayed the levels of C-reactive protein (CRP) and obtained lipid profiles. Finally, we studied the survival of patients in relationship to oxidative status (antioxidants and pro-oxidants) and inflammation markers, and added theses data to the lipid profile for each patient. RESULTS: Oxidative stress levels increased as disease stage advanced. This increase was detected early in the polyp stage, before polyps progressed to cancer, and could be measured by the increase of such new markers as MPO and oxLDL, the decrease in antioxidants, and the MOBS value. Higher levels of oxidation correlated with lower survival. CONCLUSION: The oxidation process, which can cause mutations leading to CRC, begins development in the polyp stage. This process may be detected early by monitoring serum markers such as MPO and oxLDL.

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.

Sex differences in the relationship of IL-6 signaling to cancer cachexia progression.

A devastating aspect of cancer cachexia is severe loss of muscle and fat mass. Though cachexia occurs in both sexes, it is not well-defined in the female. The Apc(Min/+) mouse is genetically predisposed to develop intestinal tumors; circulating IL-6 is a critical regulator of cancer cachexia in the male Apc(Min/+) mouse. The purpose of this study was to examine the relationship between IL-6 signaling and cachexia progression in the female Apc(Min/+) mouse. Male and female Apc(Min/+) mice were examined during the initiation and progression of cachexia. Another group of females had IL-6 overexpressed between 12 and 14 weeks or 15-18 weeks of age to determine whether IL-6 could induce cachexia. Cachectic female Apc(Min/+) mice lost body weight, muscle mass, and fat mass; increased muscle IL-6 mRNA expression was associated with these changes, but circulating IL-6 levels were not. Circulating IL-6 levels did not correlate with downstream signaling in muscle in the female. Muscle IL-6r mRNA expression and SOCS3 mRNA expression as well as muscle IL-6r protein and STAT3 phosphorylation increased with severe cachexia in both sexes. Muscle SOCS3 protein increased in cachectic females but decreased in cachectic males. IL-6 overexpression did not affect cachexia progression in female Apc(Min/+) mice. Our results indicate that female Apc(Min/+) mice undergo cachexia progression that is at least initially IL-6-independent. Future studies in the female will need to determine mechanisms underlying regulation of IL-6 response and cachexia induction.

Discrepant serum and urine ß-hCG results due to production of ß-hCG by a cribriform-morular variant of thyroid papillary carcinoma.

BACKGROUND: Although patients with medullary thyroid cancer are known to present with paraneoplastic hormone production, this is much less common with papillary thyroid cancer. METHODS: We present a patient with the cribriform morular variant of papillary thyroid cancer in association with familial adenomatous polyposis who developed a positive pregnancy test in the absence of known pregnancy. The patient had developed vaginal bleeding, and her laboratory testing was characterized by elevated serum human chorionic gonadotropin (ß-hCG) concentrations, but negative qualitative urine results. After a thorough gynecological evaluation to exclude unexpected normal, ectopic, or molar pregnancy, we pursued an evaluation for other sources of ß-hCG production. RESULTS: We showed that the elevated serum ß-hCG concentrations were not the result of heterophile antibody interferences, and ultimately we proved that her recurrent tumor produced the ectopic ß-hCG. This is the first report of ß-hCG production by papillary thyroid cancer. Thus, the possibility of ectopic production of ß-hCG by papillary thyroid cancer needs to be included in the differential diagnosis of elevated hCG concentration in the absence of pregnancy. CONCLUSIONS: This study of an unusual paraneoplastic syndrome highlights the importance of investigating discrepancies in the clinical laboratory.

Circulating gastrin concentrations in patients at increased risk of developing colorectal carcinoma.

