Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Human colon cancer cell lines with high rates of microsatellite instability were found to harbor mutations in the type II TGF-beta receptor (RII) gene. Eight such examples, due to three different mutations, were identified. The mutations were clustered within small repeated sequences in the RII gene, were accompanied by the absence of cell surface RII receptors, and were usually associated with small amounts of RII transcript. RII mutation, by inducing the escape of cells from TGF-beta-mediated growth control, links DNA repair defects with a specific pathway of tumor progression.

A benign cultured colon adenoma bears three genetically altered colon cancer oncogenes, but progresses to tumorigenicity and transforming growth factor-beta independence without inactivating the p53 tumor suppressor gene.

We describe the spontaneous progression of a colon adenoma cell line to tumorigenicity and growth factor independence. This system allows direct comparison of biologic stages of malignant progression with alterations of colon cancer suppressor genes and oncogenes. VACO-235, a human colon adenoma cell line, is at early passages nontumorigenic in the nude mouse, unable to grow in soft agar, growth stimulated by serum and EGF, and growth inhibited by TGF-beta. VACO-235 daughter passages 93 and higher have in culture spontaneously progressed to being weakly tumorigenic, but retain all other growth characteristics of VACO-235 early passages. A mouse xenograft from late passage VACO-235 was reestablished in culture as the granddaughter cell line, VACO-411. VACO-411 is highly tumorigenic, clones in soft agar, and is unresponsive to serum, EGF, and TGF-beta. Early passage VACO-235 bears a mutant K-ras allele, bears only mutant APC alleles, expresses no DCC transcripts, and expresses only wild type p53 transcripts. VACO-411 retains the identical genotype, still expressing only wild type p53. Colonic cells after ras mutation, APC mutation, and DCC inactivation remain nontumorigenic and growth factor dependent. Malignant progression involves at least two additional steps, and in VACO-411 can proceed by a novel pathway not requiring p53 inactivation.

Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer.

Serological tumor markers have proven valuable in the care of individuals with cancer for the early detection of primary cancers, early detection of cancer relapse, monitoring the response of cancers to therapy, and as predictors of cancer prognosis. Recently, the aberrant hypermethylation of the hMLH1 promoter and its consequent transcriptional silencing has been shown to be a common event in the formation of sporadic microsatellite unstable colon cancer. The silencing of hMLH1 expression appears to be controlled by the hypermethylation of a specific region in the hMLH1 promoter. We developed a methylation-specific PCR assay that assesses this region of the hMLH1 promoter. We found that this assay is able to detect methylated hMLH1 promoter DNA in the serum of some patients with microsatellite unstable colon cancers. In a panel of sera from 19 colon cancer cases, 9 with hMLH1 promoter methylation in the tumor primary, the assay proved 33% sensitive and 100% specific. This assay offers a potential means for the serum-based detection and/or monitoring of microsatellite unstable colon cancers.

Transforming growth factor-beta-induced growth inhibition in a Smad4 mutant colon adenoma cell line.

Transforming growth factor-beta (TGF-beta) inhibits growth and induces apoptosis of colon epithelial cells. Binding of TGF-beta to its receptor induces phosphorylation of the Smad proteins Smad2 and Smad3, which then form heteromeric complexes with Smad4, translocate to the nucleus, and activate gene transcription. Smad4 function has been considered an obligate requirement for TGF-beta signaling, and Smad4 mutations present in some cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe studies with a nontransformed human colon epithelial cell line that is mutant for Smad4 but remains growth-inhibited by TGF-beta. The colon cell line VACO-235 has lost one of its Smad4 alleles via a chromosome 18q deletion. The remaining allele bears two missense point mutations located in regions important for Smad4 trimer formation, which is thought necessary for Smad4 function. As expected, pSBE4-BV/Luc, a Smad4-activated transcriptional reporter, was inactive in VACO-235. Nonetheless, VACO-235 demonstrated 80% growth inhibition in response to TGF-beta, as well as retention of some TGF-beta-mediated activation of the p3TP-Lux transcriptional reporter. Transient transfection of the VACO-235 Smad4 mutant allele into a Smad4-null cell line confirmed that this allele is functionally inactive as assayed by both the pSBE4-BV and p3TP-Lux reporters. The simplest explanation of these results is that there is a non-Smad4-dependent pathway for TGF-beta-mediated signaling and growth inhibition in VACO-235 cells.

