Somatic mutations of the mitochondrial genome in human colorectal tumours.

Alterations of oxidative phosphorylation in tumour cells were originally believed to have a causative role in cancerous growth. More recently, mitochondria have again received attention with regards to neoplasia, largely because of their role in apoptosis and other aspects of tumour biology. The mitochondrial genome is particularly susceptible to mutations because of the high level of reactive oxygen species (ROS) generation in this organelle, coupled with a low level of DNA repair. However, no detailed analysis of mitochondrial DNA in human tumours has yet been reported. In this study, we analysed the complete mtDNA genome of ten human colorectal cancer cell lines by sequencing and found mutations in seven (70%). The majority of mutations were transitions at purines, consistent with an ROS-related derivation. The mutations were somatic, and those evaluated occurred in the primary tumour from which the cell line was derived. Most of the mutations were homoplasmic, indicating that the mutant genome was dominant at the intracellular and intercellular levels. We showed that mitochondria can rapidly become homogeneous in colorectal cancer cells using cell fusions. These findings provide the first examples of homoplasmic mutations in the mtDNA of tumour cells and have potential implications for the abnormal metabolic and apoptotic processes in cancer.

Mad-related genes in the human.

Resistance to the growth inhibitory effects of TGF-beta is common in human cancers. However, the mechanism(s) by which tumour cells become resistant to TGF-beta are generally unknown. We have identified five novel human genes related to a Drosophila gene called Mad which is thought to transduce signals from TGF-beta family members. One of these genes was found to be somatically mutated in two of eighteen colorectal cancers, and three of the other genes were located at chromosomal positions previously suspected to harbor tumour suppressor genes. These data suggest that this gene family may prove to be important in the suppression of neoplasia, imparting the growth inhibitory effects of TGF-beta-like ligands.

Growth stimulation by coexpression of transforming growth factor-alpha and epidermal growth factor-receptor in normal and adenomatous human colon epithelium.

Autocrine stimulation of the epidermal growth factor receptor (EGF-R), by coexpression of transforming growth factor-alpha (TGF-alpha), causes malignant transformation of some fibroblast cell lines. TGF-alpha and EGF-R are both known to be expressed in colon carcinoma tissue and have been shown coexpressed in colon carcinoma cell lines. TGF-alpha autocrine activation of EGF-R has been suggested as a potential mechanism contributing to abnormal growth control in colon cancer. We now report coexpression of TGF-alpha and EGF-R transcripts in morphologically normal colonic epithelium from five individuals, in colonic adenomas from three individuals, and in a nontumorigenic colon adenoma cell line, VACO-330. Functional studies demonstrate VACO-330 growth is stimulated by exogenous TGF-alpha and is completely abolished by a blocking anti-EGF-R antibody. Autocrine stimulation of EGF-R by TGF-alpha is therefore required for growth of the adenoma cell line. Autocrine stimulation of EGF-R by TGF-alpha does not cause malignant transformation of the colonic epithelial cell. In normal and adenomatous human colon TGF-alpha, via either an autocrine or paracrine mechanism, is likely an important physiologic stimulant of epithelial proliferation.

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.

Tumor suppressor activity of the TGF-beta pathway in human cancers.

The TGF-betas are a family of cytokines with antiproliferative activity on many cell types. Recent findings demonstrate that the TGF-beta receptor complex functions as a tumor suppressor gene in human malignancy. Somatic mutations of the type II subunit of the TGF-beta receptor (RII) have been demonstrated in several different tumor types. RII frameshift mutations within a short coding region polyadenine repeat are particularly characteristic of colon and gastric cancers that also demonstrate the phenotype of microsatellite instability (RER cancers). These and other mutations as in the type I receptor (RI) are associated with both loss of cell surface TGF beta receptors and with resistance of the cancer cells to TGF-beta-induced growth inhibition. Restoration of receptor expression by gene transfection reverses the transformed phenotype in cancer cells that lacked functional RII or RI. These receptors and, by implication, TGF-beta as well as its complete signalling pathway, are thus new and novel additions to the family of human tumor suppressor genes.

Molecular mechanisms of inactivation of TGF-beta receptors during carcinogenesis.

Signals from the TGF-betas are mediated by the TGF-beta receptors and their substrates, the Smad proteins. Inactivation of either of the two transmembrane serine/threonine kinases called the TGF-beta type I and type II receptors is now known to underlie a wide variety of human pathologies including, especially carcinogenesis. Numerous studies have now demonstrated that the TGF-beta receptor complex and its downstream signaling intermediates constitute a tumor suppressor pathway. We review here a specific pathway of mutational inactivation of the TGF-beta type II receptor resulting from microsatellite instability and demonstrate that, by contrast, the most common mechanism of loss of expression of the TGF-beta type II receptor involves transcriptional repression. This provides a new target for therapeutic intervention.

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.

Induction of nm23 gene expression in human colonic neoplasms and equal expression in colon tumors of high and low metastatic potential.

Levels of expression of the murine nm23 gene inversely correlate with metastatic potential in several rodent tumor model systems. Expression of the human nm23 homologue also is lower in human breast cancers of high metastatic potential than in breast cancers of low metastatic potential. In the present study, we examined changes in nm23 expression during colon carcinogenesis as found in 18 matched pairs of normal and neoplastic human colon tissues. We found that a 0.8-kilo-base nm23 transcript was expressed in all samples of morphologically normal colon mucosa. In 16 of 18 colon neoplasms, nm23 expression was further increased in the neoplastic, compared with the morphologically normal, colon mucosa from the same individual. Expression of nm23 was elevated over normal mucosa in 3 of 3 polyps, in 2 of 3 nonmetastatic cancers, and in 11 of the 12 cancers that were metastatic at the initial presentation. The levels of nm23 expressed were similar in the 12 metastatic colon neoplasms and in the 6 colon neoplasms of lower clinical stage. In addition, nm23 expression was maintained in culture in each of 12 cell lines initiated from human colon neoplasms and did not differ between lines established from neoplasms of high or low metastatic capability. We concluded that nm23 was expressed in normal colon mucosa. Expression of nm23 increased during early stages of colon carcinogenesis and remained increased in metastatic colon cancer. Therefore, in the colon, tissue-specific events dissociate nm23 expression from loss of tumor metastatic competence.

Cytotoxicity and mutagenicity of frameshift-inducing agent ICR191 in mismatch repair-deficient colon cancer cells.

BACKGROUND: Deficiency of DNA mismatch repair is a common feature of cancers exhibiting instability of microsatellite DNA sequences. Cancers with microsatellite instability are recognizable by their high rate of spontaneous frameshift mutations within microsatellite sequences, their resistance to killing by cytotoxic agents, and their localization to specific tissues, e.g., the proximal colon and stomach. We hypothesized that the mismatch repair deficiency of these cancers would make them vulnerable to environmental or chemical frameshift-inducing agents. This study was undertaken to test whether exogenous frameshift-inducing agents selectively induce mutations in mismatch repair-deficient cells of mutagen-exposed tissues like the colon and whether cytotoxic doses of these agents would preferentially kill those cells. METHODS: Cytotoxicity of the acridine mutagen 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxy-acridine (ICR191), a DNA frameshift inducer, was determined in the mismatch repair-deficient human colon carcinoma cell line HCT116 versus the repair-reconstituted derivative HCT116+C3. Vulnerability to the mutagenic effects of ICR191 was determined by transfection of HCT116 or HCT116+C3 cells with a frameshift reporter vector, followed by treatment of the cells with ICR191. Alternatively, the reporter vector was reacted ex vivo with ICR191, and the derivatized vector was then transfected into HCT116 or HCT116+C3 cells. RESULTS: ICR191 proved to be fivefold to 10-fold more potent in inducing mutations in mismatch repair-deficient HCT116 cells than in mismatch repair-proficient HCT116+C3 cells. Moreover, at cytotoxic doses of ICR191, repair-deficient HCT116 cells proved to be fivefold more vulnerable to killing than did HCT116+C3 cells. CONCLUSIONS: Frameshift-inducing mutagens can selectively induce mutations in mismatch repair-deficient cells versus mismatch repair-proficient cells. Environmental exposures may, therefore, favor development of cancers with microsatellite instability in tissues like the gut. Frameshift-inducing agents can, however, also preferentially kill mismatch repair-deficient cancer cells and, thus, may be promising as model therapeutic compounds.

Testing for colon neoplasia susceptibility variants at the human COX2 locus.

