Elena Pagani

MicroRNA‐155 targets the SKI gene in human melanoma cell lines

The SKI protein is a transcriptional coregulator over‐expressed in melanoma. Experimentally induced down‐regulation of SKI inhibits melanoma cell growth in vitro and in vivo. MicroRNAs (miRNAs) negatively modulate gene expression and have been implicated in oncogenesis. We previously showed that microRNA‐155 (miR‐155) is down‐regulated in melanoma cells as compared with normal melanocytes and that its ectopic expression impairs proliferation and induces apoptosis. Here, we investigated whether miR‐155 could mediate melanoma growth inhibition via SKI gene silencing. Luciferase reporter assays demonstrated that miR‐155 interacted with SKI 3′UTR and impaired gene expression. Transfection of melanoma cells with miR‐155 reduced SKI levels, while inhibition of endogenous miR‐155 up‐regulated SKI expression. Specifically designed small interfering RNAs reduced SKI expression and inhibited proliferation. However, melanoma cells over‐expressing a 3′UTR‐deleted SKI were still susceptible to the antiproliferative effect of miR‐155. Our data demonstrate for the first time that SKI is a target of miR‐155 in melanoma. However, impairment of SKI expression is not the leading mechanism involved in the growth‐suppressive effect of miR‐155 found in this malignancy.

Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T-cell leukemia line tolerant to methylating agents.

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6‐alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6‐methylguanine (O6meG) induces cell death in OGAT‐deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T‐cell leukemic Jurkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSα, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15–21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA→TGA). In addition, a (C)8→(C)7 frameshift mutation in codon 1085–1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSα. Genes Chromosomes Cancer 23:159–166, 1998.

Concomitant activation of Wnt pathway and loss of mismatch repair function in human melanoma

Constitutive activation of the Wnt pathway plays a key role in the development of colorectal cancer and has also been implicated in the pathogenesis of other malignancies. Deregulation of Wnt signaling mainly occurs through genetic alterations of APC, the β‐catenin gene (CTNNB1), AXIN1 and AXIN2, leading to stabilization of β‐catenin. Physiologically, AXIN2 is transcriptionally induced on Wnt signaling activation and acts as a negative feedback regulator of the pathway. In colorectal cancer, mutations in CTNNB1 and AXIN2 occur preferentially in tumors with inactivation of the mismatch repair (MMR) genes MSH2, MLH1, or PMS2. In this study, the expression of β‐catenin and AXIN2, and the mutational status of CTNNB1, APC, and AXIN2 were evaluated in two MMR‐deficient (PR‐Mel and MR‐Mel) and seven MMR‐proficient human melanoma cell lines. Only PR‐Mel and MR‐Mel cells showed nuclear accumulation of β‐catenin and expression of the AXIN2 gene, and hence, constitutive activation of Wnt signaling. Mutational analysis identified a somatic heterozygous missense mutation in CTNNB1 exon three and a germline heterozygous deletion within AXIN2 exon seven in PR‐Mel cells, and a somatic biallelic deletion within APC in MR‐Mel cells. Deregulation of Wnt signaling and a defective MMR system were also present in the original tumor of PR and MR patients. Thus, we describe additional melanomas with mutations in CTNNB1 and APC, identify for the first time a germline AXIN2 mutation in a melanoma patient and suggest that inactivation of the MMR system and deregulation of the Wnt/β‐catenin signaling pathway cooperate to promote melanoma development and/or progression.

Biallelic somatic inactivation of the mismatch repair gene MLH1 in a primary skin melanoma

