Stromal Indian hedgehog signaling is required for intestinal adenoma formation in mice.

BACKGROUND & AIMS: Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice. METHODS: Glioma-associated oncogene (Gli1)-CreERT2 and Patched (Ptch)-lacZ reporter mice were crossed with Apc(Min) mice to generate Gli1CreERT2-Rosa26-ZSGreen-Apc(Min) and Ptch-lacZ-Apc(Min) mice, which were used to identify hedgehog-responsive cells. Cyp1a1Cre-Apc (Apc(HET)) mice, which develop adenomas after administration of ß-naphthoflavone, were crossed with mice with conditional disruption of Ihh in the small intestine epithelium. Apc(Min) mice were crossed with mice in which sonic hedgehog (SHH) was overexpressed specifically in the intestinal epithelium. Intestinal tissues were collected and analyzed histologically and by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. We also analyzed levels of IHH messenger RNA and expression of IHH gene targets in intestinal tissues from patients with familial adenomatous polyposis (n = 18) or sessile serrated adenomas (n = 15) and normal colonic tissue from control patients (n = 12). RESULTS: Expression of IHH messenger RNA and its targets were increased in intestinal adenomas from patients and mice compared with control colon tissues. In mice, IHH signaling was exclusively paracrine, from the epithelium to the stroma. Loss of IHH from Apc(HET) mice almost completely blocked adenoma development, and overexpression of SHH increased the number and size of adenomas that developed. Loss of IHH from Apc(HET) mice changed the composition of the adenoma stroma; cells that expressed α-smooth muscle actin or desmin were lost, along with expression of cyclooxygenase-2, and the number of vimentin-positive cells increased. CONCLUSIONS: Apc mutant epithelial cells secrete IHH to maintain an intestinal stromal phenotype that is required for adenoma development in mice.

Akkermansia muciniphila and Helicobacter typhlonius modulate intestinal tumor development in mice.

Gastrointestinal tumor growth is thought to be promoted by gastrointestinal bacteria and their inflammatory products. We observed that intestine-specific conditional Apc mutant mice (FabplCre;Apc (15lox/+)) developed many more colorectal tumors under conventional than under pathogen-low housing conditions. Shotgun metagenomic sequencing plus quantitative PCR analysis of feces DNA revealed the presence of two bacterial species in conventional mice, absent from pathogen-low mice. One, Helicobacter typhlonius, has not been associated with cancer in man, nor in immune-competent mice. The other species, mucin-degrading Akkermansia muciniphila, is abundantly present in healthy humans, but reduced in patients with inflammatory gastrointestinal diseases and in obese and type 2 diabetic mice. Eradication of H.typhlonius in young conventional mice by antibiotics decreased the number of intestinal tumors. Additional presence of A.muciniphila prior to the antibiotic treatment reduced the tumor number even further. Colonization of pathogen-low FabplCre;Apc (15lox/+) mice with H.typhlonius or A.muciniphila increased the number of intestinal tumors, the thickness of the intestinal mucus layer and A.muciniphila colonization without H.typhlonius increased the density of mucin-producing goblet cells. However, dual colonization with H.typhlonius and A.muciniphila significantly reduced the number of intestinal tumors, the mucus layer thickness and goblet cell density to that of control mice. By global microbiota composition analysis, we found a positive association of A.muciniphila, and of H.typhlonius, and a negative association of unclassified Clostridiales with increased tumor burden. We conclude that A.muciniphila and H.typhlonius can modulate gut microbiota composition and intestinal tumor development in mice.

UV exposure inhibits intestinal tumor growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet.

