Human Sporadic Colorectal Adenomas Research Articles

PWE-167 Identification of Altered Keratin Level in Cancerized Colonic Fields using Isobaric Tags for Relative and Absolute Quantification (ITRAQ) for Protein Profiling

IntroductionA cancerized field is an area of abnormal tissue in the vicinity of a cancer that macroscopically appears normal, and which may be responsible for neoplastic recurrence. Evidence from keratin-8-deficient...

RUNX3 Attenuates β-Catenin/T Cell Factors in Intestinal Tumorigenesis

In intestinal epithelial cells, inactivation of APC, a key regulator of the Wnt pathway, activates beta-catenin to initiate tumorigenesis. However, other alterations may be involved in intestinal tumorigenesis. Here we found that RUNX3, a gastric tumor suppressor, forms a ternary complex with beta-catenin/TCF4 and attenuates Wnt signaling activity. A significant fraction of human sporadic colorectal adenomas and Runx3(+/-) mouse intestinal adenomas showed inactivation of RUNX3 without apparent beta-catenin accumulation, indicating that RUNX3 inactivation independently induces intestinal adenomas. In human colon cancers, RUNX3 is frequently inactivated with concomitant beta-catenin accumulation, suggesting that adenomas induced by inactivation of RUNX3 may progress to malignancy. Taken together, these data demonstrate that RUNX3 functions as a tumor suppressor by attenuating Wnt signaling.

Chromosomal Instability, Aneuploidy, and Gene Mutations in Human Sporadic Colorectal Adenomas

Chromosomal Instability, Aneuploidy, and Gene Mutations in Human Sporadic Colorectal Adenomas

Response to the Letter of B. Carvalho “Chromosomal Instability, Aneuploidy, and Gene Mutations in Human Sporadic Colorectal Adenomas” published in Cellular Oncology Vol. 27(4), 2005, p. 267

Response to the Letter of B. Carvalho “Chromosomal Instability, Aneuploidy, and Gene Mutations in Human Sporadic Colorectal Adenomas” published in Cellular Oncology Vol. 27(4), 2005, p. 267

Chromosomal instability and APC gene mutations in human sporadic colorectal adenomas.

The mechanisms by which adenomatous polyposis coli (APC) gene mutations contribute to colorectal tumourigenesis and progression are still not fully understood. Using in vitro mouse embryonic stem cells, APC mutations have been proposed to dysregulate the interactions between kinetochores and microtubules during mitosis, leading to chromosomal instability (CIN) and aneuploidy. A link between APC mutations and aneuploidy in vivo among human sporadic colorectal adenomas has not been reported previously and was therefore investigated in the present series of 61 adenomas. Multi-parameter flow cytometry, based on scattering and fluorescence from the DNA-specific 4,6-diamidino-2-phenylindole-2-hydrochloride (DAPI) dye, which separates epithelial from stromal lymphocyte nuclei, was used to evaluate the DNA index (DI) and to sort epithelial nuclei. Additionally, DNA extracted from these sorted nuclei was used to analyse APC mutations by DNA sequencing. Aneuploidy was present in 20 of 61 adenomas (33%), with 15 of these 20 cases (75%) having a near-diploid DI (DI different from 1 and less than 1.3). APC mutations were detected in 19 adenomas (31%): 12 were within or downstream of the mutation cluster region (MCR), roughly defined by codons 1200-1500, and seven were upstream of the MCR. Overall, the prevalence of aneuploidy in APC wild-type and mutated adenomas was 26% and 47%, respectively, and no statistically significant association was found between APC status and DI (p = 0.142). However, when APC mutations were subdivided into two groups, ie occurring within/downstream of the MCR and upstream of the MCR, the association of APC mutations within and downstream of the MCR with aneuploidy was statistically significant (p = 0.017). In conclusion, the present data suggest that the type of APC mutation may play a role in the origin of CIN in vivo in human sporadic colorectal adenomas and that APC mutations within and downstream of the MCR, and large-scale chromosomal alterations, may co-operate in the progression of a subgroup of adenomas.

Chromosomal Instability, Aneuploidy, and Gene Mutations in Human Sporadic Colorectal Adenomas

Whether in vivo specific gene mutations lead to chromosomal instability (CIN) and aneuploidy or viceversa is so far not proven. We hypothesized that aneuploidy among human sporadic colorectal adenomas and KRAS2 and APC mutations were not independent. Additionally, we investigated if 1p34–36 deletions by dual target FISH were associated with aneuploidy. Among 116 adenomas, 29 were DNA aneuploid by flow cytometry (25%) and 29 were KRAS2 mutated (25%). KRAS2 mutations were associated with aneuploidy (P=0.02). However, while G–C and G–T transversions were strongly associated with DNA aneuploidy (P=0.007), G–A transitions were not. Within a second series of 61 adenomas, we found, instead, that APC mutational status and aneuploidy by flow cytometry were not associated. However, a statistically significant association was found with specific APC mutations, i.e., occurring in the mutation cluster region (MCR, codons 1200–1500) or downstream (P=0.016). Finally, the correlation of 1p34–36 deletions with flow cytometric and FISH detected aneuploidy was also significant (P=0.01). Specific KRAS2 and APC mutations and loss of genes in the 1p34–36 region appear associated with aneuploidy suggesting that these events are not independent and may cooperate in inducing human sporadic colorectal adenomas. A cause effect relationship between gene mutations and aneuploidy remains, however, to be demonstrated.

Interstitial cell cyclooxygenase-2 expression is associated with increased angiogenesis in human sporadic colorectal adenomas.

Cyclooxygenase (COX)-2 plays an important role in intestinal tumorigenesis and angiogenesis in animal models. In superficial areas of human sporadic colorectal adenomas, COX-2 is expressed predominantly by interstitial macrophages, in close proximity to microvessels. The aim of this study was to investigate the association between microvessel density (MVD) and COX-2 expression in human sporadic colorectal adenomas. Immunohistochemistry and immunofluorescence for CD31 and COX-2 were performed on a well-characterized series of human sporadic colorectal adenomas (n = 37). The mean MVD and COX-2 expression level (scored 0-3) in superficial and deep interstitial cells of adenomas were assessed by two independent observers. Superficial MVD was increased in COX-2-positive adenomas, compared with COX-2-negative adenomas (p = 0.037). There was a significant correlation between superficial MVD and increasing superficial interstitial cell COX-2 expression score (p = 0.048). COX-2-expressing interstitial cells aggregated in areas of high MVD. No relationship was evident between MVD and COX-2 expression in either deep interstitial cells or epithelial cells. Multivariate analysis demonstrated that only adenoma size (p = 0.005) was a significant independent predictor of MVD. COX-2 protein expression by superficial interstitial cells in human sporadic colorectal adenomas is associated with increased angiogenesis. Promotion of angiogenesis may play a role in the pro-tumourigenic activity of COX-2 during growth of human colorectal adenomas.

Analysis of cyclooxygenase expression in human colorectal adenomas.

Evidence from rodent intestinal tumorigenesis models suggests that both cyclooxygenase-1 and cyclooxygenase-2 may play important roles in the development and progression of human sporadic colorectal adenomas. However, previous studies of cyclooxygenase isoform expression in human colorectal adenomas have produced conflicting data. Cyclooxygenase-1 expression has been poorly studied, and cyclooxygenase-2 positivity of adenomas has been variable depending on the detection technique used. It also remains unclear whether villous adenomas express cyclooxygenase-2. Cyclooxygenase isoform expression in human sporadic colorectal adenomas was analyzed by reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Variable cyclooxygenase-1 expression was detected in all adenomas (n = 9) by both reverse transcription-polymerase chain reaction and Western blot analysis. Cyclooxygenase-2 expression was detected in eight (89 percent) of nine adenomas by reverse transcription-polymerase chain reaction and immunohistochemistry. Cyclooxygenase-2 protein was not detected by Western blot analysis in any adenoma. Cyclooxygenase-2 was expressed by all histopathologic types of adenoma and localized predominantly to superficial interstitial cells, in which it was associated with increased adenoma size. Cyclooxygenase-1 is expressed at variable levels by all adenomas. Cyclooxygenase-2 is expressed by the majority of adenomas, including those of the villous type, at levels below the sensitivity of Western blot analysis.

Expression of cyclooxygenase-2 ( COX-2) mRNA in human colorectal adenomas

Cyclooxygenase-2 (COX-2) is an important target for the suppression of colorectal tumorigenesis by non-steroidal anti-inflammatory drugs (NSAIDs). To evaluate the role of COX-2 in human sporadic colorectal adenomas, COX-2 mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR) in 63 adenomas. COX-2 mRNA was detected in all the adenomas at higher levels than in normal colorectal mucosa (P<0.001). Levels of expression in the adenomas were correlated with their size ( P=0.019), but no relationships were demonstrable between COX-2 expression and adenoma location, macroscopically observed configuration or microscopic degree of dysplasia. These findings suggest that COX-2 plays an important role in the growth of sporadic colorectal adenomas.

Localization of Cyclooxygenase-2 in Human Sporadic Colorectal Adenomas

A putative target for the anti-colorectal cancer action of nonsteroidal anti-inflammatory drugs is the inducible isoform of cyclooxygenase (COX), COX-2. COX-2 is expressed within intestinal adenomas in murine polyposis models, but expression has been poorly characterized in human colorectal neoplasms. Therefore, we investigated the localization of the COX-2 protein in human sporadic colorectal adenomas. Immunohistochemistry for COX-2 and CD68 (a tissue macrophage marker) was performed on formalin-fixed, paraffin-embedded (n = 52) and frozen, acetone-fixed (n = 6) sections of human sporadic colorectal adenomas. Forty of 52 (77%) formalin-fixed adenomas expressed immunoreactive COX-2. COX-2 was localized to superficial interstitial macrophages in 39 cases (75%) and to deep interstitial macrophages in 9 cases (17%). COX-2 staining of dysplastic epithelial cells was observed in 15 cases (29%). A logistic regression analysis identified the adenoma site (P = 0.012) and histological type (P = 0.001) as independent predictors of superficial macrophage COX-2 expression. There was no relationship between the number of macrophages within an adenoma and macrophage COX-2 expression. These results indicate that COX-2 is expressed predominantly by interstitial macrophages within human sporadic colorectal adenomas. If COX-2 does indeed play a role in the early stages of colorectal carcinogenesis in man, these data suggest COX-2-mediated paracrine signaling between the macrophages and epithelial cells within adenomas.

