Preinvasive colorectal lesion transcriptomes correlate with endoscopic morphology (polypoid vs. nonpolypoid).

Improved colonoscopy is revealing precancerous lesions that were frequently missed in the past, and ∼30% of those detected today have nonpolypoid morphologies ranging from slightly raised to depressed. To characterize these lesions molecularly, we assessed transcription of 23,768 genes in 42 precancerous lesions (25 slightly elevated nonpolypoid and 17 pedunculated polypoid), each with corresponding samples of normal mucosa. Nonpolypoid versus polypoid morphology explained most gene expression variance among samples; histology, size, and degree of dysplasia were also linked to specific patterns. Expression changes in polypoid lesions frequently affected cell-cycling pathways, whereas cell-survival dysregulation predominated in nonpolypoid lesions. The latter also displayed fewer and less dramatic expression changes than polypoid lesions. Paradigmatic of this trend was progressive loss through the normal > nonpolypoid > polypoid > cancer sequence of TMIGD1 mRNA and protein. This finding, along with TMIGD1 protein expression patterns in tissues and cell lines, suggests that TMIGD1 might be associated with intestinal-cell differentiation. We conclude that molecular dysregulation in slightly elevated, nonpolypoid, precancerous colorectal lesions may be somewhat less severe than that observed in classic adenomatous polyps.

The protein tyrosine phosphatase receptor type R gene is an early and frequent target of silencing in human colorectal tumorigenesis.

BACKGROUND: Tumor development in the human colon is commonly accompanied by epigenetic changes, such as DNA methylation and chromatin modifications. These alterations result in significant, inheritable changes in gene expression that contribute to the selection of tumor cells with enhanced survival potential. RESULTS: A recent high-throughput gene expression analysis conducted by our group identified numerous genes whose transcription was markedly diminished in colorectal tumors. One of these, the protein-tyrosine phosphatase receptor type R (PTPRR) gene, was dramatically downregulated from the earliest stages of cellular transformation. Here, we show that levels of both major PTPRR transcript variants are markedly decreased (compared with normal mucosal levels) in precancerous and cancerous colorectal tumors, as well in colorectal cancer cell lines. The expression of the PTPRR-1 isoform was inactivated in colorectal cancer cells as a result of de novo CpG island methylation and enrichment of transcription-repressive histone-tail marks, mainly H3K27me3. De novo methylation of the PTPRR-1 transcription start site was demonstrated in 29/36 (80%) colorectal adenomas, 42/44 (95%) colorectal adenocarcinomas, and 8/8 (100%) liver metastases associated with the latter tumors. CONCLUSIONS: Epigenetic downregulation of PTPRR seems to be an early alteration in colorectal cell transformation, which is maintained during the clonal selection associated with tumor progression. It may represent a preliminary step in the constitutive activation of the RAS/RAF/MAPK/ERK signalling, an effect that will later be consolidated by mutations in genes encoding key components of this pathway.

The Intestinal Wnt/TCF Signature.

BACKGROUND & AIMS: In colorectal cancer, activating mutations in the Wnt pathway transform epithelial cells through the inappropriate expression of a TCF4 target gene program, which is physiologically expressed in intestinal crypts. METHODS: We have now performed an exhaustive array-based analysis of this target gene program in colorectal cancer cell lines carrying an inducible block of the Wnt cascade. Independently, differential gene-expression profiles of human adenomas and adenocarcinomas vs normal colonic epithelium were obtained. RESULTS: Expression analyses of approximately 80 genes common between these data sets were performed in a murine adenoma model. The combined data sets describe a core target gene program, the intestinal Wnt/TCF signature gene set, which is responsible for the transformation of human intestinal epithelial cells. CONCLUSIONS: The genes were invariably expressed in adenomas, yet could be subdivided into 3 modules, based on expression in distinct crypt compartments. A module of 17 genes was specifically expressed at the position of the crypt stem cell.

Transcriptome profile of human colorectal adenomas.

Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the beta-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation.

Expression of the MutL homologue hMLH3 in human cells and its role in DNA mismatch repair.

The human mismatch repair (MMR) proteins hMLH1 and hPMS2 function in MMR as a heterodimer. Cells lacking either protein have a strong mutator phenotype and display microsatellite instability, yet mutations in the hMLH1 gene account for approximately 50% of hereditary nonpolyposis colon cancer families, whereas hPMS2 mutations are substantially less frequent and less penetrant. Similarly, in the mouse model, Mlh1-/- animals are highly cancer prone and present with gastrointestinal tumors at an early age, whereas Pms2-/- mice succumb to cancer much later in life and do not present with gastrointestinal tumors. This evidence suggested that MLH1 might functionally interact with another MutL homologue, which compensates, at least in part, for a deficiency in PMS2. Sterility of Mlh1-/-, Pms2-/-, and Mlh3-/- mice implicated the Mlh1/Pms2 and Mlh1/Mlh3 heterodimers in meiotic recombination. We now show that the hMLH1/hMLH3 heterodimer, hMutLgamma, can also assist in the repair of base-base mismatches and single extrahelical nucleotides in vitro. Analysis of hMLH3 expression in colon cancer cell lines indicated that the protein levels vary substantially and independently of hMLH1. If hMLH3 participates in MMR in vivo, its partial redundancy with hPMS2, coupled with the fluctuating expression levels of hMLH3, may help explain the low penetrance of hPMS2 mutations in hereditary nonpolyposis colon cancer families.

Defective DNA mismatch repair determines a characteristic transcriptional profile in proximal colon cancers.

BACKGROUND & AIMS: Colon cancers with defective DNA mismatch repair (MMR) have peculiar molecular, pathologic, and clinical features, including high-level microsatellite instability, conspicuous lymphocytic infiltration, preferential location in the proximal colon, and better prognosis. Our aim was to characterize the transcriptional profile of this colon cancer subset. METHODS: An oligonucleotide microarray containing 12,625 probes was used to evaluate gene expression in 25 proximal colon cancers, 10 samples of normal colon mucosa, and 14 colon cancer cell lines. Transcriptional profiles of MMR-deficient cancers and cell lines were compared with those of their MMR-proficient counterparts. RESULTS: Unsupervised analysis of microarray data showed that MMR status exerts a predominant influence on the gene expression profile of proximal colon cancers. Hierarchical clustering divided the cancers into 2 groups corresponding almost perfectly with their MMR status. Supervised analysis identified numerous gene expression changes that represent a genetic signature of MMR-deficient colon cancers. Changes in genes involved in apoptosis and the immune response were consistent with the better prognosis of MMR-deficient cancers. In MMR-deficient cancers and cell lines, 4-1BBL, a crucial gene in the anti-tumor immune response, was, respectively, 2.4 and 6.0 times more expressed than in their MMR-proficient counterparts. This difference was confirmed by quantitative reverse-transcription polymerase chain reaction and flow cytometric assessment of 4-1BBL protein expression in colon cancer cell lines. Our analysis also showed novel possible gene targets of microsatellite instability. CONCLUSIONS: MMR inactivation produces distinct changes in the cellular messenger RNA pool, which is consistent with a unique tumorigenesis pathway.