BACKGROUND: Siblings and other first-degree relatives of patients with "sporadic" (i.e., apparently nonfamilial) colorectal cancer or precursor adenomatous colon polyps have an increased risk of developing colon neoplasia. This observation suggests the presence of inherited genetic determinants for sporadic colon neoplasia. Mice homozygous for a null cyclooxygenase 2 (COX2) (also called PTGS2) allele have a dramatically reduced susceptibility to the development of intestinal adenomas. In humans, use of pharmacologic inhibitors of COX2 enzyme activity are associated with reduced risk of colon neoplasia. This study examined whether the human COX2 locus may be linked to colon neoplasia in humans. METHODS: We used the affected sibling-pair method to test for linkage of the human COX2 locus to colon neoplasia. RESULTS: We examined 74 concordantly affected sibling pairs from 46 sibships with colon neoplasia. One hundred five siblings from these sibships were diagnosed with either colorectal cancer or colon adenomatous polyps before age 65 years. No linkage between COX2 and colon neoplasia was found by use of a multipoint model-free linkage analysis (estimate of allele sharing was 0.44; standard error = +/-0.04; 95% confidence interval = 0.36 to 0.52). Moreover, even allowing for heterogeneity, the potential that a COX2 colon neoplasia susceptibility variant was present within a substantial subset of these sibships was strongly excluded under either a recessive or a dominant inheritance model (95% confidence to exclude a model in which 2.7% or more of the sibling pairs harbor a dominant susceptibility allele). CONCLUSIONS: This study of concordantly affected sibling pairs thus demonstrates that variations in the COX2 gene are unlikely to be a source of individual susceptibility to colon neoplasia in humans.

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.

Short mononucleotide repeat sequence variability in mismatch repair-deficient cancers.

Mismatch repair-deficient cancers are characterized by widespread insertions and deletions in microsatellite sequences, including those comprised of mononucleotide repeats. Such alterations have been observed in relatively short mononucleotide tracts in several genes and often are interpreted to indicate that the affected genes normally act as tumor suppressors. To aid in the interpretation of such changes, we have systematically assessed their frequency within transcribed regions of the genome that are unlikely to play a tumorigenic role. The advent of the complete human genomic sequences of chromosome 22 allowed us to select 29 genes for this analysis, spaced at approximately 1-Mb intervals. Each of the selected genes had an (A)(8) or a (G)(8) tract deep within intronic sequences that was not included in the processed transcript. Surprisingly, we found that there was substantial variation in the prevalence of mutations among these tracts. Some tracts were altered in < 5% of the mismatch repair-deficient cancers studied, whereas other tracts were altered in nearly half of the cancers. In particular, (G)(8) tracts were considerably more prone to mutation than (A)(8) tracts, and the sequences or chromatin structures surrounding the mononucleotide tracts seemed to affect their mutability significantly.

Evaluation of the FHIT gene in colorectal cancers.

A variety of studies suggests that tumor suppressor loci on chromosome 3p are important in various forms of human neoplasia. Recently, a chromosome 3p14.2 gene called FHIT was discovered and proposed as a candidate tumor suppressor gene in colorectal and other cancers. We evaluated the FHIT gene in a panel of colorectal cancer cell lines and xenografts, which allowed a comprehensive mutational analysis. A transcript containing the complete coding sequence was found to be expressed at robust levels in 29 of 31 cancers tested. The complete sequence of the coding region of the gene was determined and found to be normal in all 29 of these cases. These studies suggest either that FHIT is inactivated by an unusual mechanism or that it plays a role in relatively few colorectal tumors.

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.

O6-methylguanine-DNA methyltransferase (MGMT) transfectants of a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-sensitive colon cancer cell line selectively repopulate heterogenous MGMT+/MGMT- xenografts after BCNU and O6-benzylguanine plus BCNU.

To evaluate the role of O6-alkylguanine-DNA alkyltransferase (AGT) in colon tumor chloroethylnitrosourea (CENU) resistance, AGT-deficient VACO 8 cells were transfected with a vector containing or lacking the human O6-methylguanine-DNA methyltransferase (MGMT) cDNA. VACO 8MGMT (V8MGMT) sublines possessed high levels of AGT activity in cell culture and were > 10-fold resistant to the CENU 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). V8MGMT cells, VACO 8neo cells, and mixtures of both were grown as xenografts in nude mice. MGMT expression in VACO 8 xenografts reflected the percentage of V8MGMT cells present in the tumor inoculum. Xenografts originally containing 0-10% V8MGMT cells were sensitive to BCNU, although partial resistance was observed as the percentage of V8MGMT cells increased. Tumors containing 30-100% V8MGMT cells were completely resistant to BCNU with no regressions and no growth delays. Pretreatment with O6-benzylguanine (BG) depleted tumor AGT activity for at least 6 h and sensitized xenografts containing 1 and 100% V8MGMT cells to BCNU. After BCNU or BG + BCNU, xenografts growing from inoculums containing as low as 0.1% V8MGMT cells had high AGT activities similar to that found in V8MGMT xenografts, with the majority of the cells expressing MGMT. These results provide evidence that MGMT expression influences both intrinsic and acquired colon tumor CENU resistance, that selective expansion of AGT+ colon tumor cells commonly occurs after CENU exposure, and that BG is effective in sensitizing colon tumors to CENUs, even when only a small fraction of the cells in a heterogeneous tumor express MGMT.

