Origin, prevalence and response to therapy of hepatitis C virus genotype 2k/1b chimeras.

BACKGROUND & AIMS: Little is known about the epidemiology and frequency of recombinant HCV genotype 2/1 strains, which may represent a challenge for direct antiviral therapy (DAA). This study aims to identify the epidemiology and phylogeny of HCV genotype 2/1 strains and encourages genotype screening, to select the DAA-regimen that achieves the optimal sustained virologic response. METHODS: Consecutive samples from HCV genotype 2 infected patients, according to commercial genotyping, from Germany, Italy and Israel were re-genotyped by Sanger-based sequencing. Virologic, epidemiological, and phylogenetic analyses including other published chimeras were performed. RESULTS: Sequence analysis of 442 supposed HCV genotype 2 isolates revealed 61 (genotype 2k/1b (n=59), 2a/1b (n=1) or 2b/1a (n=1)) chimeras. No chimeras were observed in Italy, but the frequency was 14% and 25% in Germany and Israel. Treatment of viral chimera with sofosbuvir/ribavirin led to virologic relapse in 25/27 patients (93%). Nearly all patients treated with genotype 1-based DAA-regimens initially (n=8/9), or after relapse (n=13/13), achieved a sustained virologic response. Most patients with 2k/1b chimeras (88%) were originally from eight different areas of the former Soviet Union. All known 2k/1b chimeras harbour the same recombination breakpoint and build one phylogenetic cluster, while all other chimeras have different phylogenies. CONCLUSIONS: The HCV genotype 2k/1b variant derives from one single recombination event most likely in the former Soviet Union, while other chimeras are unique and develop independently. A relatively high frequency has been observed along the migration flows, in Germany and Israel. In countries with little migration from the former Soviet Union the prevalence of 2k/1b chimeras is expected to be low. Treatment with sofosbuvir plus ribavirin is insufficient, but genotype 1-based regimens seem to be effective. Lay summary: The frequency of recombinant HCV is higher than expected. A novel recombinant variant (HCV genotype 2a/1b) was identified. Screening for recombinant viruses would contribute to increased response rates to direct antiviral therapy.

Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1L749P and MLH1Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα.

Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1.

INTRODUCTION: Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated. METHODS: Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively. RESULTS: MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability. CONCLUSIONS: These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1.

A CRM1-dependent nuclear export pathway is involved in the regulation of MutLα subcellular localization.

MutLα plays an essential role in DNA mismatch repair (MMR) and is additionally involved in other cellular mechanisms such as the regulation of cell cycle checkpoints and apoptosis. Therefore, not only germline MMR gene defects but also the subcellular localization of MutLα might be of importance for the development of Lynch syndrome. Recently, we showed that MutLα contains functional nuclear import sequences and is most frequently localized in the nucleus. Here, we demonstrate that MutLα can move bidirectionally towards the nuclear membrane. Using MutLα transfected HEK293T cells we observed a significant shift of MLH1 and PMS2 from the nucleus to the cytoplasm after irradiation or cisplatin treatment. We analyzed both proteins for potential nuclear export sequences (NES) and identified one functional Rev-type NES (578LFDLAMLAL) in the C-terminal part of MLH1 that facilitates export via the CRM1/exportin pathway. Moreover, an MLH1-NES mutation detected in a patient with Lynch syndrome showed normal MMR activity but led to significantly impaired cytoplasmic transport after actinomycin D treatment. These results indicate that MutLα is able to shuttle from the nucleus to the cytoplasm, probably signaling DNA damages to downstream pathways. In conclusion, not only a defective MMR but also impaired nucleo-cytoplasmic shuttling might result in the onset of Lynch syndrome.

Cytoskeletal scaffolding proteins interact with Lynch-Syndrome associated mismatch repair protein MLH1.

The involvement of MLH1 in several mismatch repair-independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two-hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the "General Repository for Interaction Datasets" highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin beta 4 and Actin gamma, Cathepsin B and Annexin A2 as well as Spectrin alpha and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1-deficient and -proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1-Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.

An interstitial deletion at 3p21.3 results in the genetic fusion of MLH1 and ITGA9 in a Lynch syndrome family.

PURPOSE: Germline mutations in DNA mismatch repair genes, mainly MLH1 or MSH2, have been shown to predispose with high penetrance for the development of the clinical phenotype of hereditary nonpolyposis colorectal cancer (Lynch syndrome). Here, we describe the discovery and first functional characterization of a novel germline MLH1 mutant allele. EXPERIMENTAL DESIGN: A large kindred including 54 potential carriers was investigated at the molecular level by using different types of PCR experiments, gene cloning, transfection studies, Western blot experiments, and mismatch repair assays to identify and characterize a novel MLH1 mutant allele. Twenty-two of 54 putative carriers developed colon cancer or other tumors, including breast cancer. RESULTS: The identified MLH1 mutant allele emerged from an interstitial deletion on chromosome 3p21.3, leading to an in-frame fusion of MLH1 (exons 1-11) with ITGA9 (integrin alpha 9; exons 17-28). The deleted area has a size of about 400 kb; codes for LRRFIP2 (leucine-rich repeat in flightless interaction protein 2), GOLGA4 (Golgi autoantigen, golgin subfamily a, 4), and C3orf35/APRG1 (chromosome 3 open reading frame 35/AP20 region protein 1); and partly disrupts the AP20 region implicated in major epithelial malignancies. Tumor cells lost their second MLH1 allele. The MLH1*ITGA9 fusion protein provides no capability for DNA mismatch repair. Murine fibroblasts, expressing a doxycycline-inducible MLH1*ITGA9 fusion gene, exhibit a loss-of-contact inhibition phenotype. CONCLUSIONS: This is the first description of a functional gene fusion of the human MLH1 gene, resulting in the loss of mismatch repair capabilities. The MLH1*ITGA9 fusion allele, together with deletions of the AP20 region, presumably defines a novel subclass of Lynch syndrome patients, which results in an extended tumor spectrum known from hereditary nonpolyposis colorectal cancer and Muir-Torre syndrome patients.

Promoter methylation of MLH1, PMS2, MSH2 and p16 is a phenomenon of advanced-stage HCCs.

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future.

C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.