A transforming growth factor beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability.

We have recently demonstrated that mutation of the transforming growth factor-beta (TGF-beta) receptor type II (RII) gene is characteristic of colon cancers exhibiting microsatellite instability or replication errors (RER+). Moreover, we have shown that RII mutations in these RER+ colon cancers are characteristically frameshift mutations within a 10-bp polyadenine repeat present in the RII-coding region. We now show that RII gene mutations in this polyadenine repeat are also commonly present in RER+ gastric cancers (71%). In contrast, we find these same RII gene mutations are distinctly uncommon in RER+ endometrial cancers (17%, P < 0.02). These results suggest that RII gene mutations confer a growth advantage and are selected for in RER+ cancers of both the upper and lower gastrointestinal tract. The genesis of RER+ endometrial tumors must, however, be by a different route.

Mutational inactivation of transforming growth factor beta receptor type II in microsatellite stable colon cancers.

We previously demonstrated that mutational inactivation of transforming growth factor beta type II receptors (RIIs) is very common among the 13% of human colon cancers with microsatellite instability. These mutations principally cluster in the BAT-RII polyadenine sequence repeat. Among microsatellite stable (MSS) colon cancers, we now find that non-BAT-RII point mutations inactivate RII in another 15% of cases, thus doubling the known number of colon cancers in which RII mutations are pathogenetic. Functional analysis confirms that these mutations inactivate RII signaling. Moreover, another 55% of MSS colon cancers demonstrate a transforming growth factor beta signaling blockade distal to RII. The transforming growth factor beta pathway and RII in particular are major targets for inactivation in MSS colon cancers as well as in colon cancers with microsatellite instability.

Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability.

Inactivation of DNA-mismatch repair underlies the genesis of microsatellite unstable (MSI) colon cancers. hPMS2 is one of several genes encoding components of the DNA-mismatch repair complex, and germline hPMS2 mutations have been found in a few kindreds with hereditary nonpolyposis colorectal carcinoma (HNPCC), in whom hereditary MSI colon cancers develop. However, mice bearing null hPMS2 genes do not develop colon cancers and hPMS2 mutations in sporadic human colon cancers have not been described. Here we report that in Vaco481 colon cancer the hPMS2 gene is inactivated by somatic mutations of both hPMS2 alleles. The cell line derived from this tumor is functionally deficient in DNA mismatch repair. This deficiency can be biochemically complemented by addition of a purified hMLH1-hPMS2 (hMutLalpha) complex. The hPMS2 deficient Vaco481 cancer cell line demonstrates microsatellite instability, an elevated HPRT gene mutation rate, and resistance to the cytotoxicity of the alkylator MNNG. We conclude that somatic inactivation of hPMS2 can play a role in development of sporadic MSI colon cancer expressing the full range of cancer phenotypes associated with inactivation of the mismatch repair system.

Increased transversions in a novel mutator colon cancer cell line.

We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.

Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability.

Colon cancer and an increasing number of other cancers have been found to exhibit instability of DNA microsatellite sequences. Such tumors have been designated as replication errors (RER) tumors. However, as microsatellites are only rarely found within coding regions of the genome, instability of these sequences cannot directly contribute to carcinogenesis. Recently, we have shown RER colon cancers also demonstrate a marked 100-fold increase in mutation rates measured within an expressed gene, hprt, suggesting the mutator phenotype in these tumors extends beyond microsatellite sequences. To determine whether the RER phenotype indeed destabilizes non-repetitive DNA sequences we have sequenced hprt gene mutations recovered from the RER colon cancer cell line RKO. Greater than 10% of hprt mutants proved to be a single 3 bp deletion located in a nonrepetitive ATTAT sequence motif. Additionally, 1-4 bp deletions or insertions were found to be randomly located throughout the hprt gene. Lastly, one third of hprt mutations proved to be transitions or transversions. The microsatellite instability demonstrated in RKO is thus a global mutator phenotype which destabilizes DNA sequences both inside and outside of repetitive sequence elements and which augments base substitutions as well as frameshifts. These findings extend the characteristics of mutations associated with RER tumors and suggest additional mechanisms by which mutator phenotypes may alter oncogenes and tumor suppressor genes.

