Genetic changes in the transforming growth factor beta (TGF-beta) type II receptor gene in human gastric cancer cells: correlation with sensitivity to growth inhibition by TGF-beta.

We have found several genetic changes in the TGF-beta-type II receptor gene in human gastric cancer cell lines resistant to the growth inhibitory effect of TGF-beta. Southern blot analysis showed deletion of the type II receptor gene in two of eight cell lines and amplification in another two lines. The single cell line we studied that is sensitive to growth inhibition by TGF-beta showed no structural abnormalities of the type II receptor gene. Some of the gastric cancer cells resistant to the growth inhibitory effect of TGF-beta express either truncated or no detectable TGF-beta type II receptor mRNAs, whereas the one that retains responsiveness to the growth inhibitory effect of TGF-beta expresses a full-size type II receptor mRNA. Immunoprecipitation followed by Western blot analysis showed parallel changes in TGF-beta type II receptor expression. Our results suggest that one of the possible mechanisms of escape from autocrine or paracrine growth control by TGF-beta during carcinogenesis could involve genetic changes in the TGF-beta type II receptor gene itself or altered expression of its mRNA.     

Loss of activin receptor type 2 protein expression in microsatellite unstable colon cancers

BACKGROUND & AIMS: Colorectal tumors manifesting high-frequency microsatellite instability (MSI-H) develop genetically as a consequence of mutations in genes harboring repetitive DNA sequences. The activin type 2 receptor (ACVR2), possessing 2 polyadenine coding sequences, was identified as a mutational target, but it is not clear if expression is abrogated. Here, we analyzed MSI-H colorectal cancers for ACVR2 mutation and expression to assess if biallelic inactivation occurs. METHODS: All 54 MSI-H colon cancers and 20 random microsatellite stable (MSS) tumors from a population-based cohort of 503 patients were analyzed for mutations in 2 A(8) tracts (exon 3 and 10) of ACVR2 and the A(10) tract of transforming growth factor beta receptor 2 (TGFBR2). Additionally, we sequenced exon 10 of ACVR2 in select cancers. ACVR2 expression was determined by immunohistochemistry using an antibody targeting an epitope beyond the predicted truncated protein. RESULTS: Forty-five of 54 MSI-H cancers (83%) showed mutation (A(8) to A(7)) in the polyadenine tract of exon 10 compared with no MSS tumors. Of tumors with mutant ACVR2, 62% lacked protein expression but all MSS and MSI-H tumors with wild-type ACVR2 expressed protein. We found no evidence of loss of heterozygosity at the ACVR2 locus in MSS tumors. Comparatively, 69% of MSI-H cancers had frameshift mutation in TGFBR2. CONCLUSIONS: ACVR2 mutations are highly frequent in MSI-H colon cancers and in most cases cause loss of ACVR2 expression, indicating biallelic inactivation of the gene. Loss of activin signaling through mutation of ACVR2, similar to observations with TGFBR2, may be important in the genesis of MSI-H colorectal cancer.     

Replication error deficient and proficient colorectal cancer gene expression differences caused by 3'UTR polyT sequence deletions

Replication error deficient (RER+) colorectal cancers are a distinct subset of colorectal cancers, characterized by inactivation of the DNA mismatch repair system. These cancers are typically pseudodiploid, accumulate mutations in repetitive sequences as a result of their mismatch repair deficiency, and have distinct pathologies. Regulatory sequences controlling all aspects of mRNA processing, especially including message stability, are found in the 3'UTR sequence of most genes. The relevant sequences are typically A/U-rich elements or U repeats. Microarray analysis of 14 RER+ (deficient) and 16 RER- (proficient) colorectal cancer cell lines confirms a striking difference in expression profiles. Analysis of the incidence of mononucleotide repeat sequences in the 3'UTRs, 5'UTRs, and coding sequences of those genes most differentially expressed in RER+ versus RER- cell lines has shown that much of this differential expression can be explained by the occurrence of a massive enrichment of genes with 3'UTR T repeats longer than 11 base pairs in the most differentially expressed genes. This enrichment was confirmed by analysis of two published consensus sets of RER differentially expressed probesets for a large number of primary colorectal cancers. Sequence analysis of the 3'UTRs of a selection of the most differentially expressed genes shows that they all contain deletions in these repeats in all RER+ cell lines studied. These data strongly imply that deregulation of mRNA stability through accumulation of mutations in repetitive regulatory 3'UTR sequences underlies the striking difference in expression profiles between RER+ and RER- colorectal cancers.     

Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells

Epithelial (E)-cadherin and its associated cytoplasmic proteins (alpha-, beta-, and gamma-catenins) are important mediators of epithelial cell-cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin-catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.     

Loss of receptors for transforming growth factor beta in human T-cell malignancies.

Ki-1 (CD30)+ cutaneous T-cell lymphomas CTCLs) are slowly progressive lymphomas in which initial spontaneous regression is often observed. To better understand the mechanisms of spontaneous regression and eventual tumor progression in Ki-1+ CTCLs, type beta transforming growth factor (TGF-beta)-mediated growth inhibition of clonally related cell lines derived from two time points, before and after tumor progression, was studied. TGF-beta 1 inhibited colony-forming efficiency (CFE) of a cell line (Mac-1) derived from clinically indolent Ki-1+ CTCLs but failed to inhibit CFE of Mac-2A and -2B cell lines from advanced CTCLs. To determine the basis for TGF-beta 1 resistance in advanced CTCL cells, we looked for possible defects in the expression of cell surface TGF-beta receptors. Mac-1 cells were found to express TGF-beta receptors I and II, which mediate growth inhibition, and the TGF-beta-binding proteoglycan betaglycan. In contrast, receptors I and II were not detected in CTCL lines Mac-2A and -2B even though these cell lines did express betaglycan. Various treatments that unmask or induce TGF-beta receptors in other cells failed to show evidence for these receptors in advanced CTCL cells. Loss of TGF-beta receptor expression in these cells correlated with a marked decrease in TGF-beta receptor II mRNA levels. Loss of cell surface TGF-beta receptors was also found in two of five other patients with T-cell lymphomas including the Sezary syndrome and a noncutaneous T-cell lymphoma, suggesting that loss of TGF-beta receptor expression may be a recurrent feature of human T-cell malignancies.     

APC mutations in sporadic colorectal tumors: A mutational "hotspot" and interdependence of the "two hits"

Although APC mutations occur at a high frequency in colorectal cancers, few studies have performed a comprehensive analysis by screening the whole gene for mutations and assessing allelic loss. APC seems to act as a tumor-suppressor gene in a "nonclassical" fashion: data from familial adenomatous polyposis (FAP) show that the site of the germ-line mutation determines the type of "second hit" in FAP tumors, and simple protein inactivation is selected weakly, if at all. In this study, we screened the entire coding region of APC for mutations and assessed allelic loss in a set of 41 colorectal cancer cell lines. Of 41 cancers, 32 (83%) showed evidence of APC mutation and/or allelic loss. We identified several APC mutations and found a "hotspot" for somatic mutation in sporadic colorectal tumors at codon 1,554. Our results suggest that APC mutations occur in the great majority of colorectal cancers, the exceptions almost all being RER+ tumors, which may substitute for altered APC function by mutations in beta-catenin and/or at other loci. When combined with previously published data, our results show that there is interdependence of the "two hits" at APC in sporadic colorectal tumors as well as in FAP. APC mutations in the "mutation cluster region," especially those close to codon 1,300, are associated with allelic loss, whereas tumors with mutations outside this region tend to harbor truncating mutations. The causes of this phenomenon are probably selection for retained N-terminal and lost C-terminal APC functions, effects on beta-catenin levels, and APC protein stability.     

Genetic epidemiology of mutated K-ras proto-oncogene, altered suppressor genes, and microsatellite instability in colorectal adenomas

BACKGROUND: The genetic epidemiology of colorectal adenomas has not been studied prospectively in colonoscopy patients without cancer. AIMS: To study genetic alterations in colorectal adenomas and correlate these with patient demographics and adenoma characteristics. METHODS: Mutations and allelic deletions in 201 adenomas from 60 patients were compared with demographic features, adenoma characteristics, and family history. RESULTS: The most common alteration was K-ras proto-oncogene mutation, present in 35% of adenomas and 65% of patients. Patients 65 years of age and older had a decreased probability of K-ras mutations (26% versus 45%). Overexpression of p53 gene product was present in only 6% of adenomas but was more frequent in villous or tubulovillous adenomas (19% versus 3%). Allelic loss of chromosome 18q was present in only 2% of adenomas and was significantly less frequent than p53 overexpression. DNA replication errors (RER) were present in 7% of adenomas and 15% of patients, including multiple adenomas in four patients (two with hereditary non-polyposis colorectal cancer syndrome). Only 36% of RER positive adenomas had alteration of BAT-26 alleles, none had alteration of BAT-25, and only one (8%) had mutation in the transforming growth factor beta type II receptor gene. RER positive adenomas were more likely to have a K-ras mutation. In patients with multiple adenomas, there was concordance of p53 overexpression and RER but not of K-ras mutations. CONCLUSIONS: Genetic progression in colorectal adenomas is heterogeneous, involving factors related to patient age and the presence of RER for the occurrence of ras mutations, but different intraindividual characteristics for the occurrence of p53 alterations and RER.     

Molecular genetics of Marfan syndrome

PURPOSE OF REVIEW: Marfan syndrome, the founding member of connective tissue disorders, is characterized by involvement of three major systems (skeletal, ocular, and cardiovascular) due to alteration in microfibrils. FBN1 at 15q21.1 was found to cause Marfan syndrome in 1991, and in 2004 TGFBR2 at 3p24.1 was newly identified as the Marfan syndrome type II gene. Several studies implied that fibrillin-1 and transforming growth factor-beta (TGF-beta) signaling are functionally related in extracellular matrix. Identification of TGFBR2 mutations in Marfan syndrome type II provided the direct evidence of the relation in humans. RECENT FINDINGS: More than 500 FBN1 mutations have been found in Marfan syndrome, tentative genotype - phenotype correlations have emerged, and mouse models are providing insight into pathogenic mechanisms. TGFBR2 mutations are still limited, however, in 2005 were also reported to cause a new aneurysm syndrome. Functional association between fibrillin-1 and TGF-beta signaling in extracellular matrix has been presented. SUMMARY: This review focuses on recent molecular genetics advances in Marfan syndrome and overlapping connective tissue disorders. Mutation spectrum of FBN1 and TGFBR2 in relation to phenotype is presented. Functional relation between fibrillin-1 and TGF-beta signaling is discussed. Future prospects in the study of Marfan syndrome are presented.     

Replication error phenotype, clinicopathological variables, and patient outcome in Dukes' B stage II (T3,N0,M0) colorectal cancer

AIMS: To examine the relation between the replication error (RER) phenotype and other genetic events, clinical features, and long term survival in patients with Dukes' B stage II (T3,N0,M0) colorectal cancer. METHODS: RER phenotype was investigated in 159 patients by PCR amplification of microsatellite marker loci on chromosomes 5q, 17p, 17q, and 18q from tumour DNA extracted from archival tissue. Data on activating c-Ki-ras mutations were available from a previous study. Immunohistochemical detection of p53 and c-erbB-2 expression was performed on paraffin wax embedded tissue. RESULTS: Of 159 colorectal cancers studied, 22 (14%) were RER+ while 137 (86%) were RER- for two or more loci. RER+ tumours were more commonly located in the right colon, tended to be larger than RER- tumours, and were more often poorly differentiated than RER- cancers. No significant associations were seen between RER status and the presence of a mutant c-Ki-ras gene, or between RER status and p53, c-erbB-2, or c-myc gene expression. Univariate survival analysis showed that outcome was similar in RER+ and RER- cases. Multivariate survival analysis showed that the relative risk of death for patients with RER+ cancers was 0.95 that of patients with RER- cancers. CONCLUSIONS: The results suggest that, while the RER phenotype may be associated with some differences in tumour pathology (site, size, differentiation), it is not associated with the genetic alterations studied or with significant differences in long term survival.     

