Human Non-polyposis Colorectal Cancer Research Articles

Nuclear import of human MLH1, PMS2, and MutLα: Redundancy is the key

DNA mismatch repair maintains genomic stability by correcting errors that have escaped polymerase proofreading. Defects on mismatch repair genes lead to an increased mutation rate, microsatellite instability and predisposition to human non-polyposis colorectal cancer (HNPCC). Human MutLalpha is a heterodimer formed by the interaction of MLH1 and PMS2 that coordinates a series of key events in mismatch repair. It has been proposed that nuclear import of MutLalpha may be the first regulatory step on the activation of the mismatch repair pathway. Using confocal microscopy and mismatch repair deficient cells, we have identified the sequence determinants that drive nuclear import of human MLH1, PMS2, and MutLalpha. Transient transfection of the individual proteins reveals that MLH1 has a bipartite and PMS2 has a single monopartite nuclear localization signal. Although dimerization is not required for nuclear localization, the MutLalpha heterodimer is imported more efficiently than the MLH1 or PMS2 monomers. Interestingly, the bipartite localization signal of MLH1 can direct import of MutLalpha even when PMS2 encompasses a mutated localization signal. Hence we conclude that the presence of redundant nuclear localization signals guarantees nuclear transport of MutLalpha and, consequently, efficient mismatch repair.

Microsatellite stable genome in an epithelioid hemangioendothelioma: An example of a microsatellite stable tumor

Microsatellite stable genome in an epithelioid hemangioendothelioma: An example of a microsatellite stable tumor

Non Hodgkin Lymphomas Showing Microsatellite Instability (MSI-H NHL) Are Characterized by Both Specific Biological and Clinical Features.

Cancers showing microsatellite instability (MSI-H) constitute a frequent subset of human tumors characterized by mismatch repair (MMR) deficiency. This phenotype is well known to occur in the Human Non Polyposis Colorectal Cancer (HNPCC) syndrome, as well as in about 10–15% of sporadic colorectal, gastric and endometrial cancers. Of interest, we recently demonstrated that it was also strikingly associated with a subset of Non Hodgkin Lymphomas (NHL) developed in either HIV positive (HIV-RL, e.g. HIV-related Lymphomas) or iatrogenically immunosuppressed (PTLD, e.g. Post-Transplantation Lymphoprolipherative Disorders) patients. We here further characterize this new subset of immunodeficiency-related lymphomas (ID-RL) at both the biological and clinical levels. By using specific markers of the MSI-H phenotype, we extended our molecular screening to a series of 161 NHL developed in either immunocompetent (n = 136) or immunodeficient (n =25, including 4 HIV-RL and 21 PTLD) hosts. Clinically, MSI-H NHL were found to be:mostly PTLD (12/114, e.g 10%), and more rarely HIV-RL (4/131, e.g. 3%) (p = 0;05; fisher's exact test):mostly T-cell NHL (4/17, e.g. 24% of T-cell ID-RL, as compared to 10/224, e.g 4% of B-cell ID-RL);late occurring PTLD after organ transplantation associated or not with EBV (52% EBV negative and 48% EBV positive).Biologically, MSI-H NHL presented with:primary MMR defects involving MLH1, MSH2 or MSH6;secondary mutational events at target genes for instability, as already reported (RAD-50, BAX, CASP-5 and others…);frequent V599E activating mutation within the BRAF gene (3/9, e.g. 30%);stable cytogenetic profiles characterized by only few chromosomal rearrangements if any.Taken together, these data show that oncogenic mechanisms involved in MSI-H NHL differ from those already known to be associated with other ID-RL, e.g. Chromosomal Instability or EBV-related pathways, and that they should now be considered as a distinct tumor entity.