Prostate cancer and other xenografts from cells in peripheral blood of patients.

Good models for the investigation of human prostate cancer are few. Cells from approximately 9.2-21 ml of peripheral blood from patients with metastatic prostate cancer or metastatic colon cancer were injected s.c. into nude mice. Prostate cancer from 2 of 11 patients and colon cancer from 1 of 3 patients were found to be growing as metastases in the lungs of the nude mice. To our knowledge, this is the first report of the formation of xenografts from carcinoma cells taken directly from the peripheral blood of patients. Expanding circulating cancer cells with this approach may have important translational applications including: (a) development of models of human cancers; and (b) sampling of cancers from specific patients for novel molecular and therapeutic approaches.

MMTV promoter hypomethylation is linked to spontaneous and MNU associated c-neu expression and mammary carcinogenesis in MMTV c-neu transgenic mice.

The erbB family of receptor tyrosine kinases is frequently implicated in neoplasia. Amplification and overexpression of erbB2/neu has been found in 20 to 40% of human breast cancers. Previous studies using MMTV/c-neu transgenic mice have linked rat neu overexpression to mammary tumor development. In this study, we provide evidence that rat neu overexpression in mammary tumors of MMTV/c-neu transgenic mice is always associated with demethylation of the MMTV promoter, whereas the normal mammary glands of these transgenic mice always contain specific methylated regions of the MMTV promoter. In addition, after exposure to N-methyl-N-nitrosourea (MNU), the latency of mammary tumor development is significantly reduced and again is also associated with MMTV promoter demethylation. Thus, the transition from methylation to hypomethylation of the MMTV promoter induces high-level expression of c-neu and appears to be a prerequisite for transformation from normal to malignant mammary epithelium, either spontaneously or after carcinogen exposure. Expression of transgenic c-neu from the demethylated MMTV promoter appears to be an early event that allows outgrowth of mammary epithelium predisposed to malignant transformation.

Increased mutation rate at the hprt locus accompanies microsatellite instability in colon cancer.

Hereditary Non-Polyposis Colon Cancer (HNPCC) tumors and some sporadic colon cancers acquire somatic changes in the length of microsatellite sequences. We hypothesized that this 'replication error' (RER) phenotype in these cancers reflects a more general defect which should result in hypermutability of expressed genes. To test this hypothesis mutations of hprt were studied in RER and non-RER tumor cell lines. Increased mutation rates of greater than 100-fold were found in RER compared to non-RER lines. Heterogeneity within the RER group suggests the likely existence of different classes of RER tumors. One non-RER cell line demonstrated a greater than 10-fold increase in mutation rate, suggesting that a novel mutator phenotype may exist in some non-RER tumors.

Chromosome number and structure both are markedly stable in RER colorectal cancers and are not destabilized by mutation of p53.

Fourteen colorectal cancer cell lines, categorized according to the presence or absence of microsatellite instability, were further analysed for chromosomal stability by karyotyping. NonRER (microsatellite stable) cell lines typically displayed highly aberrant karyotypes with alterations not only of chromosome number but also of chromosome structure including chromosomal deletions, inversions, and translocations. RER (microsatellite unstable) cell lines, in contrast, displayed significantly fewer alterations of chromosome number. Moreover, RER cell lines also displayed significantly fewer cytogenetically evident alterations of chromosome structure. Compared to NonRER colon cancers, RER colon cancers are significantly less likely to have undergone chromosomal gain, loss, or breakage. Characterization of p53 gene status by gene sequencing was performed in an attempt to determine if p53 gene status correlated with the chromosomal stability of the RER cancers. Gene mutations in p53 were present in all of the NonRER colon cancers. However, p53 gene mutations were also found present in four of nine of the RER colon cancers. Unexpectedly, RER colon cancers bearing mutant p53 demonstrated the same stability of chromosome number, and the same stability of chromosome structure, as the RER colon cancers with wild-type p53. Therefore, in RER colon cancers specific p53 independent mechanisms actively maintain the stability of both chromosome number and structure.

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.