Allele loss, replication errors and loss of expression of E-cadherin in colorectal cancers.

BACKGROUND: Loss of E-cadherin expression has been implicated in the development of invasive characteristics in colorectal carcinomas. Failure to express E-cadherin may result from mutations of the E-cadherin gene (HSECAD). AIMS: To examine the correlation between E-cadherin expression and genetic changes at HSECAD; and to examine differences in E-cadherin expression and genetic changes at HSECAD between three different groups of colorectal cancer--replication error positive (RER+) sporadic cancers, RER--sporadic cancers and ulcerative colitis associated cancers. SUBJECTS AND METHODS: Sixty eight colorectal cancers (22 RER+ sporadic cancers, 32 RER- sporadic cancers and 14 ulcerative colitis associated cancers) were studied using immunohistochemistry and for allele loss at the HSECAD locus. Exon 16 of HSECAD contains several mononucleotide repeat tracts which are very similar to microsatellite repeats and which may be susceptible to replication errors (manifest as new alleles). All cases were also examined for new alleles in exon 16. RESULTS: Absent or decreased E-cadherin protein expression was found in 27 (38%) of 68 colorectal cancers and the pattern of expression did not differ significantly among the three tumour groups. Allele loss occurred at HSECAD in four (10%) of 40 informative cancers and there were no differences between the three subgroups. New alleles at exon 16 were detected in three (14%) of 22 RER+ tumours; no new alleles were detected in RER- or ulcerative colitis associated cancers. Overall, there was no correlation between allelic loss or exon 16 replication errors and immunohistochemical E-cadherin expression. CONCLUSIONS: (1) Loss of E-cadherin expression probably does not occur as a result of mutation at the HSECAD locus in colorectal cancers. (2) There is no difference in the frequency of loss of heterozygosity at HSECAD among RER+, RER- and ulcerative colitis associated colorectal cancers.     

Expression of transforming growth factor β receptors in normal human colon and sporadic adenocarcinomas

Background & Aims: An absence or a presence of mutated transforming growth factor (TGF)-β receptors is a possible hypothesis explaining the resistance of cancer cells to the growth-inhibitory effect of TGF-β. Mutations involving microsatellite-like regions of the type II TGF-β receptor have been described in subgroups of colorectal cancers. The aim of this study was to investigate the expression and distribution of TGF-β receptors in sporadic colorectal cancers and normal tissues. Methods: Thirty-three sporadic colorectal cancers and 20 normal colonic tissues were explored by immunohistochemistry for the expression of type I and type II TGF-β receptors. Eighteen tumor and 20 normal samples were used for radioactive thermocycling and sequencing of the two microsatellite-like regions of the type II receptor. Results: Both receptors were overexpressed in tumors compared with normal samples. There was a relationship between the abundance of type II receptor expression and the degree of differentiation of the tumors but not the Dukes' staging or the localization of the neoplasias. No mutation was observed in the microsatellite-like regions of receptor II in any of the samples. Conclusions: Sporadic colorectal cancers do not show an absence or a presence of mutated TGF-β receptors that could explain a resistance to TGF-β–mediated growth inhibition. The pathways to tumorigenesis of sporadic colorectal cancers may be different from those of some hereditary ones.GASTROENTEROLOGY 1998;114:1211-1220     

Mechanisms of inactivation of mismatch repair genes in human colorectal cancer cell lines: the predominant role of hMLH1.

