Beta-catenin inhibits melanocyte migration but induces melanoma metastasis.

The canonical Wnt signalling pathway induces the ß-catenin/lymphoid enhancer factor transcription factors. It is activated in various cancers, most characteristically carcinomas, in which it promotes metastatic spread by increasing migration and/or invasion. The Wnt/ß-catenin signalling pathway is frequently activated in melanoma, but the presence of ß-catenin in the nucleus does not seem to be a sign of aggressiveness in these tumours. We found that, unlike its positive role in stimulating migration and invasion of carcinoma cells, ß-catenin signalling decreased the migration of melanocytes and melanoma cell lines. In vivo, ß-catenin signalling in melanoblasts reduced the migration of these cells, causing a white belly-spot phenotype. The inhibition by ß-catenin of migration was dependent on MITF-M, a key transcription factor of the melanocyte lineage, and CSK, an Src-inhibitor. Despite reducing migration, ß-catenin signalling promoted lung metastasis in the NRAS-driven melanoma murine model. Thus, ß-catenin may have conflicting roles in the metastatic spread of melanoma, repressing migration while promoting metastasis. These results highlight that metastasis formation requires a series of successful cellular processes, any one of which may not be optimally efficient.

BCR-ABL1 kinase inhibits uracil DNA glycosylase UNG2 to enhance oxidative DNA damage and stimulate genomic instability.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50-90% of patients developing TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species, which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34(+) CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na(+)/K(+)ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.

Brain atrophy evolution and lesion load accrual in multiple sclerosis: a 2-year follow-up study.

BACKGROUND: To investigate in a large cohort of patients with multiple sclerosis (MS), lesion load and atrophy evolution, and the relationship between clinical and magnetic resonance imaging (MRI) correlates of disease progression. METHODS: Two hundred and sixty-seven patients with MS were studied at baseline and two years later using the same MRI protocol. Abnormal white matter fraction, normal appearing white matter fraction, global white matter fraction, gray matter fraction and whole brain fraction, T2-hyperintense, and T1-hypointense lesions were measured at both time points. RESULTS: The majority of patients were clinically stable, whereas MRI-derived brain tissue fractions were significantly different after 2 years. The correlation between MRI data at baseline and their variation during the follow-up showed that lower basal gray matter atrophy was significantly related with higher progression of gray matter atrophy during follow-up. The correlation between MRI parameters and disease duration showed that gray matter atrophy rate decreased with increasing disease duration, whereas the rate of white matter atrophy had a constant pattern. Lower basal gray matter atrophy was associated with increased probability of developing gray matter atrophy at follow-up, whereas gray matter atrophy progression over 2 years and new T2 lesion load were risk factors for whole brain atrophy progression. CONCLUSIONS: In MS, brain atrophy occurs even after a relatively short period of time and in patients with limited progression of disability. Short-term brain atrophy progression rates differ across tissue compartments, as gray matter atrophy results more pronounced than white matter atrophy and appears to be a early phenomenon in the MS-related disease progression.

Activation of NF-kappaB by Akt upregulates Snail expression and induces epithelium mesenchyme transition.

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of mesenchymal characteristics and invasive properties by tumour cells. The loss of cell-cell contacts may be the first step of the epithelium mesenchyme transition (EMT) and involves the functional inactivation of the cell-cell adhesion molecule E-cadherin. Repression of E-cadherin expression by the transcription factor Snail is a central event during the loss of epithelial phenotype. Akt kinase activation is frequent in human carcinomas, and Akt regulates various cellular mechanisms including EMT. Here, we show that Snail activation and consequent repression of E-cadherin may depend on AKT-mediated nuclear factor-kappaB (NF-kappaB) activation, and that NF-kappaB induces Snail expression. Expression of the NF-kappaB subunit p65 is sufficient for EMT induction, validating this signalling module during EMT. NF-kappaB pathway activation is associated with tumour progression and metastasis of several human tumour types; E-cadherin acts as a metastasis suppressor protein. Thus, this signalling and transcriptional network linking AKT, NF-kappaB, Snail and E-cadherin during EMT is a potential target for antimetastatic therapeutics.

