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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;Apc15lox/+ 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. Clin Cancer Res; 18(9); 2613–24. ©2012 AACR.

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mice, which develop intestinal tumors, and their parents were put on a vitamin D-deficient diet. Next to acontrol 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: mean6SD from 7.761.9 in controls to 75615nmol/l with vitamin D supplementation (no gender difference), and to 31613 nmol/l in males and 85617 nmol/l in femalesupon UV irradiation. The tumor load (area covered by tumors) at 7.5 months of age was significantly reduced in both the vita-min D-supplemented group (130625 mm

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;Apc15lox/+ 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. Clin Cancer Res; 18(9); 2613–24. ©2012 AACR.

Objective: In the past years, the canonical Wnt/beta-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. In this pathway, glycogen synthase kinase-3 beta (GSK3 beta) down-regulates transduction of the canonical Wnt signal by promoting degradation of beta-catenin. In this study we wanted to further investigate the role of Gsk3 beta in cartilage maintenance. Design: Therefore, we have treated chondrocytes ex vivo and in vivo with GIN, a selective GSK3 beta inhibitor. Results: In E17.5 fetal mouse metatarsals, GIN treatment resulted in loss of expression of cartilage markers and decreased chondrocyte proliferation from day 1 onward. Late (3 days) effects of GIN included cartilage matrix degradation and increased apoptosis. Prolonged (7 days) GIN treatment resulted in resorption of the metatarsal. These changes were confirmed by microarray analysis showing a decrease in expression of typical chondrocyte markers and induction of expression of proteinases involved in cartilage matrix degradation. An intra-articular injection of GIN in rat knee joints induced nuclear accumulation of beta-catenin in chondrocytes 72 h later. Three intra-articular GIN injections with a 2 days interval were associated with surface fibrillation, a decrease in glycosaminoglycan expression and chondrocyte hypocellularity 6 weeks later. Conclusions: These results suggest that, by down-regulating beta-catenin, Gsk3 beta preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term beta-catenin up-regulation in cartilage secondary to Gsk3 beta inhibition may be sufficient to induce osteoarthritis-like features in vivo. (C) 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

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(delta 15/+) 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.

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.

Item does not contain fulltext 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.

Item does not contain fulltext 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.

Previously, we have mimicked human neurofibromatosis type 2 (NF2) in conditional Nf2 mutant (P0Cre;Nf2flox2/flox2) mice. Schwannomas, characteristic for NF2, were found at low frequency in older mice. Here, we report that these mice, upon additional hemizygosity for p53, rapidly develop multiple tumours showing features consistent with malignant peripheral nerve sheath tumours. Thus, p53 hemizygosity promotes tumorigenesis of mutant Nf2 peripheral nerve cells. In contrast, young P0Cre;Nf2flox2/+;p53+/- cis mice mainly succumb to Nf2/p53-related osteogenic tumours. Therefore, Cre-mediated early biallelic loss of Nf2 function in neural crest-derived cells hemizygous for p53 results in resistance to osteogenic tumours and increased susceptibility to peripheral nerve sheath tumours.

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), p0.0005; 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.

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.

Desmoid tumours (also called deep or aggressive fibromatoses) are potentially life-threatening fibromatous lesions. Hereditary desmoid tumours arise in individuals affected by either familial adenomatous polyposis (FAP) or hereditary desmoid disease (HDD) carrying germline mutations in APC. Most sporadic desmoids carry somatic mutations in CTNNB1. Previous studies identified losses on 5q and 6q, and gains on 8q and 20q as recurrent genetic changes in desmoids. However, virtually all genetic changes were derived from sporadic tumours. To investigate the somatic alterations in FAP-associated desmoids and to compare them with changes occurring in sporadic tumours, we analysed 17 FAP-associated and 38 sporadic desmoids by array comparative genomic hybridisation and multiple ligation-dependent probe amplification. Overall, the desmoids displayed only a limited number of genetic changes, occurring in 44% of cases. Recurrent gains at 8q (7%) and 20q (5%) were almost exclusively found in sporadic tumours. Recurrent losses were observed for a 700 kb region at 5q22.2, comprising the APC gene (11%), a 2 Mb region at 6p21.2-p21.1 (15%), and a relatively large region at 6q15-q23.3 (20%). The FAP-associated desmoids displayed a significantly higher frequency of copy number abnormalities (59%) than the sporadic tumours (37%). As predicted by the APC germline mutations among these patients, a high percentage (29%) of FAP-associated desmoids showed loss of the APC region at 5q22.2, which was infrequently (3%) seen among sporadic tumours. Our data suggest that loss of region 6q15-q16.2 is an important event in FAP-associated as well as sporadic desmoids, most likely of relevance for desmoid tumour progression.

The canonical Wnt pathway plays a key regulatory role in osteoblastogenesis and bone mass acquisition through its main effector, β-catenin. Adenomatous polyposis coli (APC) represents the key intracellular gatekeeper of β-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

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 β-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.