Fifteen to twenty-five percent of sporadic colorectal carcinomas are replication error (RER) positive. Because the frequency of mutations in the mismatch repair genes (hMLH1 and hMSH2) is low in these tumors, we have investigated the role of mutational inactivation, methylation of the promoter region, and loss of heterozygosity (LOH) as a possible explanation for the mutator phenotype of RER+ colorectal cancer cell lines. Genomic DNA was extracted from a panel of 49 human colorectal cancer cell lines. The RER status was determined by amplification of BAT-26. All exons of hMLH1 and hMSH2 were amplified with the PCR and screened by using single-strand conformational polymorphism and direct sequencing. The methylation status was ascertained by methylation-specific PCR after bisulfite modification of DNA. Western blotting for hMLH1 was performed on methylated cell lines before and after the addition of the demethylating agent 5-azacytidine. LOH was sought by GENESCAN analysis of amplified CA repeat markers and indirectly by determining the number of homozygotes in the cell lines and human random controls. Twelve cell lines from ten tumors (24%) were RER+. Hypermethylation of the hMLH1 promoter occurred in five of ten (50%) RER+ tumors, whereas three of thirty-two (6%) RER tumors showed partial methylation. None of the fully methylated cell lines expressed hMLH1, although all reexpressed hMLH1 after treatment with 5-azacytidine. There was no LOH in the RER+ tumors in either hMLH1 or hMSH2. Our results suggest that mutations of hMLH1 together with hypermethylation of the promoter region, but not LOH, are the cause of the mutator phenotype in the majority (70%) of RER+ tumors.     

Mutational analysis of activin/transforming growth factor-beta type I and type II receptor kinases in human pituitary tumors

Genetic mutation or loss of activin/transforming growth factor-beta (TGFbeta) receptor function has been shown in human lymphoid, breast, and colorectal tumors as well as Hep2B and Mv1Lu cell lines. Although activin stimulates FSHbeta biosynthesis and secretion, a large percentage of human gonadotroph tumors have previously been demonstrated to be nonresponsive to characterized activin effects. This phenotype may be indicative of loss of functional cell surface receptors and/or intracellular signaling mediators of activin responses. Several studies examining the structure/function of type I and II receptors specific for ligands in the TGFbeta superfamily have delineated the critical regions for receptor intracellular kinase function. In the case of TGFbeta, inactivating mutations in these regions have been shown to render these receptors kinase deficient by a dominant negative phenotype and result in resistance to growth arrest. We therefore hypothesized that activin/TGFbeta cell surface receptors may act as tumor suppressors in human pituitary tumors, and that inactivating genetic mutations in the intracellular kinase region of this gene family may release pituicytes from normal growth suppression by activin through a similar mechanism. We used single stranded conformational polymorphism analysis to examine 2 intracellular regions required for type I receptor signaling by human Alk1-5 type I receptors as well as the entire coding region of 2 activin type II receptors and the TGFbeta type II receptor in 64 human pituitary tumors. A novel polymorphism was found in 45% of tumors at codon P117 of the ActRIIA gene and was used as a positive control for single stranded conformational polymorphism. One patient with a gonadotroph tumor had a confirmed A482V germline mutation in the Alk1 gene within kinase subdomains X-XI. No other mutations were detected in any tumor studied. These data suggest that somatic mutations within these intracellular kinase regions of type I/type II receptors are rare in human pituitary tumors.     

Lack of responsiveness to TGF-beta1 in a thyroid carcinoma cell line with functional type I and type II TGF-beta receptors and Smad proteins, suggests a novel mechanism for TGF-beta insensitivity in carcinoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine. In the present study we have investigated the expression of TGF-beta receptors (TbetaR's) and SMAD proteins in non-neoplastic and neoplastic thyroid follicle cells. We found expression of all TbetaR's (type I, II and III) and SMAD proteins analysed (Smad2, Smad3, Smad4, Smad6 and Smad7). Five out of six human anaplastic thyroid carcinoma cell lines were growth inhibited by addition of TGF-beta1, and therefore considered to be TGF-responsive. One cell line however, HTh 7, did not respond to TGF-beta1 with growth inhibition, induction of the extracellular matrix protein fibronectin or immediate early genes junB, Smad6 and Smad7 mRNA. Analysis of the TGF-beta intracellular signalling pathway in HTh 7 cells showed that receptors were capable of signalling, e.g. Smad2 phosphorylation and SMAD nuclear translocation. In summary, our data shows abundant expression of TGF-beta signalling components in thyroid follicle cells, and the escape from TGF-beta sensitivity in one anaplastic thyroid carcinoma despite an apparently functional TGF-beta/SMAD-signalling pathway, indicating a novel mechanism for TGF-beta insensitivity.     