Mortality from colorectal cancer increases with latitude and decreases with ambient UV radiation. We investigated whether moderate UV dosages could inhibit intestinal tumor development and whether this corresponded with UV-induced vitamin D. FabplCre;Apc(15lox/+) mice, which develop intestinal tumors, and their parents were put on a vitamin D-deficient diet. Next to a control group, one group was vitamin D supplemented and another one group was daily UV irradiated from 6 weeks of age. Vitamin D statuses after 6 weeks of treatment were markedly increased: mean ± SD from 7.7 ± 1.9 in controls to 75 ± 15 nmol/l with vitamin D supplementation (no gender difference), and to 31 ± 13 nmol/l in males and 85 ± 17 nmol/l in females upon UV irradiation. The tumor load (area covered by tumors) at 7.5 months of age was significantly reduced in both the vitamin D-supplemented group (130 ± 25 mm(2), p = 0.018) and the UV-exposed group (88 ± 9 mm(2), p < 0.0005; no gender differences) compared to the control group (202 ± 23 mm(2)). No reductions in tumor numbers were found. Only UV exposure appeared to reduce progression to malignancy (p = 0.014). Our experiments clearly demonstrate for the first time an inhibitory effect of moderate UV exposure on outgrowth and malignant progression of primary intestinal tumors, which at least in part can be attributed to vitamin D.

WNT signaling and cartilage: of mice and men.

In adult articular cartilage, the extracellular matrix is maintained by a balance between the degradation and the synthesis of matrix components. Chondrocytes that sparsely reside in the matrix and rarely proliferate are the key cellular mediators for cartilage homeostasis. There are indications for the involvement of the WNT signaling pathway in maintaining articular cartilage. Various WNTs are involved in the subsequent stages of chondrocyte differentiation during development, and deregulation of WNT signaling was observed in cartilage degeneration. Even though gene expression and protein synthesis can be activated upon injury, articular cartilage has a limited ability of self-repair and efforts to regenerate articular cartilage have so far not been successful. Because WNT signaling was found to be involved in the development and maintenance of cartilage as well as in the degeneration of cartilage, interfering with this pathway might contribute to improving cartilage regeneration. However, most of the studies on elucidating the role of WNT signaling in these processes were conducted using in vitro or in vivo animal models. Discrepancies have been found in the role of WNT signaling between chondrocytes of mouse and human origin, and extrapolation of results from mouse models to the human situation remains a challenge. Elucidation of detailed WNT signaling functions will provide knowledge to improve cartilage regeneration.

Apc bridges Wnt/ß-catenin and BMP signaling during osteoblast differentiation of KS483 cells.

The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of ß-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of ß-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc(si) cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/ß-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc(si) cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

A new conditional Apc-mutant mouse model for colorectal cancer.

Mutations of the adenomatous polyposis coli (APC) gene predispose individuals to familial adenomatous polyposis (FAP), characterized by multiple tumours in the large intestine. Most mouse models heterozygous for truncating mutant Apc alleles mimic FAP, however, the intestinal tumours occur mainly in the small intestine. To model large intestinal tumours, we generated a new conditional Apc-mutant allele, Apc(15lox), with exon 15 flanked by loxP sites. Similar survival of Apc(1638N/15lox) and Apc(1638N/+) mice indicated that the normal function of Apc was not impaired by the loxP sites. Deletion of exon 15, encoding nearly all functional Apc domains and containing the polyadenylation signal, resulted in a mutant allele expressing low levels of a 74 kDa truncated Apc protein. Germ line Cre-mediated deletion of exon 15 resulted in Apc(Delta15/+) mice, showing a severe Apc(Min/+)-like phenotype characterized by multiple tumours in the small intestine and early lethality. In contrast, conditional Cre-mediated deletion of exon 15 specifically directed to the epithelia of distal small and large intestine of FabplCre;Apc(15lox/+) mice led to longer survival and to tumours that developed predominantly in the large intestine, mimicking human FAP-associated colorectal cancer and sporadic colorectal cancer. We conclude that the FabplCre;Apc(15lox/+) mouse should be an attractive model for studies on prevention and treatment of colorectal cancer.

5-Aminosalicylic acid inhibits colitis-associated but not sporadic colorectal neoplasia in a novel conditional Apc mouse model.

Genetic predisposition, life-style habits and inflammatory bowel diseases (IBD)-related colitis are a main risk factor for colorectal cancer (CRC). 5-Aminosalicylic acid (5-ASA, mesalazine) is a mainstay therapy in IBD and believed to reduce the risk for developing CRC. We aimed to determine the ability of 5-ASA enemas to inhibit the development of sporadic and colitis-related neoplasia in mice. FabplCre;Apc(15lox/+) mice, which spontaneously develop sporadic colorectal tumours, were treated at 5 weeks of age with 5-ASA or placebo enemas for 3 weeks and examined for colorectal tumourigenesis at 8 weeks of age. Colitis-related tumour development was investigated in these mice by administration of dextran sodium sulphate, inducing intestinal inflammation and accelerating colorectal tumourigenesis, combined with treatment of 5-ASA or placebo enemas during and/or after colitis induction. 5-ASA significantly reduced colitis-accelerated neoplasia development by 50%, from 19.4 +/- 2.7 to 9.4 +/- 2.4 (mean tumour numbers +/- SEM, P = 0.02), in the distal part of the large intestine covered by the enema. 5-ASA was only effective when given during and/or after the intestinal inflammatory period. 5-ASA did not reduce, however, sporadic neoplasia development in the FabplCre;Apc(15lox/+) mice. 5-ASA tended to reduce proliferation of epithelial cells in the colitis-associated colorectal tumours but not in the sporadic colorectal tumours. In conclusion, 5-ASA medication inhibits the development of colitis-associated tumours in FabplCre;Apc(15lox/+) mice when administered during and/or after the induction of inflammation. 5-ASA does not reduce, however, sporadic tumour development in this mouse model.

Adenomatous polyposis coli-mediated control of beta-catenin is essential for both chondrogenic and osteogenic differentiation of skeletal precursors.

BACKGROUND: During skeletogenesis, protein levels of beta-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of beta-catenin turnover by down-regulating intracellular levels of beta-catenin. RESULTS: To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of beta-catenin was used, our approach resulted in the accumulation of wild type beta-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells. CONCLUSION: Our data indicate that a tight Apc-mediated control of beta-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner.

The Complete Genome Sequence of the Murine Pathobiont Helicobacter typhlonius.

BACKGROUND: Immuno-compromised mice infected with Helicobacter typhlonius are used to model microbially inducted inflammatory bowel disease (IBD). The specific mechanism through which H. typhlonius induces and promotes IBD is not fully understood. Access to the genome sequence is essential to examine emergent properties of this organism, such as its pathogenicity. To this end, we present the complete genome sequence of H. typhlonius MIT 97-6810, obtained through single-molecule real-time sequencing. RESULTS: The genome was assembled into a single circularized contig measuring 1.92 Mbp with an average GC content of 38.8%. In total 2,117 protein-encoding genes and 43 RNA genes were identified. Numerous pathogenic features were found, including a putative pathogenicity island (PAIs) containing components of type IV secretion system, virulence-associated proteins and cag PAI protein. We compared the genome of H. typhlonius to those of the murine pathobiont H. hepaticus and human pathobiont H. pylori. H. typhlonius resembles H. hepaticus most with 1,594 (75.3%) of its genes being orthologous to genes in H. hepaticus. Determination of the global methylation state revealed eight distinct recognition motifs for adenine and cytosine methylation. H. typhlonius shares four of its recognition motifs with H. pylori. CONCLUSION: The complete genome sequence of H. typhlonius MIT 97-6810 enabled us to identify many pathogenic features suggesting that H. typhlonius can act as a pathogen. Follow-up studies are necessary to evaluate the true nature of its pathogenic capabilities. We found many methylated sites and a plethora of restriction-modification systems. The genome, together with the methylome, will provide an essential resource for future studies investigating gene regulation, host interaction and pathogenicity of H. typhlonius. In turn, this work can contribute to unraveling the role of Helicobacter in enteric disease.

Proximal fluid proteome profiling of mouse colon tumors reveals biomarkers for early diagnosis of human colorectal cancer.

PURPOSE: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. This study aimed to identify novel protein biomarkers for the early diagnosis of CRC. EXPERIMENTAL DESIGN: Proximal fluids are a rich source of candidate biomarkers as they contain high concentrations of tissue-derived proteins. The FabplCre;Apc(15lox/+) mouse model represents early-stage development of human sporadic CRC. Proximal fluids were collected from normal colon and colon tumors and subjected to in-depth proteome profiling by tandem mass spectrometry. Carcinoembryonic antigen (CEA) and CHI3L1 human serum protein levels were determined by ELISA. RESULTS: Of the 2,172 proteins identified, quantitative comparison revealed 192 proteins that were significantly (P < 0.05) and abundantly (>5-fold) more excreted by tumors than by controls. Further selection for biomarkers with highest specificity and sensitivity yielded 52 candidates, including S100A9, MCM4, and four other proteins that have been proposed as candidate biomarkers for human CRC screening or surveillance, supporting the validity of our approach. For CHI3L1, we verified that protein levels were significantly increased in sera from patients with adenomas and advanced adenomas compared with control individuals, in contrast to the CRC biomarker CEA. CONCLUSION: These data show that proximal fluid proteome profiling with a mouse tumor model is a powerful approach to identify candidate biomarkers for early diagnosis of human cancer, exemplified by increased CHI3L1 protein levels in sera from patients with CRC precursor lesions.

APC mutations are associated with increased bone mineral density in patients with familial adenomatous polyposis.

The canonical Wnt pathway plays a key regulatory role in osteoblastogenesis and bone mass acquisition through its main effector, ß-catenin. Adenomatous polyposis coli (APC) represents the key intracellular gatekeeper of ß-catenin turnover, and heterozygous germ-line mutations in the APC gene cause familial adenomatous polyposis (FAP). Whether APC mutations affect bone mass has not been previously investigated. We conducted a cross-sectional study evaluating skeletal status in FAP patients with a documented APC mutation. Twenty-two FAP patients with a mean age of 42 years (54.5% women) were included in this study. Mean bone mineral density (BMD) Z-scores were significantly increased above normal at all measured sites: lumbar spine (p < .01), total hip (p < .01), femoral neck (p < .05), and trochanter (p < .01). Z-scores were +1 or greater in 14 patients (63.6%) and +2 or greater in 5 (22.7%). Mean values of bone turnover markers were within normal ranges. There was a significant positive correlation between procollagen type I N-terminal propeptide (P1NP) and ß-crosslaps (ß-CTX) (r = 0.70, p < .001) and between these markers and sclerostin and BMD measurements. We demonstrate that FAP patients display a significantly higher than normal mean BMD compared with age- and sex-matched healthy controls in the presence of a balanced bone turnover. Our data suggest a state of "controlled" activation of the Wnt signaling pathway in heterozygous carriers of APC mutations, most likely owing to upregulation of cytoplasmic ß-catenin levels.

Germline mutation of INI1/SMARCB1 in familial schwannomatosis.

Patients with schwannomatosis develop multiple schwannomas but no vestibular schwannomas diagnostic of neurofibromatosis type 2. We report an inactivating germline mutation in exon 1 of the tumor-suppressor gene INI1 in a father and daughter who both had schwannomatosis. Inactivation of the wild-type INI1 allele, by a second mutation in exon 5 or by clear loss, was found in two of four investigated schwannomas from these patients. All four schwannomas displayed complete loss of nuclear INI1 protein expression in part of the cells. Although the exact oncogenetic mechanism in these schwannomas remains to be elucidated, our findings suggest that INI1 is the predisposing gene in familial schwannomatosis.

Association of C-MYC amplification with progression from the in situ to the invasive stage in C-MYC-amplified breast carcinomas.

Human carcinoma in situ of the breast already demonstrates genomic changes found in invasive lesions. However, no specific genetic alterations have previously been identified that are associated with progression from the in situ to the invasive stage. By comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis of an invasive breast carcinoma with a large associated in situ component, high-level amplification of C-MYC was found in the invasive component only. To determine the frequency of this correlation in a panel of 188 invasive breast carcinomas, 18 additional cases with C-MYC amplification were identified. Nine of these cases had a detectable adjacent in situ component. FISH analysis demonstrated increased (>5) C-MYC signals per nucleus in seven invasive components and increased (>4) C-MYC/centromere 8 signal ratios in five of these. None of the associated in situ components demonstrated these increases. The minimal amplified region was defined at 8q24.13-8qter. C-MYC amplification was correlated with overexpression of C-MYC and two of its target genes, TERT and FBL. Thus, C-MYC amplification is the first identified genetic alteration that is associated with progression from the in situ to the invasive stage of breast carcinoma.