Inactivation of mismatch repair (MMR) genes has been linked to the hereditary nonpolyposis colon cancer syndrome and to a subset of sporadic cancers. A phenotypic characteristic of tumors with defective MMR is microsatellite instability (MSI). Although MSI has been reported in a proportion of cutaneous melanomas, inactivation of MMR genes in this tumor type has not been detected thus far. We recently described a human melanoma cell line, PR‐Mel, and a cutaneous metastasis from the same patient, which displayed a MMR defect, and showed high MSI. Here we report that in the PR‐Mel cell line both MLH1 alleles are somatically inactivated. One allele is lost through a chromosomal deletion of the region 3p21–24, whereas the remaining allele harbors a G → A transition at position −1 of the acceptor splice site of intron 15, leading to the in‐frame skipping of exon 16. The primary melanoma of the PR patient shows loss of heterozygosity at the BAT21 microsatellite marker, located in the MLH1 gene, and does not express the MLH1 and PMS2 proteins. Moreover, it harbors the same mutation detected in the PR‐Mel cells. These results demonstrate that biallelic inactivation of MLH1 had occurred in the primary melanoma of the PR patient and suggest that disruption of MMR might have had a role in the development of the melanoma. This is the first report in which genetic defects leading to disruption of MMR function in a human melanoma have been identified.

DNA repair enzymes and cytotoxic effects of temozolomide: Comparative studies between tumor cells and normal cells of the immune system

Abstract O6-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair system (MRS) play a crucial role in the susceptibility of tumor cells to the cytotoxic effects of agents that generate O6-methylguanine in DNA, including the triazene compound temozolomide (TMZ). Studies performed with peripheral blood mononuclear cells (MNC) showed that TMZ was scarcely active on lymphocyte functions not dependent on cell proliferation (e.g. NK activity and cytokine-mediated induction of CD1b molecule in adherent MNC). In contrast, TMZ depressed proliferation and lymphokine activated killer (LAK) cell generation in response to IL-2. In this case, a reasonably good inverse relationship was found between OGAT levels of MNC and their susceptibility to TMZ. This study also analyzed the ratio of the toxic effect of TMZ on MNC and on tumor cells (i.e. “Tumor-Immune Function Toxicity Index”, TIFTI). A particularly favorable TIFTI can be obtained when OGAT levels are extremely high in MNC and markedly low in tumor cells. This holds true for MRS-proficient neo-plastic cells, but not for MRS-deficient tumors. In conclusion, strategies aimed at modulating OGAT and MRS may improve the clinical response to TMZ. However, the use of OGAT inhibitors to potentiate the antitumor activity of TMZ might result in a concomitant increase of the immunosuppressive effects of the drug, thus reducing the relative TIFTI.

Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro.

Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, we tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 µM orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis.

Abstract 150: Regulation of TGFB receptor by miR21 in Sezary syndrome

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Sezary syndrome (SS) represents a very aggressive form of Cutaneous T Cell Lymphomas with a median overall survival of 5.1 years (range, 0.4-18.6 years). As it appears evident there are in this disease either short survivors than long survivors. Previous studies of prognostic indicators in SS showed that circulating Sezary cell count, high CD4/CD8 ratio, advanced age, high lactate dehydrogenase serum level and a high white blood cell count were associated with an unfavorable outcome. Up to now, few data have been provided concerning possible associations between immunological and genetic markers, who might provide also clues on tumor progression in this disease. In the last 10 years we have observed more than 50 Sezary Syndromes in our Institute and phenotyped them with new immunologic markers. We have also performed genetic analysis with Single Nucleotide polymorphysms (SNPs) for evaluation of genomic imbalances, mRNA expression and lately microRNA(miRNA) expression profiling. In this study we have analyzed the expression profile of 470 miRNAs using Agilent platform array in 22 SS patients. We investigated the relationship between the expression level of miRNAs and the clinical outcome of SS patients by Kaplan-Meier method and risk assessment by multivariate analysis. We also functionally investigated the role of miR-21, mapping in one of the region more frequently amplified in SS and some of its targets. We identified 45 miRNAs differentially expressed between SS and healthy controls. Using predictive analysis, a list of 19 miRNAs, including miR-21 and miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs able to discriminate patients with unfavorable and favorable outcome. We show that miR-21 knockdown increases apoptosis and modulates TGF beta receptor 2 expression in vitro. The antipoptotic effect appears to be regulated through PTEN. Conversely, we were not able to observe a direct miR-21 regulation on PDCD4 gene mapping to chromosome 10q24, a frequently imbalanced region in SS. In conclusion we have identified a new prognostic miRNAs signature in SS and characterized the role of miR-21, one of the most involved in cancer, recognized as a disease and prognostic classifier in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 150. doi:10.1158/1538-7445.AM2011-150