Regulatory mechanisms for transforming growth factor beta as an autocrine inhibitor in human hepatocellular carcinoma: implications for roles of smads in its growth

Transforming growth factor beta (TGF-beta) initiates signaling through heteromeric complexes of transmembrane type I and type II serine/threonine kinase receptors. Activated TGF-beta type I receptor phosphorylates receptor-regulated Smads (2 and 3). Antagonistic Smad 7 forms stable association with the activated TGF-beta type I receptor, blocking phosphorylation of receptor-regulated Smads. On the other hand, elevated serum concentration of TGF-beta along with resistance to its growth-inhibitory effect is commonly observed in human hepatocellular carcinoma (HCC) patients. In this study, we investigated the mechanisms of resistance to tumor-derived TGF-beta in human HCC and hepatoblastoma-derived cell lines, focusing on the roles of receptor-regulated Smads and antagonistic Smad 7. HuH-7 and HepG2 cells showed poor response to TGF-beta-mediated growth inhibition. Because neutralization of TGF-beta in the medium or blockage of signal transduction pathway by inductions of dominant negative Smad 2/3 resulted in a stimulation of cell growth, tumor-derived TGF-beta signal acts on cell growth negatively. However, Smad 7 induced by TGF-beta negatively regulated Smad 2 action and rendered most Smad 2 proteins in the cytoplasm. Taken together, these results indicate that endogenous TGF-beta-mediated induction of Smad 7 results in a higher "threshold" for the antiproliferative signals mediated by receptor-regulated Smads, and can be involved in reduced responsiveness to the cytokine in some human HCC cells.     

Reduced expression of cyclooxygenase 2 proteins in hereditary nonpolyposis colorectal cancers relative to sporadic cancers.

BACKGROUND & AIMS: Cyclooxygenase (COX) enzymes catalyze the conversion of arachidonic acid to prostaglandins. Evidence suggests that nonsteroidal anti-inflammatory drugs reduce the risk of colorectal cancer (CRC) and that this effect is mediated through COX inhibition. We analyzed and compared expression of the inducible COX-2 isoform in colorectal neoplasms from patients with hereditary nonpolyposis colorectal cancer (HNPCC), familial adenomatous polyposis (FAP), and sporadic CRC. Given that COX-2 is induced by transforming growth factor (TGF)-beta and that TGF-beta type II receptor (RII) mutations are found in HNPCCs, we determined the relationship between RII status and COX-2 expression. METHODS: COX-2 protein expression was determined in colorectal epithelia using immunohistochemistry and Western blotting. Patients with HNPCC had known mutations in hMLH1 or hMSH2 genes and/or met the Amsterdam criteria. In CRCs from HNPCC cases, mutations were sought in the coding region of the RII gene using the polymerase chain reaction. RESULTS: COX-2 was detected in adenomas from 2 of 3 HNPCC, 6 of 7 FAP, and 5 of 8 sporadic cases. In CRCs, COX-2 staining was found in 16 of 24 (67%) HNPCC vs. 24 of 26 (92%) sporadic cases (P = 0.035) and in 2 of 2 FAP cases. Staining intensity was reduced in HNPCCs compared with sporadic CRCs (P = 0.035). Staining localized to the cytoplasm of neoplastic cells; normal epithelial cells were negative for COX-2. Overexpression of COX-2 in CRCs relative to normal mucosa was confirmed by Western blotting. TGF-beta RII mutations were detected in 12 of 14 HNPCCs examined, including 3 of 4 COX-2-negative and 9 of 10 COX-2-positive cancers. CONCLUSIONS: The frequency and intensity of COX-2 expression was significantly reduced in HNPCCs relative to sporadic CRCs, and was not a consequence of RII mutations. Given that many HNPCCs express COX-2, inhibition of this enzyme may be an important strategy to prevent CRC in these patients.     

Microsatellite mutation of type II transforming growth factor-beta receptor is rare in atherosclerotic plaques

A somatic mutation within a microsatellite polyA tract in the coding region of the type II transforming growth factor (TGF)-beta receptor gene was reported to occur in human atherosclerotic and restenotic lesions. This mutation occurs frequently in colorectal cancer with the replication error repair phenotype and results in loss of sensitivity to the growth inhibitory effects of TGF-beta in cells from the tumors. The mutation was proposed to account for the clonal expansion of vascular smooth muscle cells observed in atherosclerotic plaques, through loss of the growth inhibitory effect of TGF-beta. The frequency of the mutation and the extent of clonal expansion of the mutated cells have major implications for the mechanism of atherogenesis and therapeutic strategies. We analyzed a set of 22 coronary arterial and 9 aortic samples containing early to advanced atherosclerotic lesions for the mutation in the type II TGF-beta receptor polyA tract. Only 1 coronary arterial sample from an advanced lesion showed detectable amounts of the mutation, present at a low level (8% of the DNA sample). The data imply that the mutation occurs only at low frequency and is not a major mechanistic contributor to the development of atherosclerosis.     

SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway

Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.     

ALK5- and TGFBR2-independent role of ALK1 in the pathogenesis of hereditary hemorrhagic telangiectasia type 2

ALK1 belongs to the type I receptor family for transforming growth factor-beta family ligands. Heterozygous ALK1 mutations cause hereditary hemorrhagic telangiectasia type 2 (HHT2), a multisystemic vascular disorder. Based largely on in vitro studies, TGF-beta1 has been considered as the most likely ALK1 ligand related to HHT, yet the identity of the physiologic ALK1 ligand remains controversial. In cultured endothelial cells, ALK1 and another TGF-beta type I receptor, ALK5, regulate angiogenesis by controlling TGF-beta signal transduction, and ALK5 is required for ALK1 signaling. However, the extent to which such interactions between these 2 receptors play a role in pathogenesis of HHT is unknown. We directly addressed these issues in vivo by comparing the phenotypes of mice in which the Alk1, Alk5, or Tgfbr2 gene was conditionally deleted in restricted vascular endothelia using a novel endothelial Cre transgenic line. Alk1-conditional deletion resulted in severe vascular malformations mimicking all pathologic features of HHT. Yet Alk5- or Tgfbr2-conditional deletion in mice, or Alk5 inhibition in zebrafish, did not affect vessel morphogenesis. These data indicate that neither ALK5 nor TGFBR2 is required for ALK1 signaling pertinent to the pathogenesis of HHT and suggest that HHT might not be a TGF-beta subfamily disease.     

Beta 2-microglobulin gene mutations: a study of established colorectal cell lines and fresh tumors.

The technique of single-strand conformation polymorphism (SSCP) was used to screen a series of 37 established colorectal cell lines, 22 fresh tumor samples, and 22 normal DNA samples for mutations in the beta 2-microglobulin gene. Exon 1 (including the leader peptide sequence) and exon 2 were screened separately. Six of 37 colorectal cell lines and 1 of 22 fresh tumors were shown to contain mutations, whereas no mutations were detected in the normal DNA samples. Sequencing of these mutations showed that an 8-bp CT repeat in the leader peptide sequence was particularly variable, since 3 of the cell lines and one fresh tumor sample have deletions in this region. In the related cell lines, DLD-1 and HCT-15, two similar mutations were identified, a C-->A substitution in codon 10 and a G-->T mutation in the splice sequence of intron 1. Expression of beta 2-microglobulin was examined using a series of monoclonal antibodies in an ELISA system. Reduced expression correlated with a mutation in one allele of beta 2-microglobulin, whereas loss of expression was seen in instances where a line was homozygous for a mutation or heterozygous for two mutations.