Spotlight

Cheng et al., pp.345–353 Prolonged exposure to estrogens can induce tumors in laboratory animals and has been associated with breast and uterine cancers in humans. The proliferative effect of estrogen on breast epithelium is part of the mechanism. In addition, recently surfaced evidence demonstrated that estrogen metabolites can cause DNA damage and mutations, an important step in tumor initiation. Oxidation of two catechol estrogen (CE) metabolites, 4-hydroxyestradiol and 4-hydroxyestrone, generates quinone intermediates. They can react with purine bases to form DNA adducts that generate highly mutagenic apurinic sites; CEs can also generate oxygen radicals and DNA double strand breaks (DSB). Cheng et al. examined whether breast tumorigenesis is linked to inherited variants in five genes that are involved in detoxifying pathways of estrogen metabolites. Women harboring a greater number of high-risk genotypes of CE-metabolizing genes faced an increased risk of breast cancer. This trend was more pronounced in women who had never been pregnant or older at their first full-term pregnancy and therefore at greater risk for breast cancer. Cheng also examined whether the relationship between cancer risk and the genotypic polymorphism of CE-metabolizing genes was modified by the genotypes of DSB repair genes. They found that a joint effect was significantly associated with breast cancer development. This study supports the hypotheses that increased estrogen exposure confers a higher risk of breast cancer, in part caused by DSB in DNA. Brennetot et al., pp.446–450 High-frequency microsatellite instability (MSI-H) is due to either insertion or deletion of repetitive sequences during DNA replication and failure of the mismatch repair system (MMR) to correct these errors. As a consequence, MMR-deficient tumors accumulate thousands of small mutations genome-wide. Almost all tumors from patients with human non-polyposis colorectal cancer (HNPCC) are MMR deficient, as are 10–15% of sporadic colorectal, gastric and endometrial cancers.2 Allelic profiles of BAT-26 and BAT-25 obtained by independent fluorescent PCR of these markers. MSI-H tumors differ from microsatellite-stable (MSS) tumors in molecular and in clinicopathological attributes. Generally, patients with MSI-H tumors are reported to have a better clinical outcome than patients with MMR-proficient tumors. Hence, screening of all human tumors where the MSI phenotype is frequent would not only allow the treatment to be custom-tailored but also assist in the diagnosis of ambiguous HNPCC cases. The present study demonstrates that together the markers BAT-25 and BAT-26, two mononucleotide repeats widely used as part of a larger set of markers, are sensitive enough to establish the MSI status of human tumors. Furthermore, they can detect instability when the tumor cell content of a sample is as low as 5–10%. This observation is very important in the context of proposed regular MSI screening for all colorectal, gastric and endometrial cancers. Not only might fewer markers than the routinely suggested panel of five suffice to reliably establish the microsatellite status of tumors, the labor-intensive and time-consuming process of enriching tumor cells in contaminated samples may only be required in a small subset of biopsies. Merchan et al., pp.490–498 Most angiogenesis inhibitors were developed intentionally to interrupt new blood vessel formation. In an unexpected twist, the antiangiogenic activity of several cytotoxic agents was discovered accidentally, among them the widely used chemotherapeutic paclitaxel. In addition to its well-known microtubule-interfering effect, paclitaxel inhibits endothelial cell (EC) migration, invasion, capillary tube formation and proliferation at low concentrations in vitro. In vivo, it significantly reduces microvessel density and decreases vascular endothelial growth factor (VEGF) production. While paclitaxel's effects on ECs are well studied and clinical trials investigating its antiangiogenic potential are underway, the underlying mechanism has not yet been elucidated. Based on the latest results by Merchan et al. it appears that an approximately fivefold increased ability of human ECs to accumulate the drug when compared to other cell types is responsible for the enhanced sensitivity of ECs. However, at the same time low concentrations of paclitaxel upregulate the expression of Cox-2, an enzyme also known for its angiogenesis-stimulating activity. Combined treatment with the specific Cox-2 inhibitor celecoxib resolved the undesirable dichotomy and resulted in more pronounced antiendothelial effects than applying either compound alone. These findings could be extended to a clinical setting, where four patients with prostate cancer or malignant melanoma were treated with prolonged intravenous infusions of paclitaxel and celecoxib. The potent antiangiogenic activity observed in plasma specimens suggests that a combination treatment may be an attractive strategy to capitalize on the intrinsic antiangiogenic and cytotoxic effects of each agent alone.

