No abstract

Animal models for human colorectal cancer recapitulate multistep carcinogenesis that is typically initiated by activation of the Wnt pathway. Although potential roles of both genetic and environmental modifiers have been extensively investigated in vivo, it remains elusive whether epithelial cells definitely require interaction with stromal cells or microflora for tumor development. Here we show that tumor development could be simply induced independently of intestinal microenvironment, even with WT murine primary intestinal cells alone. We developed an efficient method for lentiviral transduction of intestinal organoids in 3D culture. Despite seemingly antiproliferative effects by knockdown of adenomatous polyposis coli (APC), we managed to reproducibly induce APC-inactivated intestinal organoids. As predicted, these organoids were constitutively active in the Wnt signaling pathway and proved tumorigenic when injected into nude mice, yielding highly proliferative tubular epithelial glands accompanied by prominent stromal tissue. Consistent with cellular transformation, tumor-derived epithelial cells acquired sphere formation potential, gave rise to secondary tumors on retransplantation, and highly expressed cancer stem cell markers. Inactivation of p53 or phosphatase and tensin homolog deleted from chromosome 10, or activation of Kras, promoted tumor development only in the context of APC suppression, consistent with earlier genetic studies. These findings clearly indicated that genetic cooperation for intestinal tumorigenesis could be essentially recapitulated in intestinal organoids without generating gene-modified mice. Taken together, this in vitro model for colon cancer described herein could potentially provide unique opportunities for carcinogenesis studies by serving as a substitute or complement to the currently standard approaches.colon carcinogenesis | shRNA | primary culture | Matrigel | validation

Colon cancers have been shown to develop after accumulation of multiple genetic and epigenetic alterations with changes in global gene expression profiles, contributing to the establishment of widely diverse phenotypes. Transcriptional and posttranscriptional regulation of gene expression by small RNA species, such as the small interfering RNA and microRNA and the RNA-induced silencing complex (RISC), is currently drawing major interest with regard to cancer development. SND1, also called Tudor-SN and p100 and recently reported to be a component of RISC, is among the list of highly expressed genes in human colon cancers. In the present study, we showed remarkable up-regulation of SND1 mRNA in human colon cancer tissues, even in early-stage lesions, and also in colon cancer cell lines. When mouse Snd1 was stably overexpressed in IEC6 rat intestinal epithelial cells, contact inhibition was lost and cell growth was promoted, even after the cells became confluent. Intriguingly, IEC6 cells with high levels of Snd1 also showed an altered distribution of Ecadherin from the cell membrane to the cytoplasm, suggesting loss of cellular polarity. Furthermore, the adenomatous polyposis coli (Apc) protein was coincidentally down-regulated, with no significant changes in the Apc mRNA level. Immunohistochemical analysis using chemically induced colonic lesions developed in rats revealed overexpression of Snd1 not only in colon cancers but also in aberrant crypt foci, putative precancerous lesions of the colon. Up-regulation of SND1 may thus occur at a very early stage in colon carcinogenesis and contribute to the posttranscriptional regulation of key players in colon cancer development, including APC and B-catenin. [Cancer Res 2007;67(19):9568-76]

Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.

Many mutagenic heterocyclic amines (HAs) have been isolated from cooked foods and pyrolysates of amino acids and proteins, and the carcinogenicity of 10 of these HAs in rodents and of 1 in monkeys has been reported. Quantification of these carcinogenic HAs in various kinds of cooked foods indicated that the level of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was highest (0.56-69.2 ng/g), that of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was second highest (0.64-6.44 ng/g), and those of other HAs were 0.03-2.50 ng/g. Heterocyclic amines were found in urine samples of 10 healthy volunteers consuming a normal diet, but HAs were not detectable in urine samples of three patients receiving parenteral alimentation. These results strongly suggest that humans are continuously exposed to HAs derived from food in the normal diet. Based on quantitative data on the levels of HAs in cooked foods and urine samples, the daily exposures to PhIP and MeIQx were estimated to be 0.1-13.8 micrograms and 0.2-2.6 micrograms per person, respectively. These levels of carcinogenic HAs are in the same range as those of other carcinogens such as N-nitrosodimethylamine and benzo[a]pyrene to which humans are exposed.