BACKGROUND AND AIM: An increase in circulating concentrations of gastrin or gastrin precursors such as progastrin and glycine-extended gastrin has been proposed to promote the development of colorectal carcinomas (CRC). The aim of this study was to investigate whether or not circulating gastrin concentrations were increased in patients with an increased risk of developing CRC. METHOD: Patients were divided according to their risk into the five following groups: familial adenomatous polyposis (n = 20), hereditary non-polyposis colorectal cancer (n = 53), cluster of common colorectal cancers (n = 13), personal history and/or family history of adenomatous polyps or CRC (n = 150) and controls (n = 42). Radioimmunoassay with four region-specific gastrin antisera was used to measure progastrin, glycine-extended gastrin (gastrin-gly), amidated gastrin (gastrin-amide), and total gastrin in peripheral blood taken at the time of colonoscopy. RESULTS: Compared with the control group, familial adenomatous polyposis patients had significantly higher median values of total gastrin (29.8 pM vs 16.9 pM, P = 0.003) and gastrin-amide (17.1 pM vs 12.0 pM, P = 0.015). Patients with a personal or family history of adenomatous polyps or CRC also had higher circulating concentrations of total gastrin (21.8 pM) compared with controls (P < 0.05), while patients from all groups who presented with an adenomatous polyp on the day of colonoscopy had higher concentrations of total gastrin, progastrin, and gastrin-amide than patients without polyps. CONCLUSION: Concentrations of gastrin precursors are increased in particular groups with an increased risk of developing CRC.

Genetic instability in lymphoblastoid cell lines expressing biallelic and monoallelic variants in the human MUTYH gene.

The MUTYH DNA glycosylase counteracts mutagenesis by removing adenine misincorporated opposite DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated polyposis (MAP). The impact on genetic instability of the p.Tyr179Cys and p.Arg245His MUTYH variants was evaluated in lymphoblastoid cell lines (LCLs) derived from MAP patients and their relatives in comparison to wild-type LCLs. No difference in MUTYH expression was identified between wild type and LCLs with the p.Tyr179Cys, while the p.Arg245His mutation was associated with an unstable MUTYH protein. LCLs homozygous for the p.Tyr179Cys or the p.Arg245His variant contained increased DNA 8-oxodG levels and exhibited a mutator phenotype at the PIG-A gene. The extent of the increased spontaneous mutation frequency was 3-fold (range 1.6- to 4.6-fold) in four independent LCLs carrying the p.Tyr179Cys variant, while a larger increase (6-fold) was observed in two p.Arg245His LCLs. A similar hypermutability and S-phase delay following treatment with KBrO3 was observed in LCLs homozygous for either variant. When genetic instability was investigated in monoallelic p.Arg245His carriers, mutant frequencies showed an increase which is intermediate between wild-type and homozygous cells, whereas the mutator effect in heterozygous p.Tyr179Cys LCLs was similar to that in homozygotes. These findings indicate that the type of MUTYH mutation can affect the extent of genome instability associated with MUTYH inactivation. In addition, the mild spontaneous mutator phenotype observed in monoallelic carriers highlights the biological importance of this gene in the protection of the genome against endogenous DNA damage.

Markers of the APC/ß-catenin signaling pathway as potential treatable, preneoplastic biomarkers of risk for colorectal neoplasms.

BACKGROUND: Malfunctioning of the adenomatous polyposis coli (APC)/ß-catenin signaling pathway is both an early and common event in sporadic colorectal cancer. To assess the potential of APC/ß-catenin signaling pathway markers as treatable, preneoplastic biomarkers of risk for colorectal neoplasms, we conducted a pilot colonoscopy-based case-control study (51 cases and 154 controls) of incident, sporadic colorectal adenoma. METHODS: We evaluated APC, ß-catenin, and E-cadherin expression in normal mucosa from the rectum and ascending and sigmoid colon using automated immunohistochemical and quantitative image analysis. Diet, lifestyle, and medical history were assessed with validated questionnaires. RESULTS: In the normal rectal mucosa, the ratio of the proportion of APC expression in the upper 40% of crypts with total ß-catenin expression (APC/ß-catenin score) was 14.3% greater in controls than in cases [P = 0.02; OR, 0.40; 95% confidence interval (CI), 0.14-1.14]. Compared with controls, in cases, APC expression was 3.2% lower, ß-catenin expression was 3.0% higher, and E-cadherin expression was 0.7% lower; however, none of these differences were statistically significant. The APC/ß-catenin score statistically significantly differed according to categories of plausible risk factors for colorectal cancer [e.g., it was 17.7% higher among those with 25(OH) vitamin D(3) concentrations ≥ 27 ng/mL]. CONCLUSIONS: These preliminary data suggest that the combined expression of APC and ß-catenin in the normal rectal mucosa may be associated with risk for incident, sporadic colorectal neoplasms, as well as with modifiable risk factors for colorectal neoplasms. IMPACT: Our results may help advance the development of treatable, preneoplastic biomarkers of risk for colorectal neoplasms.