Differential stromal and epithelial localization of cyclooxygenase-2 (COX-2) during colorectal tumorigenesis.

The purpose of the following study is to describe the localization of COX-2 protein and COX-2 mRNA during human colorectal tumorigenesis and to identify potential cellular targets for COX-2 inhibition in chemopreventive strategies. Immunohistochemistry with digital image analysis was used to determine COX-2 protein expression in histologic sections containing synchronous normal colorectal mucosa, adenomas and carcinomas, from 17 previously untreated patients. Epithelial and stromal COX-2 mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), on laser-capture microdissected samples from the same histologies. The stromal compartment in normal colorectal mucosa and adenomas showed higher levels of COX-2 protein expression compared to colorectal carcinomas (p < .0001). Conversely, epithelial COX-2 protein was significantly increased only after development of the invasive phenotype (p < .0001). RT-PCR demonstrated higher stromal COX-2 mRNA expression compared to that within the epithelium for colorectal adenomas and carcinomas. In conclusion, stromal COX-2 may be the target for chemopreventive agents in the early stages of colorectal carcinogenesis.

Brain atrophy and lesion load in a large population of patients with multiple sclerosis.

OBJECTIVE: To measure white matter (WM) and gray matter (GM) atrophy and lesion load in a large population of patients with multiple sclerosis (MS) using a fully automated, operator-independent, multiparametric segmentation method. METHODS: The study population consisted of 597 patients with MS and 104 control subjects. The MRI parameters were abnormal WM fraction (AWM-f), global WM-f (gWM-f), and GM fraction (GM-f). RESULTS: Significant differences between patients with MS and control subjects included higher AWM-f and reduced gWM-f and GM-f. MRI data showed significant differences between patients with relapsing-remitting and secondary progressive forms of MS. Significant correlations between MRI parameters and between MRI and clinical data were found. CONCLUSIONS: Patients with multiple sclerosis have significant atrophy of both white matter (WM) and gray matter (GM); secondary progressive patients have significantly more atrophy of both WM and GM than do relapsing-remitting patients and a significantly higher lesion load (abnormal WM fraction); lesion load is related to both WM and even more to GM atrophy; lesion load and WM and GM atrophy are significantly related to Expanded Disability Status Scale score and age at onset (suggesting that the younger the age at disease onset, the worse the lesion load and brain atrophy); and GM atrophy is the most significant MRI variable in determining the final disability.

Atypical forms of multiple sclerosis or different phases of a same disease?

Multiple sclerosis (MS) patients complain with the first symptoms of the disease in a range period which varies from childhood to adult life. The extent to which clinical presentation, disease course and demographic features may differ between childhood and adult onset has been the object of investigation. This paper aims to demonstrate that the different clinical phenotypes in young and old patients might simply reflect different phases of a same pathological process.

The transition from relapsing-remitting MS to irreversible disability: clinical evaluation.

The development of a progressive course is by far the most deleterious event in the case of a multiple sclerosis (MS) patient. It occurs in about 90% of relapsing remitting patients by 20-25 years from onset. The clinical transition to secondary progressive MS is phenotypically distinctive and both patients and physicians acknowledge that a fundamental shift in the degree of responsiveness to anti-inflammatory therapies has occurred. This review discusses the clinical studies that provided important findings relating to our understanding on different mechanisms that may contribute to irreversible disability in MS.

Course and prognosis in early-onset MS: comparison with adult-onset forms.

OBJECTIVES: To establish the prognostic role of clinical and demographic factors in a hospital-based cohort of MS patients categorized by age at clinical onset and clinical course. METHODS: Eighty-three patients with MS had a clinical onset of the disease in childhood (age <16 years; early-onset MS [EOMS]) and 710 in adult age (between 16 and 65 years; adult-onset MS [AOMS]). Patients were followed for a mean period of observation of 5 years. Univariate and multivariate analyses of clinical and demographic predictors for rapid progression and disability were performed using a stepwise Cox regression model with time-dependent covariates. RESULTS: In EOMS, the Expanded Disability Status Scale (EDSS) evaluated at last clinical examination was lower than in AOMS, despite a longer disease duration. The probability to reach growth disability and progression was significantly lower in EOMS than in AOMS. Median times to reach EDSS score of 4 and secondary progression were longer in EOMS than in AOMS, but the age at both endpoints was significantly lower in EOMS. In EOMS and AOMS, an irreversible disability was related to a secondary progressive course, a sphincteric system involvement at onset, and an older age at onset (in EOMS only for the group >14 years); in AOMS, other unfavorable factors were a pyramidal involvement at onset and a high relapse frequency in the first 2 years. The risk of entering secondary progression was significantly influenced by a high number of relapses in EOMS and by a higher age at onset and a short interattack interval in AOMS. CONCLUSION: A slower rate of progression of disease characterized EOMS patients, suggesting more plasticity to recover in developing CNS, but the early clinical manifestation cannot be considered a positive prognostic factor.

Functional interactions and signaling properties of mammalian DNA mismatch repair proteins.

The mismatch repair (MMR) system promotes genomic fidelity by repairing base-base mismatches, insertion-deletion loops and heterologies generated during DNA replication and recombination. This function is critically dependent on the assembling of multimeric complexes involved in mismatch recognition and signal transduction to downstream repair events. In addition, MMR proteins coordinate a complex network of physical and functional interactions that mediate other DNA transactions, such as transcription-coupled repair, base excision repair and recombination. MMR proteins are also involved in activation of cell cycle checkpoint and induction of apoptosis when DNA damage overwhelms a critical threshold. For this reason, they play a role in cell death by alkylating agents and other chemotherapeutic drugs, including cisplatin. Inactivation of MMR genes in hereditary and sporadic cancer is associated with a mutator phenotype and inhibition of apoptosis. In the future, a deeper understanding of the molecular mechanisms and functional interactions of MMR proteins will lead to the development of more effective cancer prevention and treatment strategies.

Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites.

Whereas in Escherichia coli DNA mismatch repair is directed to the newly synthesized strand due to its transient lack of adenine methylation, the molecular determinants of strand discrimination in eukaryotes are presently unknown. In mammalian cells, cytosine methylation within CpG sites may represent an analogous and mechanistically plausible means of targeting mismatch correction. Using HeLa nuclear extracts, we conducted a systematic analysis in vitro to determine whether cytosine methylation participates in human DNA mismatch repair. We prepared a set of A.C heteroduplex molecules that were either unmethylated, hemimethylated or fully methylated at CpG sequences and found that the methylation status persisted under the assay conditions. However, no effect on either the time course or the magnitude of mismatch repair events was evident; only strand discontinuities contributed to strand bias. By western analysis we demonstrated that the HeLa extract contained MED1 protein, which interacts with MLH1 and binds to CpG-methylated DNA; supplementation with purified MED1 protein was without effect. In summary, human DNA mismatch repair operates independently of CpG methylation status, and we found no evidence supporting a role for CpG hemimethylation as a strand discrimination signal.

Role of MED1 (MBD4) Gene in DNA repair and human cancer.

The human protein MED1, also known as MBD4, was isolated in a yeast two-hybrid screening as an interactor of the mismatch repair protein MLH1. MED1 contains an N-terminal 5-methylcytosine binding domain (MBD), which allows binding to methylated DNA, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. This suggests that DNA methylation may play a role in human DNA repair. MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, 5-fluorouracil and, weakly, 3,N(4)-ethenocytosine paired with guanine. The glycosylase activity of MED1 prefers substrates in which the G:T mismatch is present in the context of methylated or unmethylated CpG sites. Since G:T mismatches can originate via spontaneous deamination of 5-methylcytosine to thymine, MED1 appears to act as a caretaker of genomic fidelity at CpG sites. Mutagenesis caused by these deamination events is a frequent mechanism of genetic instability in cancer; thus, based on the biochemical activity of its gene product, MED1 is a candidate tumor suppressor gene. Indeed, frameshift mutations of the MED1 gene have been reported in human colorectal, gastric, endometrial, and pancreatic cancer. In the future, efforts should be directed toward investigations of the functional role of the MED1 gene in the pathogenesis, prevention, and treatment of human cancer.

Microsatellite instability is an independent indicator of recurrence in sporadic stage I-II endometrial adenocarcinoma.

PURPOSE: The aim of this study was to define the prognostic role of microsatellite status in 65 stage I-II primary sporadic endometrioid endometrial adenocarcinoma (EEA) patients. PATIENTS AND METHODS: Familiarity for neoplasia was ascertained in all patients on the basis of a questionnaire. Microsatellite status was assessed by matching normal and tumoral DNA probed for five dinucleotide repeats and one mononucleotide repeat marker. Microsatellite status was analyzed in relation to clinicopathologic characteristics of the patients and length of disease-free survival (DFS). RESULTS: Eleven tumors (17%) of 65 had instability at two or more loci and were considered as unstable or microsatellite instability (MI). Tumors with no instability or instability at one locus were classified as microsatellite stable (MS). The percentage of MI was significantly higher in poorly than in well to moderately differentiated tumors (50% v 9%; P =.003). The 5-year DFS rate of MI patients was 63% (95% confidence interval [CI], 35% to 91%) versus 96% (95% CI, 91% to 101%) of MS patients (P =.0004). In multivariate analysis, only the presence of MI, stage II of disease, and depth of myometrial invasion greater than 50% retained independent prognostic roles. CONCLUSION: The assessment of microsatellite status may provide useful information for preoperative prognostic characterization of stage I-II primary sporadic EEA patients in which more individualized treatment options can be attempted.

Axonal damage in multiple sclerosis plaques: a combined magnetic resonance imaging and 1H-magnetic resonance spectroscopy study.

The purpose of this study was to compare magnetic resonance imaging (MRI) features and proton MR spectroscopy (1H-MRS) patterns of multiple sclerosis (MS) plaques in order to define the metabolic substrate in different lesion subtypes. Combined MRI and single-voxel 1H-MRS investigation was performed in 54 MS patients (47 relapsing remitting (RR) and seven secondary progressive (SP)). Sixty-seven MS lesions were selected. Thirty-seven lesions were Gadolinium (Gd) enhancing (nine isointense and 28 hypointense on pre-contrast T(1)-weighted scans) and 30 Gd unenhancing (six isointense and 24 hypointense on pre- and post-contrast T(1)-weighted scans). Choline (Cho), creatine (Cr), N-acetyl aspartate (NAA) and lactate were evaluated in 1H spectra acquired from MS plaques and from normal white matter (NWM) of 22 neurological controls. MS lesions of RR patients were characterized by a significant increase of Cho/Cr and decrease of NAA/Cr and NAA/Cho ratios. No significant metabolite changes were found in lesions of SP patients. Gd enhancing plaques showed lactate signal with higher frequency (37.8%) than Gd unenhancing plaques (16.7%) (p=0.04). A significant increase of Cho/Cr was found in Gd enhancing lesions when compared to controls (p<0.01), and to Gd unenhancing lesions (p<0.05). In particular, there was evidence of a significant increase of Cho/Cr in pre-contrast T(1) hypointense Gd enhancing lesions (p<0.01 vs. controls). The Gd unenhancing lesions (p<0.01), in particular the T(1) hypointense group (p<0.05), showed a significant decrease of NAA/Cr only when compared to controls. These data confirm that in vivo MRS indicates key pathological features of MS plaques. The increased Cho/Cr ratio found in Gd-enhancing plaques, in particular in the T(1) hypointense lesions, may reflect increased membrane cell turnover. The T(1) hypointense Gd unenhancing plaques better reflect axonal damage, as suggested by the decrease of NAA/Cr. Nevertheless, the lack of statistical differences in NAA/Cr between plaque subgroups suggests that axonal impairment might occur even in the early stages.

Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways.

Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase encoded by the c-Met protooncogene. HGF/Met signaling has multifunctional effects on various cell types. We sought to determine the role of HGF/Met in apoptosis and identify signal transducers involved in this process. In experiments with human SK-LMS-1 leiomyosarcoma cells, we show that the Akt kinase is activated by HGF in a time- and dose-dependent manner by phosphatidylinositol 3-kinase (PI3-kinase). Akt is also activated by active tumorigenic forms of Met, i.e., ligand-independent Tpr-Met, a truncated and constitutively dimerized form of Met, and a mutationally activated version of Met corresponding to that found in human hereditary papillary renal carcinoma. In NIH 3T3 cells transfected with wild-type Met, HGF inhibits apoptosis induced by serum starvation and UV irradiation. HGF-induced survival correlates with Akt activity and is inhibited by the specific PI3-kinase inhibitor LY294002, indicating that HGF inhibits cell death through the PI3-kinase/Akt signal transduction pathway. Furthermore, transiently transfected Tpr-Met activates Akt (both Akt1 and Akt2) and protects cells from apoptosis. Mitogen-activated protein kinase (MAPK) also is activated by HGF and rescues cells from apoptosis, although the cytoprotective effect is less marked than for PI3-kinase/Akt. Blocking MAPK with the specific MAPK kinase inhibitor PD098059 impairs the ability of HGF to promote cell survival. Similar results were obtained with NIH 3T3 cells expressing the fusion protein Trk-Met and stimulated with nerve growth factor, the Trk ligand. These results demonstrate that HGF/Met is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways.

Mutations of the 'minor' mismatch repair gene MSH6 in typical and atypical hereditary nonpolyposis colorectal cancer.

Mutations of the mismatch repair (MMR) genes MLH1 and MSH2 are associated with hereditary nonpolyposis colorectal cancer (HNPCC), a highly penetrant autosomal dominant condition characterized by hypermutability of short tandemly repeated sequences in tumor DNA. Mutations of another MMR gene, MSH6, seem to be less common than MLH1 and MSH2 defects, and have been mostly observed in atypical HNPCC families, characterized by a weaker tumor family history, higher age at disease onset, and low degrees of microsatellite instability (MSI), predominantly involving mononucleotide runs. We have investigated the MSH6 gene sequence in the peripheral blood of 4 HNPCC and 20 atypical HNPCC probands. Two frameshift mutations within exon 4 were detected in 2 patients. One mutation was found in a proband from a typical HNPCC family, who had developed a colorectal cancer (CRC), a gastric cancer and a rectal adenoma. The CRC and the adenoma showed mild MSI limited to mononucleotide tracts, while the gastric carcinoma was microsatellite stable. The other mutation was detected in an atypical HNPCC proband, whose CRC showed widespread MSI involving both mono- and dinucleotide repeats. The phenotypic variability associated with MSH6 constitutional mutations represents a complicating factor for the optimization of strategies aimed at identifying candidates to MSH6 genetic testing.

Investigation of the substrate spectrum of the human mismatch-specific DNA N-glycosylase MED1 (MBD4): fundamental role of the catalytic domain.

The human DNA repair protein MED1 (also known as MBD4) was isolated as an interactor of the mismatch repair protein MLH1 in a yeast two-hybrid screening. MED1 has a tripartite structure with an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. Indeed, MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine. The glycosylase activity of MED1 preferentially targets G:T mismatches in the context of CpG sites; this indicates that MED1 is involved in the repair of deaminated 5-methylcytosine. Interestingly, frameshift mutations of the MED1 gene have been reported in human colorectal, endometrial, and pancreatic cancers. For its putative role in maintaining genomic fidelity at CpG sites, it is important to characterize the biochemical properties and the substrate spectrum of MED1. Here we show that MED1 works under a wide range of temperature and pH, and has a limited optimum range of ionic strength. MED1 has a weak glycosylase activity on the mutagenic adduct 3,N(4)-ethenocytosine, a metabolite of vinyl chloride and ethyl carbamate. The differences in glycosylase activity on G:U and G:T substrates are not related to differences in substrate binding and likely result from intrinsic differences in the chemical step. Finally, the isolated catalytic domain of MED1 retains the preference for G:T and G:U substrates in the context of methylated or unmethylated CpG sites. This suggests that the catalytic domain is fundamental, and the 5-methylcytosine binding domain dispensable, in determining the substrate spectrum of MED1.

Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase.

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.