Specific K-ras2 Mutations in Human Sporadic Colorectal Adenomas Are Associated with DNA Near-Diploid Aneuploidy and Inhibition of Proliferation

Recent studies indicate that p21ras proteins mediate their multiple cell functions through interactions with multiple effectors and that the number of new effectors is growing. We recently reported that K-ras2 mutations in human colorectal adenomas were associated with chromosome instability and proliferation changes. In the present study, we extend these previous observations. Hereditary and multiple (n > or = 5) adenomas and adenomas with early cancer were excluded. Dysplasia was moderate in 91 cases and high in 25, and the median adenoma size was 1.5 cm. K-ras2 spectrum analysis was done by sequence-specific oligonucleotide hybridization using nuclear suspensions provided by analysis and sorting of multiparameter flow cytometry. In particular, tissue inflammatory cells were separated for DNA diploid tumors, whereas DNA aneuploid epithelial subclones were analyzed separately. K-ras2 mutations and DNA aneuploidy were both detected in 29 of 116 (25%) cases. DNA aneuploid index was in the near-diploid region in the majority of cases. DNA aneuploidy was strongly associated with G-->C/T transversions. An association was also found between low S-phase values and G-->A transitions. These findings were confirmed using multivariate logistic regression analysis to account for the effects of size, dysplasia, site, type, age, and sex. These data suggest that specific K-ras2 mutations in a subgroup of human sporadic colorectal adenomas play a role in chromosome instability and, contrary to expectations, are associated with inhibition of proliferation.

APC mutations in human colorectal adenomas: analysis of the mutation cluster region with temperature gradient gel electrophoresis and clinicopathological features.

To date, the earliest mutations in colorectal adenomas have been found in the adenomatous polyposis coli (APC) gene, considered the 'gatekeeper' in tumourigenesis. To study the types of APC gene mutations and their relation to clinicopathological features which are associated with malignant potential, the mutation cluster region of the APC gene was analysed in a series of 32 human sporadic colorectal adenomas from 22 patients. DNA was extracted from frozen sections and two overlapping fragments which cover the mutation cluster region were amplified using the polymerase chain reaction (PCR). Mutations were screened with temperature gradient gel electrophoresis and identified by DNA sequencing. Mutations were found in 14 samples (44 per cent) from 11 patients. The changes could be characterized as point mutations (n = 7), deletions of one or more nucleotides (n = 6), and insertions (n = 1). From five patients, multiple adenoma samples were obtained and the adenomas from two of these patients showed a heterogeneous mutation pattern in the APC gene. All detected mutations are predicted to result in a truncated APC protein. Genetic alterations in the mutation cluster region of the APC gene occurred significantly more frequently in large adenomas and showed a trend towards an increase in villous adenomas and adenomas with moderate and severe dysplasia. It is concluded that an increased proportion of APC gene mutations were found in the adenomas showing clinicopathological features associated with increased proliferation but not with characteristics of malignant potential. The results suggest that in a significant proportion of adenomas, other (genetic) alterations are involved in early tumourigenesis.

Correlation between 1p deletions and aneusomy in human colorectal adenomas.

Human sporadic colorectal adenomas are characterized by a relatively high occurrence of aneuploidy. Similarly, 1p deletions have been reported to be an early event in colorectal tumorigenesis, while chromosome 7, 17 and 18 gain/losses were also found. The present study investigated 1p deletions, the numerical aberrations of chromosomes 1, 7, 17 and 18, and the nuclear DNA content as obtained by flow cytometry in a series of 34 human sporadic colorectal adenomas. From these adenomas, 51 intra-adenoma regions were microdissected according to 2 degrees of dysplasia and presence of foci of early cancer. Isolated epithelial nuclei were analyzed by fluorescence in situ hybridization interphase cytogenetics using centromeric probes for chromosomes 7, 17 and 18 and, in a double-target analysis, a centromeric probe for chromosome 1 simultaneously with a telomeric probe mapping to the 1p36 band. Aneuploidy incidence due to presence of numerical aberrations for at least one among the investigated chromosomes and/or abnormal flow-cytometric DNA content was 35%, while 1p deletion incidence was 38%. The correlation of 1p deletions with aneuploidy was statistically highly significant (p = 0.003), suggesting that loss of genes in this region may be implicated in chromosome instability.