Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival.

Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. We originally identified KIAA1199 (now officially called CEMIP) as a transcript highly induced in colon cancer: initially designating the transcript as Colon Cancer Secreted Protein 1. We molecularly characterized CEMIP expression both at the mRNA and protein level and found it is a secreted protein induced an average of 54-fold in colon cancer. Knockout of CEMIPreduced the ability of human colon cancer cells to form xenograft tumors in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. We find CEMIP mRNA overexpression correlates with poorer patient survival. In stage III only (n = 31) or in combined stage II plus stage III colon cancer cases (n = 73), 5-year overall survival was significantly better (p = 0.004 and p = 0.0003, respectively) among patients with low CEMIP expressing tumors than those with high CEMIP expressing tumors. These results demonstrate that CEMIP directly facilitates colon tumor growth, and high CEMIP expression correlates with poor outcome in stage III and in stages II+III combined cohorts. We present CEMIP as a candidate prognostic marker for colon cancer and a potential therapeutic target.

Ugene, a newly identified protein that is commonly overexpressed in cancer and binds uracil DNA glycosylase.

Expression microarrays identified a novel transcript, designated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in malignant colon cancers. These findings were validated by real-time PCR and Northern blot analysis in an independent panel of colon cancer cases. In addition, Ugene expression was found to be elevated in many other common cancer types, including breast, lung, uterus, and ovary. Immunofluorescence of V5-tagged Ugene revealed it to have a nuclear localization. In a pull-down assay, uracil DNA glycosylase 2 (UNG2), an important enzyme in the base excision repair (BER) pathway, was identified as a partner protein that binds to Ugene. Coimmunoprecipitation and Western blot analysis confirmed the binding between the endogenous Ugene and UNG2 proteins. Using deletion constructs, we find that Ugene binds to the first 25 amino acids of the UNG2 NH(2) terminus. We suggest that Ugene induction in cancer may contribute to the cancer phenotype by interacting with the BER pathway.

Germline mutations and variants in the succinate dehydrogenase genes in Cowden and Cowden-like syndromes.

Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid, and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent of SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. SDHB-D may be candidate susceptibility genes for some PTEN mutation-negative individuals with CS-like cancers. To address this hypothesis, germline SDHB-D mutation analysis in 375 PTEN mutation-negative CS/CS-like individuals was performed, followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation-negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Among these, 10 (13.5%) had germline mutations/variants in SDHB (n = 3) or SDHD (7), not found in 700 controls (p < 0.001). Compared to PTEN mutation-positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH versus 30/107 PTEN, p < 0.001), thyroid (5/10 versus 15/106, p < 0.001), and kidney (2/10 versus 4/230, p = 0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants show increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid, and renal cancers beyond those conferred by germline PTEN mutations. SDH testing should be considered for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid, and/or renal cancers.

Differential expression of PTEN-targeting microRNAs miR-19a and miR-21 in Cowden syndrome.

Germline mutations in the gene encoding phosphatase and tensin homolog deleted on chromosome ten (PTEN [MIM 601728]) are associated with a number of clinically distinct heritable cancer syndromes, including both Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS). Seemingly identical pathogenic PTEN mutations have been observed in patients with CS and BRRS, as well as in patients with incomplete features of CS, referred to as CS-like (CSL) patients. These observations indicate that additional, unidentified, genetic and epigenetic factors act as phenotypic modifiers in these disorders. These genetic factors could also contribute to disease in patients with CS, CSL, or BRRS without identifiable PTEN mutations. Two potential modifiers are miR-19a and miR-21, which are previously identified PTEN-targeting miRNAs. We investigated the role of these miRNAs by characterizing their relative expression levels in PTEN-mutation-positive and PTEN-mutation-negative patients with CS, CSL, or BRRS. Interestingly, we observed differential expression of miR-19a and miR-21 in our PTEN-mutation-positive patients. Both were found to be significantly overexpressed within this group (p < 0.01) and were inversely correlated with germline PTEN protein levels. Similarly, the relative expression of miR-19a and miR-21 was differentially expressed in a series of PTEN-mutation-negative patients with CS or CSL with variable clinical phenotypes and decreased full-length PTEN protein expression. Among PTEN-mutation-positive patients with CS, both miRNAs were significantly overexpressed (p = 0.006-0.013). Taken together, our study results suggest that differential expression of PTEN-targeting miR-19a and miR-21 modulates the PTEN protein levels and the CS and CSL phenotypes, irrespective of the patient's mutation status, and support their roles as genetic modifiers in CS and CSL.

Genotype-phenotype correlation in combined deficiency of factor V and factor VIII.

Combined deficiency of factor V and factor VIII (F5F8D) is caused by mutations in one of 2 genes, either LMAN1 or MCFD2. Here we report the identification of mutations for 11 additional F5F8D families, including 4 novel mutations, 2 in MCFD2 and 2 in LMAN1. We show that a novel MCFD2 missense mutation identified here (D81Y) and 2 previously reported mutations (D89A and D122V) abolish MCFD2 binding to LMAN1. Measurement of platelet factor V (FV) levels in 7 F5F8D patients (4 with LMAN1 and 3 with MCFD2 mutations) demonstrated similar reductions to those observed for plasma FV. Combining the current data together with all previous published reports, we performed a genotype-phenotype analysis comparing patients with MCFD2 mutations with those with LMAN1 mutations. A previously unappreciated difference is observed between these 2 classes of patients in the distribution of plasma levels for FV and factor VIII (FVIII). Although there is considerable overlap, the mean levels of plasma FV and FVIII in patients with MCFD2 mutations are significantly lower than the corresponding levels in patients with LMAN1 mutations. No differences in distribution of factor levels are observed by sex. These data suggest that MCFD2 may play a primary role in the export of FV and FVIII from the ER, with the impact of LMAN1 mediated indirectly through its interaction with MCFD2.

SNP arrays in heterogeneous tissue: highly accurate collection of both germline and somatic genetic information from unpaired single tumor samples.

SNP arrays provide reliable genotypes and can detect chromosomal aberrations at a high resolution. However, tissue heterogeneity is currently a major limitation for somatic tissue analysis. We have developed SOMATICs, an original program for accurate analysis of heterogeneous tissue samples. Fifty-four samples (42 tumors and 12 normal tissues) were processed through Illumina Beadarrays and then analyzed with SOMATICs. We demonstrate that tissue heterogeneity-related limitations not only can be overcome but can also be turned into an advantage. First, admixture of normal cells with tumor can be used as an internal reference, thereby enabling highly sensitive detection of somatic deletions without having corresponding normal tissue. Second, the presence of normal cells allows for discrimination of somatic from germline aberrations, and the proportion of cells in the tissue sample that are harboring the somatic events can be assessed. Third, relatively early versus late somatic events can also be distinguished, assuming that late events occur only in subsets of cancer cells. Finally, admixture by normal cells allows inference of germline genotypes from a cancer sample. All this information can be obtained from any cancer sample containing a proportion of 40-75% of cancer cells. SOMATICs is a ready-to-use open-source program that integrates all of these features into a simple format, comprehensively describing each chromosomal event.

Identification of susceptibility genes for cancer in a genome-wide scan: results from the colon neoplasia sibling study.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in Americans and is the second leading cause of cancer mortality. Only a minority ( approximately 5%) of familial CRC can be explained by known genetic variants. To identify susceptibility genes for familial colorectal neoplasia, the colon neoplasia sibling study conducted a comprehensive, genome-wide linkage scan of 194 kindreds. Clinical information (histopathology, size and number of polyps, and other primary cancers) was used in conjunction with age at onset and family history for classification of the families into five phenotypic subgroups (severe histopathology, oligopolyposis, young, colon/breast, and multiple cancer) prior to analysis. By expanding the traditional affected-sib-pair design to include unaffected and discordant sib pairs, analytical power and robustness to type I error were increased. Sib-pair linkage statistics and Haseman-Elston regression identified 19 linkage peaks, with interesting results for chromosomes 1p31.1, 15q14-q22, 17p13.3, and 21. At marker D1S1665 (1p31.1), there was strong evidence for linkage in the multiple-cancer subgroup (p = 0.00007). For chromosome 15q14-q22, a linkage peak was identified in the full-sample (p = 0.018), oligopolyposis (p = 0.003), and young (p = 0.0009) phenotypes. This region includes the HMPS/CRAC1 locus associated with hereditary mixed polyposis syndrome (HMPS) in families of Ashkenazi descent. We provide compelling evidence linking this region in families of European descent with oligopolyposis and/or young age at onset (

Frequency of germline genomic homozygosity associated with cancer cases.

CONTEXT: Cancer is a multigenic disease resulting from both germline susceptibility and somatic events. While studying loss of heterozygosity (LOH) in cancer tissues, we anecdotally observed a low frequency of heterozygosity in cancer patients compared with controls, raising the question whether homozygosity could play a role in cancer predisposition. OBJECTIVES: To determine the frequency of germline homozygosity in a large series of patients with 3 different types of solid tumors compared with population-based controls. DESIGN, SETTING, AND PATIENTS: Germline and corresponding tumor DNA isolated from 385 patients with carcinomas (147 breast, 116 prostate, and 122 head and neck carcinomas) were subjected to whole genome (345-microsatellite marker) LOH analysis. MAIN OUTCOME MEASURES: Frequency of homozygosity at microsatellite markers in cancer cases vs controls and frequency of somatic LOH in cancers at loci with the highest homozygosity. RESULTS: We identified 16 loci in common among the 3 cancer types, with significantly increased germline homozygosity frequencies in the cancer patients compared with controls (P < .001). In the cases who happened to be germline heterozygous at these 16 loci, we found a mean (SD) LOH frequency of 58% (4.2%) compared with 50% (7.5%) at 197 markers without increased germline homozygosity (P < .001). Across the genome, this relationship holds as well (r = 0.46; 95% confidence interval, 0.37-0.53; P < .001). We validated the association of specific loci with high germline homozygosity frequencies in an independent, single-nucleotide polymorphism-based, public data set of 205 lung carcinomas from white individuals (P < .05 to P < .001) as well as the correlation between genome-wide germline homozygosity and LOH frequencies (r = 0.21; 95% confidence interval, 0.18-0.24; P < .001). CONCLUSIONS: In our study of 4 different types of solid tumors (our data for 3 types validated in a fourth type), increased germline homozygosity occurred at specific loci. When the germline was heterozygous at these loci, high frequencies of LOH/allelic imbalance occurred at these loci in the corresponding carcinomas.

Breast-cancer stromal cells with TP53 mutations and nodal metastases.

BACKGROUND: The importance of cross-talk between a cancer and its microenvironment has been increasingly recognized. We hypothesized that mutational inactivation of the tumor-suppressor gene TP53 and genomic alterations in stromal cells of a tumor's microenvironment contribute to the clinical outcome. METHODS: We performed TP53 mutation analysis and genomewide analysis of loss of heterozygosity and allelic imbalance on DNA from isolated neoplastic epithelial and stromal cells from 43 patients with hereditary breast cancer and 175 patients with sporadic breast cancer. Compartment-specific patterns and TP53 mutations were analyzed. Associations between compartment-specific TP53 status, loss of heterozygosity or allelic imbalance, and clinical and pathological characteristics were computed. RESULTS: TP53 mutations were associated with an increased loss of heterozygosity and allelic imbalance in both hereditary and sporadic breast cancers, but samples from patients with hereditary disease had more frequent mutations than did those from patients with sporadic tumors (74.4% vs. 42.3%, P=0.001). Only 1 microsatellite locus (2p25.1) in stromal cells from hereditary breast cancers was associated with mutated TP53, whereas there were 66 such loci in cells from sporadic breast cancers. Somatic TP53 mutations in stroma, but not epithelium, of sporadic breast cancers were associated with regional nodal metastases (P=0.003). A specific set of five loci linked to an increased loss of heterozygosity and allelic imbalance in the stroma of sporadic tumors was associated with nodal metastases in the absence of TP53 mutations. No associations were found between any of the clinical or pathological features of hereditary breast cancer with somatic TP53 mutations. CONCLUSIONS: Stroma-specific loss of heterozygosity or allelic imbalance is associated with somatic TP53 mutations and regional lymph-node metastases in sporadic breast cancer but not in hereditary breast cancer.

Microenvironmental genomic alterations and clinicopathological behavior in head and neck squamous cell carcinoma.

CONTEXT: Carcinogens associated with head and neck squamous cell carcinoma (SCC) genesis should inflict genomic alterations not only on the epithelium but also the mesenchyme of the aerodigestive tract. Therefore, the apparently nonmalignant stroma surrounding the tumor epithelium can acquire genomic alterations and contribute to cancer initiation and progression. OBJECTIVES: To determine compartment-specific loci of loss of heterozygosity or allelic imbalance (LOH/AI) and to identify which genomic alterations restricted to the stroma cell population contribute to aggressiveness of head and neck SCC disease. DESIGN, SETTING, AND PATIENTS: Tumor epithelium and surrounding stroma were isolated from 122 US patients with oral cavity and oropharyngeal or hypopharyngeal SCC and subjected to whole-genome LOH/AI analysis using 366 microsatellite markers. Samples, collected between 2001 and 2004, were pulled and transferred in batches of 10 to 30 between 2002 and 2005. Laser capture microdissection DNA extraction and technical genotyping occurred on a rolling model between 2002 and November 2005. MAIN OUTCOME MEASURES: Compartment-specific frequency and distribution of LOH/AI were determined, and hot spots of genomic alterations identified. Compartment-specific LOH/AI events were correlated with presenting clinicopathologic characteristics. RESULTS: Tumor-associated stroma of head and neck SCC from smokers were found to have a high degree of genomic alterations. A correlation between tumor aggressiveness could be found for a specific set of 5 loci. Three stroma-specific loci (D4S2417, D3S360, and D19555) were associated with tumor size (pT) and regional nodal metastases (pN). Furthermore, 2 epithelial-specific LOH/AI hot spots were positively correlated with pN status and clinical stage. CONCLUSIONS: Stroma-specific genetic alterations are associated with smoking-related head and neck SCC genesis. These findings suggest novel prognostic or diagnostic biomarkers and identify potential new molecular targets for therapeutic and preventive intervention.

15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis.

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a prostaglandin-degrading enzyme that is highly expressed in normal colon mucosa but is ubiquitously lost in human colon cancers. Herein, we demonstrate that 15-PGDH is active in vivo as a highly potent suppressor of colon neoplasia development and acts in the colon as a required physiologic antagonist of the prostaglandin-synthesizing activity of the cyclooxygenase 2 (COX-2) oncogene. We first show that 15-PGDH gene knockout induces a marked 7.6-fold increase in colon tumors arising in the Min (multiple intestinal neoplasia) mouse model. Furthermore, 15-PGDH gene knockout abrogates the normal resistance of C57BL/6J mice to colon tumor induction by the carcinogen azoxymethane (AOM), conferring susceptibility to AOM-induced adenomas and carcinomas in situ. Susceptibility to AOM-induced tumorigenesis is mediated by a marked induction of dysplasia, proliferation, and cyclin D1 expression throughout microscopic aberrant crypt foci arising in 15-PGDH null colons and is concomitant with a doubling of prostaglandin E(2) in 15-PGDH null colonic mucosa. A parallel role for 15-PGDH loss in promoting the earliest steps of colon neoplasia in humans is supported by our finding of a universal loss of 15-PGDH expression in microscopic colon adenomas recovered from patients with familial adenomatous polyposis, including adenomas as small as a single crypt. These models thus delineate the in vivo significance of 15-PGDH-mediated negative regulation of the COX-2 pathway and moreover reveal the particular importance of 15-PGDH in opposing the neoplastic progression of colonic aberrant crypt foci.

Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene.

BACKGROUND: Increased DNA methylation is an epigenetic alteration that is common in human cancers and is often associated with transcriptional silencing. Aberrantly methylated DNA has also been proposed as a potential tumor marker. However, genes such as vimentin, which are transcriptionally silent in normal epithelium, have not until now been considered as targets for cancer-associated aberrant methylation and for use as cancer markers. METHODS: We applied methylation-specific polymerase chain reaction to the vimentin gene, which is transcriptionally silent in normal colonocytes, and compared methylation of vimentin exon 1 in cancer tissues and in fecal DNA from colon cancer patients versus control samples from healthy subjects. RESULTS: Vimentin exon-1 sequences were unmethylated in 45 of 46 normal colon tissues. In contrast, vimentin exon-1 sequences were methylated in 83% (38 of 46) and 53% (57 of 107) of tumors from two independently collected groups of colon cancer patients. When evaluated as a marker for colon cancer detection in fecal DNA from another set of colon cancer patients, aberrant vimentin methylation was detected in fecal DNA from 43 of 94 patients, for a sensitivity of 46% (95% confidence interval [CI] = 35% to 56%). The sensitivity for detecting stage I and II cancers was 43% (26 of 60 case patients) (95% CI = 31% to 57%). Only 10% (20 of 198 case patients) of control fecal DNA samples from cancer-free individuals tested positive for vimentin methylation, for a specificity of 90% (95% CI = 85% to 94%). CONCLUSIONS: Aberrant methylation of exon-1 sequences within the nontranscribed vimentin gene is a novel molecular biomarker of colon cancer and can be successfully detected in fecal DNA to identify nearly half of individuals with colon cancer.

Colon cancer secreted protein-2 (CCSP-2), a novel candidate serological marker of colon neoplasia.

Cancers of the colon and rectum are the second leading cause of cancer death among adult Americans. When detected at early stages, colon cancer is highly curable. Colonoscopy, an effective but invasive screening test, has been limited in its public acceptance. The goal of this study was to identify novel serum markers of colon cancers and precancerous colon adenomas as potential candidates for noninvasive detection of early colon neoplasms. Employing expression microarrays, we identified colon cancer secreted protein-2 (CCSP-2) as a novel transcript whose expression is generally absent in normal colon and other normal body tissues, but that is induced an average of 78-fold in Stage II, III, and IV colon cancers, as well as in colon adenomas and colon cancer cell lines. These findings were validated by real-time PCR analysis in an independent panel of colon cancer cases. Moreover, CCSP-2 was shown to encode a secreted protein that circulates stably and is detectable in the blood of mice bearing human cancer xenografts transfected with epitope-tagged CCSP-2. As a novel secreted protein that is markedly induced in colon adenomas and cancers, CCSP-2 is a novel candidate for development as a diagnostic serum marker of early stage colon cancer.

15-Hydroxyprostaglandin dehydrogenase, a COX-2 oncogene antagonist, is a TGF-beta-induced suppressor of human gastrointestinal cancers.

Marked increased expression of cyclooxygenase 2 (COX-2), a prostaglandin-synthesizing enzyme that is pharmacologically inhibited by nonsteroid anti-inflammatory-type drugs, is a major early oncogenic event in the genesis of human colon neoplasia. We report that, in addition to inducing expression of COX-2, colon cancers further target the prostaglandin biogenesis pathway by ubiquitously abrogating expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme that physiologically antagonizes COX-2. We find that 15-PGDH transcript and protein are both highly expressed by normal colonic epithelia but are nearly undetectable in colon cancers. Using gene transfection to restore 15-PGDH expression in colon cancer cells strongly inhibits the ability of these cells to form tumors in immune-deficient mice and demonstrates 15-PGDH to have functional colon cancer tumor suppressor activity. In interrogating the mechanism for 15-PGDH expression loss in colon cancer, we determined that colonic 15-PGDH expression is directly controlled and strongly induced by activation of the TGF-beta tumor suppressor pathway. These findings thus delineate an enzymatic pathway that induces colon cancer suppression, a pathway that is activated by TGF-beta and mediated by 15-PGDH.

C8orf4 is a transforming growth factor B induced transcript downregulated in metastatic colon cancer.

Transforming growth factor (TGF) beta mediates a tumor suppressor pathway in human colon epithelial cells. We were interested in identifying and characterizing novel genes regulated by the TGF beta pathway in the colon. We employed expression microarrays to identify transcripts induced by TGF beta in Vaco 330, a colon adenoma cell line. We then used expression microarrays to determine which of these TGF beta induced transcripts are down-regulated in metastatic colon cancer. Northern analysis and real-time reverse transcription PCR confirmed and quantified our findings from the microarrays. These analyses highlighted C8orf4 as induced by TGF beta in colon cells. Moreover, C8orf4 is expressed in most normal colon mucosa samples, and is not expressed in most colon cancer metastases or colon cancer cell lines. Colon cancer primary tumors showed reduced expression of C8orf4 relative to normal mucosa, possibly reflecting contributions of C8orf4 expression in stromal cells. C8orf4 is a gene regulated by TGF beta signaling and loss in advanced colon cancer suggests C8orf4 may play a role in colon cell differentiation or growth regulation.

A subset of familial colorectal neoplasia kindreds linked to chromosome 9q22.2-31.2.

Colorectal cancer is the second most leading cause of cancer death among adult Americans. Two autosomal dominant hereditary forms of the disease, familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer, together account for perhaps 5% of all cases. However, in approximately 20% of additional colon cancer cases, the affected individuals report a family history of colon cancer in a first-degree relative. Similar familial clusters of colon cancer and early-onset colon adenomas have also been reported. To determine whether such familial aggregations arise by chance or reflect a hereditary colon cancer susceptibility, we conducted a whole genome scan to test for genetic linkage in 53 kindreds in which two or more siblings were affected by age 65 or younger with colon cancer or with advanced colon adenomas that were >1 cm in size or that showed high-grade dysplasia. In this cohort we found genetic linkage of disease (P = 0.00045) to chromosomal region 9q22.2-31.2 in a pattern consistent with autosomal dominant disease alleles. These data suggest that a single locus can contribute to disease susceptibility in a subset of patients with nonsyndromic forms of familial colorectal neoplasia.

PMEPA1, a transforming growth factor-beta-induced marker of terminal colonocyte differentiation whose expression is maintained in primary and metastatic colon cancer.

To identify potential effectors of transforming growth factor (TGF)-beta-mediated suppression of colon cancer, we used GeneChip expression microarrays to identify TGF-beta-induced genes in VACO 330, a nontransformed TGF-beta-sensitive cell line derived from a human adenomatous colon polyp. PMEPA1 was identified as a gene highly up-regulated by TGF-beta treatment of VACO 330. Northern blot analysis confirmed TGF-beta induction of PMEPA1 in VACO 330, as well as a panel of three other TGF-beta-sensitive colon cell lines. PMEPA1 induction could be detected as early as 2 h after TGF-beta treatment and was not inhibited by pretreatment of cells with cycloheximide, suggesting that PMEPA1 is a direct target of TGF-beta signaling. Wild-type PMEPA1 and an alternative splice variant lacking the putative transmembrane domain were encoded by the PMEPA1 locus and were shown by epitope tagging to encode proteins with differing subcellular localization. Both variants were found to be expressed in normal colonic epithelium, and both were shown to be induced by TGF-beta. Consistent with TGF-beta playing a role in terminal differentiation of colonocytes, in situ hybridization of normal colonic epithelium localized PMEPA1 expression to nonproliferating, terminally differentiated epithelium located at the top of colonic crypts. Intriguingly, in situ hybridization and Northern blot analysis showed that the expression of PMEPA1 was well maintained both in colon cancer primary tumors and in colon cancer liver metastases. PMEPA1 is thus a novel TGF-beta-induced marker of a differentiated crypt cell population. Moreover, as PMEPA1 expression is maintained, presumptively in a TGF-beta-independent manner after malignant transformation and metastasis, it demonstrates that even late colon cancers retain a strong capacity to execute many steps of the normal colonic differentiation program.

Silence of chromosomal amplifications in colon cancer.

Oncogene activation by gene amplification is a major pathogenetic mechanism in human cancer. Using comparative genomic hybridization, we determined that metastatic human colon cancers commonly acquire numerous extra copies of chromosome arms 7p, 8q, 13q, and 20q. We then examined the consequence of these amplifications on gene expression using DNA microarrays. Of 55,000 transcripts profiled, 2,146 were determined to map to one of the four common colon cancer amplicons and to also be expressed in normal or malignant colon tissues. Of these, only 81 transcripts (3.8%) demonstrated a 2-fold increase over normal expression among cancers bearing the corresponding chromosomal amplification. Chromosomal amplifications are common in colon cancer metastasis, but increased expression of genes within these amplicons is rare.