Spontaneous apoptosis in human colon tumor cell lines and the relation of wt p53 to apoptosis.

OBJECTIVE: To examine spontaneous apoptosis of cultured human colon tumor cell lines in vitro and to investigate the role of wild type (wt) p53 in regulation of apoptosis induced by DNA-damaging treatment. METHODS: A model system of human tumor progression involving three cell lines was used in this study for examination of apoptosis. They were originally established from human colon villous adenoma, including an early passage of non-tumorigenic cell line, V235E; a late passage of weakly tumorigenic cell line, V235L; and a spontaneous progressing highly tumorigenic cell line. V411. All of them maintain wt p53 expression. For identification of apoptosis, two tests were performed: 1. morphology study using acridine orange (AO)/ethidium bromide (EB) stainning by fluorescence microscopy; 2. DNA electrophoresis on agarose gel. P53 and WAF-1 (a downstream gene of p53) expressions were analysed at mRNA level using Northern blot technique. Apoptotic index of cell lines examined was measured by DNA fluorescence assay. RESULTS: Spontaneous apoptosis was demonstrated in cell lines of all stages of progression by both morphology and DNA agarose gel electrophoresis. Apoptosis was further induced in V411 after treatment of cells with 137Cs gamma-irradiation and accompanied by increases in p53 and WAF-1 expression. In contrast, a mutant p53 bearing human colon cancer cell line, sw480, lacked spontaneous apoptosis, and upon irradiation neither induction of apoptosis nor increase expression of p53 and WAF-1 were seen. CONCLUSIONS: Apoptosis can be maintained in some human tumor cell lines despite transformation and carcinogenesis. Wt p53 and WAF-1 products are two of the potential mediators which effect apoptosis. Additionally, since apoptosis was enhanced by irradiation in V411, but not in sw480, it suggests that wt p53 cancer cells are more sensitive to DNA-damaging treatment than mutant p53 cancer cells. These finding may have implications for cancer therapy.

Mutant K-ras oncogenes in colon cancers Do not predict Patient's chemotherapy response or survival

One half of human colon cancers bear mutant c-K-ras oncogenes. Mutant K-ras oncogenes are associated with shortened survival in non-small cell lung cancers, and, in cell line models, with resistance to cis-platinum and to ionizing radiation. This study examines whether mutant K-ras alleles in colon cancer alter patients' response to chemotherapy or survival. We studied 37 patients who received chemotherapy with 5-fluorouracil and leucovorin, Exon 1 of the c-K-ras gene was PCR amplified from DNA extracted from paraffin-embedded tumor blocks. The presence of mutant or wild-type c-K-ras alleles was determined by dideoxy sequencing of the PCR-amplified c-K-ras DNA. c-K-ras mutations at codons 12 or 13 were present in 19 and absent in 18 cases. Responses to chemotherapy were equally likely in patients with either wild-type or mutant c-K-ras, occurring in 28% of patients with wild-type ras and 32% of patients with mutant ras (P = 0.8). Survival was also indistinguishable among both groups. Median survival from diagnosis was 35 months for ras wild-type patients and 31 months for ras mutant patients (P = 0.96). Median survival from starting chemotherapy was 14 months for ras wild-type patients and 17 months for ras mutant patients (P = 0.26). Patients with colon cancers bearing either wild-type or mutant c-K-ras alleles are indistinguishable in overall survival and are equally likely to respond to 5-fluorouracil-based chemotherapy.

Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers.

Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI-positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism.