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.

Increased nm23-H1 and nm23-H2 messenger RNA expression and absence of mutations in colon carcinomas of low and high metastatic potential.

BACKGROUND: The murine nm23 gene suppresses the metastatic behavior of malignant rodent tumor lines, and reduced nm23 expression correlates with increased likelihood of lymph node metastases in human breast cancers. More recent data have demonstrated the existence of two human nm23 gene homologues, nm23-H1 and nm23-H2, and have shown that deletion of nm23-H1 alleles occurs in some colon carcinomas associated with poor prognosis. These findings suggest that nm23-H1 encodes for suppression of colon carcinoma metastasis. In contrast, we have previously reported that total nm23 messenger RNA (mRNA) expression is increased to similar levels in colon tumors of both high and low metastatic potential. PURPOSE: This study was designed to reconcile our previous findings with the recent report of nm23-H1 allelic deletion in human colon cancers associated with poor prognosis. Our purpose was to examine human colon cancers for inactivation of two candidate metastasis suppressor genes, nm23-H1 and nm23-H2, either by mutation or by loss of gene transcription. METHODS: We used ribonuclease protection assays to analyze human colon tumors for the level of nm23-H1 (43 samples) and nm23-H2 (41 samples) transcript (mRNA) expression and the presence of mutations that could inactivate potential suppressor function. RESULTS: We detected only wild-type nm23-H1 and nm23-H2 mRNA. Expression of nm23-H1 mRNA increased in 33 of 41 colon tumors, and expression of nm23-H2 mRNA was elevated in 28 of 41 colon tumors relative to that in matched normal mucosa. Increases in these mRNA levels were similar in tumors of both low and high metastatic potential. CONCLUSIONS: These results suggest that, despite correlation of nm23-H1 allelic deletions with colon cancers associated with poor prognosis, nm23-H1 and nm23-H2 alleles do not directly mediate metastasis suppression in colon carcinoma. Our results leave unexplained the observation that nm23-H1 allelic deletion correlates with metastatic potential of colon carcinomas. IMPLICATIONS: These findings also contrast with the demonstration of nm23 metastasis suppressor activity in murine melanoma and with the correlation of loss of nm23 expression in breast cancer with poor prognosis. It may be that metastasis suppression by the nm23 gene is a tissue-specific phenomenon.

Mutation of the type II transforming growth factor-beta receptor is coincident with the transformation of human colon adenomas to malignant carcinomas.

The transforming growth factor-beta (TGF-beta) type II receptor (RII) is a colon cancer suppressor gene that is inactivated by mutation in 90% of human colon cancers arising via the microsatellite instability (MSI) pathway of carcinogenesis. To determine the pathophysiological consequence of RII mutations, we have determined the timing of their onset among 22 MSI human colon adenomas of varying stages. No RII mutations were detected in any early MSI adenoma, including all those with simple tubular or villous histology. The earliest RII mutation detected was in a region of high-grade dysplasia but was absent from the surrounding simple adenoma. Six additional RII mutations were all found in highly progressed adenomas that contained regions of frankly invasive adenocarcinoma. These RII mutations were detected in both the advanced adenomas and their adjacent regions of carcinoma. RII mutation is a late event in MSI adenomas and correlates tightly with progression of these adenomas to cancer.

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.

Microsatellite instability and mutations of the transforming growth factor beta type II receptor gene in colorectal cancer.

The TGF beta type II receptor (RII) was found to be mutated within a polyadenine tract in 100 of 111 (90%) colorectal cancers with microsatellite instability. Other polyadenine tracts of similar length were mutated in these samples but not as frequently as RII. In most cases, the polyadenine tract mutations affected both alleles of RII, and in four tumors heterozygous for the polyadenine mutations, three had additional mutations that were expected to inactivate the other RII allele. These genetic data support the idea that RII behaves like a tumor suppressor during CR cancer development and is a critical target of inactivation in mismatch repair-deficient tumors.

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.

Mutation Detection inTGF-ß Receptors.

The transforming growth factor ß (TGF-ß) receptors are an important class of tumor suppressor gene. TGF-ß markedly inhibits the growth of many epithelial cell types; whereas in contrast, cancers of many different tissue types are commonly TGF-ß resistant (10). Many cancer cell lines are resistant to the growth suppressive effects of TGF-ß and display evidence of disruption of TGF-ß signal transduction (10,4,10). To date, this resistance appears to often result from mutations in the TGF-ß receptors or the smad family of TGF-ß signaling proteins (5,11,12,14,16).

Demonstration that mutation of the type II transforming growth factor beta receptor inactivates its tumor suppressor activity in replication error-positive colon carcinoma cells.

Escape from negative growth regulation by transforming growth factor beta (TGF-beta) as a result of the loss of TGF-beta type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-beta 1 treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-beta 1-neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-beta autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

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.