Effects of celecoxib on prostanoid biosynthesis and circulating angiogenesis proteins in familial adenomatous polyposis.

Vascular cyclooxygenase (COX)-2-dependent prostacyclin (PGI(2)) may affect angiogenesis by preventing endothelial activation and platelet release of angiogenic factors present in platelet α-granules. Thus, a profound inhibition of COX-2-dependent PGI(2) might be associated with changes in circulating markers of angiogenesis. We aimed to address this issue by performing a clinical study with celecoxib in familial adenomatous polyposis (FAP). In nine patients with FAP and healthy controls, pair-matched for gender and age, we compared systemic biosynthesis of PGI(2), thromboxane (TX) A(2), and prostaglandin (PG) E(2), assessing their urinary enzymatic metabolites, 2,3-dinor-6-keto PGF(1α) (PGI-M), 11-dehydro-TXB(2) (TX-M), and 11-α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), respectively. The impact of celecoxib (400 mg b.i.d. for 7 days) on prostanoid biosynthesis and 14 circulating biomarkers of angiogenesis was evaluated in FAP. Intestinal tumorigenesis was associated with enhanced urinary TX-M levels, but unaffected by celecoxib, suggesting the involvement of a COX-1-dependent pathway, presumably from platelets. This was supported by the finding that in cocultures of a human colon adenocarcinoma cell line (HT-29) and platelets enhanced TXA(2) generation was almost completely inhibited by pretreatment of platelets with aspirin, a preferential inhibitor of COX-1. In FAP, celecoxib profoundly suppressed PGE(2) and PGI(2) biosynthesis that was associated with a significant increase in circulating levels of most proangiogenesis proteins but also the antiangiogenic tissue inhibitor of metalloproteinase 2. Urinary PGI-M, but not PGE-M, was negatively correlated with circulating levels of fibroblast growth factor 2 and angiogenin. In conclusion, inhibition of tumor COX-2-dependent PGE(2) by celecoxib may reduce tumor progression. However, the coincident depression of vascular PGI(2), in a context of enhanced TXA(2) biosynthesis, may modulate the attendant angiogenesis, contributing to variability in the chemopreventive efficacy of COX-2 inhibitors such as celecoxib.

Intestinal inflammatory cytokine response in relation to tumorigenesis in the Apc(Min/+) mouse.

The etiology of colon cancer is a complex phenomenon that involves both genetic and environmental factors. However, only about 20% have a familial basis with the largest fraction being attributed to environmental causes that can lead to chronic inflammation. While the link between inflammation and colon cancer is well established, the temporal sequence of the inflammatory response in relation to tumorigenesis has not been characterized. We examined the timing and magnitude of the intestinal inflammatory cytokine response in relation to tumorigenesis in the Apc(Min/+) mouse. Apc(Min/+) mice and wildtype mice were sacrificed at one of 4 time-points: 8, 12, 16, and 20 weeks of age. Intestinal tissue was analyzed for polyp burden (sections 1, 4 and 5) and mRNA expression and protein concentration of MCP-1, IL-1ß, IL-6 and TNF-α (sections 2 and 3). The results show that polyp burden was increased at 12, 16 and 20 weeks compared to 8 weeks (P<0.05). Gene expression (mRNA) of MCP-1, IL-1ß, IL-6 and TNF-α was increased in sections 2 and 3 starting at week 12 (P<0.05), with further increases in MCP-1, IL-1ß and IL-6 at 16 weeks (P<0.05). Protein concentration for these cytokines followed a similar pattern in section 3. Similarly, circulating MCP-1 was increased at 12 weeks (P<0.05) and then again at 20 weeks (P<0.05). In general, overall polyp number and abundance of large polyps were significantly correlated with the inflammatory cytokine response providing further support for a relationship between polyp progression and these markers. These data confirm the association between intestinal cytokines and tumorigenesis in the Apc(Min/+) mouse and provide new information on the timing and magnitude of this response in relation to polyp development. These findings may lead to the development of inflammatory mediators as important biomarkers for colon cancer progression. Further, these data may be relevant in the design of future investigations of therapeutic interventions to effectively target inflammatory processes in rodent models.