Genetic instability in human mismatch repair deficient cancers

Cancers showing microsatellite instability (MSI-H) are frequent tumors characterized by inactivating alterations of mismatch repair (MMR) genes that lead to an incapacity to recognize and repair errors that occur during DNA replication. These cancers can be inherited as in the human non-polyposis colorectal cancer syndrome, or can occur sporadically in 10–15% of colorectal, gastric and endometrial cancers. MSI-H tumors have different clinicopathological features compared to cancers without this phenotype, termed MSS, and the repertoire of genetic events involved in tumoral progression of both phenotypes is thought to be different. In MSI-H tumors, most of the genetic changes occur at both non-coding and coding microsatellites that are particularly prone to errors during replication due to their repetitive sequence. This mechanism appears to be the main “genetic pathway” by which functional changes with putative oncogenic effects are accumulated in these tumors.

Cell type specificity in alternative splicing of the human mismatch repair gene hMSH2.

Human non-polyposis colorectal cancer is caused by germline mutations in the DNA mismatch repair genes hMSH2 and hMHL1. Several alternatively spliced mRNA species of these genes are present in peripheral blood lymphocytes of normal individuals, which can confound RT-PCR based techniques of mutation detection. Using RT-PCR, we compared the pattern of alternative splicing in whole peripheral blood lymphocytes (PBLs), separated T and B cells, lymphoblastoid cell lines (LCLs) from the same individuals, and a variety of tissues. Alternatively spliced forms of hMLH1 lacking exons 9/10, 10/11 and 9/10/11 were found to have similar patterns of expression in T cells, B cells, and LCLs. By contrast, a subset of hMSH2 transcripts, some of which were produced by utilisation of novel splicing motifs, were generally expressed in T but not in B cells. LCLs derived from the same blood samples showed no expression of any hMSH2 splicing variants. The hMSH2 delta ex13 transcript, while absent from LCLs, was expressed in whole PBLs and both T and B cell fractions. This transcript was furthermore largely undetectable in tissues other than mononuclear blood cells. These data provide evidence for tissue specificity in the regulation of alternative splicing in hMSH2. In particular we show that LCLs generally do not express alternatively spliced forms of hMSH2 mRNA and are thus suited for RT-PCR based mutation screening in that gene.

Pathological exon skipping in an HNPCC proband withMLH1 splice acceptor site mutation

One of the most commonly mutated mismatch repair genes in human nonpolyposis colorectal cancer (HNPCC) is MLH1. We identified a splice site mutation in MLH1 in a colorectal cancer proband (T-to-A at position -11 of intron 1 splice acceptor) and investigated its functional consequences by RT-PCR, using lymphocyte mRNA from the proband, two noncarrying siblings, and one unrelated individual. Subcloning of PCR products followed by sequencing of individual clones revealed increased transcript heterogeneity in the mutation carrier, attributable to the presence of a variety of mRNA forms lacking exon 2, or combinations of exons 2, 4, 6, 9, and 10. The full-length transcript subcloned from the mutation carrier was detected with a much reduced frequency, suggesting that only the wild-type allele produced functional MLH1 mRNA. The three noncarriers expressed some previously described transcripts and several novel variants, but none that lacked exon 2. The results are consistent with the hypothesis that this splice site mutation causes skipping of MLH1 exon 2 in a large proportion of mRNA transcripts derived from the mutated allele. Such an observation strengthens the case for identifying the mutation as pathogenic in this HNPCC family, which is of interest given the rarity of exon skipping defects resulting from splice acceptor site mutations outside the invariant AG dinucleotide.

Decreased expression of MLH1, MSH2, PMS1 and PMS2 in pigmented lesions indicates accumulation of failed DNA repair along with malignant transformation and tumour progression

The tumorigenesis of human nonpolyposis colorectal cancer was reported to be connected with the mutations in DNA mismatch repair genes. The main aim of this study was to check the epression of 4 proteins MLH1, MSH2, PMS1 and PMS2 responsible for mismatch DNA repair in naevi and melanomas. Fifty-one naevi, 78 primary melanomas, 30 lymphatic and 7 organ melanoma metastases were stained for the presence of MLH1, MSH2, PMS1 and PMS2. All proteins were preserved in 88% of naevi and only in 37% of primary melanomas, 17% of lymphatic metastases and in none of the distant metastases. The difference of expression of all 4 proteins between naevi and melanomas was highly significant (p<0.01). MLH1 and MSH2 correlated significantly with each other as well with the follow-up of patients. On the basis of our results one can conclude that the defect of mismatch DNA repair plays an important role in both tumorigenesis of melanoma and metastatic spread of tumour.