Characterization of MTAP Gene Expression in Breast Cancer Patients and Cell Lines

MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors´ MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNBC.     

Diaphyseal medullary stenosis with malignant fibrous histiocytoma: a hereditary bone dysplasia/cancer syndrome maps to 9p21-22

Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal dominant bone dysplasia/cancer syndrome of unknown etiology. This rare hereditary cancer syndrome is characterized by bone infarctions, cortical growth abnormalities, pathological fractures, and eventual painful debilitation. Notably, 35% of individuals with DMS develop MFH, a highly malignant bone sarcoma. A genome scan for the DMS-MFH gene locus in three unrelated families with DMS-MFH linked the syndrome to a region of approximately 3 cM on chromosome 9p21-22, with a maximal two-point LOD score of 5.49 (marker D9S171 at recombination fraction [theta].05). Interestingly, this region had previously been shown to be the site of chromosomal abnormalities in several other malignancies and contains a number of genes whose protein products are involved in growth regulation. Identification of this rare familial sarcoma-causing gene would be expected to simultaneously define the cause of the more common nonfamilial, or sporadic, form of MFH-a tumor that constitutes approximately 6% of all bone cancers and is the most frequently occurring adult soft-tissue sarcoma.     

PRMT5 silencing selectively affects MTAP-deleted mesothelioma: In vitro evidence of a novel promising approach

Malignant mesothelioma (MM) is an aggressive asbestos-related cancer of the serous membranes. Despite intensive treatment regimens, MM is still a fatal disease, mainly due to the intrinsic resistance to current therapies and the lack of predictive markers and new valuable molecular targets. Protein arginine methyltransferase 5 (PRMT5) inhibition has recently emerged as a potential therapy against methylthioadenosine phosphorylase (MTAP)-deficient cancers, in which the accumulation of the substrate 5'-methylthioadenosine (MTA) inhibits PRMT5 activity, thus sensitizing the cells to further PRMT5 inhibition. Considering that the MTAP gene is frequently codeleted with the adjacent cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in MM, we assessed whether PRMT5 could represent a therapeutic target also for this cancer type. We evaluated PRMT5 expression, the MTAP status and MTA content in normal mesothelial and MM cell lines. We found that both administration of exogenous MTA and stable PRMT5 knock-down, by short hairpin RNAs (shRNAs), selectively reduced the growth of MTAP-deleted MM cells. We also observed that PRMT5 knock-down in MTAP-deficient MM cells reduced the expression of E2F1 target genes involved in cell cycle progression and of factors implicated in epithelial-to-mesenchymal transition. Therefore, PRMT5 targeting could represent a promising new therapeutic strategy against MTAP-deleted MMs.     

Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion- and epilepsy-associated brain tissue

The chloride channel 2 (CLCN2) gene codes for a protein organized in N- and C-terminal regions with regulatory functions and a transmembrane region which forms the ring of the pore. Mutations in the gene have previously been described in patients with idiopathic familial epilepsy. In this study we looked for new isoforms of CLCN2 and we estimated expression levels by real time PCR in brain tissue containing epileptic foci. Samples used in this study were first analyzed and selected to exclude mutations in the coding region of the gene. Four isoforms (skipping exons 3, 16, 22 and 6/7) were identified and quantified by Real Time PCR and compared with total expression of the gene. Expression of the region common to all CLCN2 isoforms was 50% less in epilepsy-associated brain tissue than in controls. The ratio of the various isoforms was slightly greater in epileptic than control tissue. The greatest difference was recorded in the temporal lobe for the isoform with skipped exon 22. Analysis of these isoforms in brain tissue containing epileptic foci suggests that CLCN2 could be implicated in epilepsy, even in the absence of mutations.     

Primary structure and developmental acidic to basic transition of 13 alternatively spliced mouse fast skeletal muscle troponin T isoforms

Large samples of original cDNAs encoding neonatal and adult mouse fast skeletal muscle troponin T (fTnT) have been isolated and characterized. The results demonstrate expression relationships of 8 alternatively spliced exons of the fTnT gene and reveal the primary structure of as many as 13 fTnT isoforms that diverge into acidic and basic classes due to differential mRNA splicing in the N-terminal variable region. In the C-terminal variable region encoded by the mutually exclusive exons 16 and 17, the splicing pathway and structure of exon 16 appears to be adult fTnT-specific, suggesting an adaptation to the functional demands of mature fast skeletal muscle. The cloned cDNAs were expressed in E. coli as standards to identify a high M_r to low M_r, acidic to basic fTnT isoform transition in postnatal developing skeletal muscles. Different from the developmental cardiac TnT switch generated by alternative splicing of a single exon, the fTnT isoform transition is an additive effect of alternative splicing of multiple N-terminal-coding exons, especially exons 4, 8 and fetal that are expressed at higher frequencies in the neonatal than in the adult muscle. The developmental fTnT isoform primary structure transition in both N- and C-terminal variable regions suggest a physiological importance of the apparently complex TnT isoform expression.     

Exploring a Local Genetic Interaction Network Using Evolutionary Replay Experiments

Understanding how genes interact is a central challenge in biology. Experimental evolution provides a useful, but underutilized, tool for identifying genetic interactions, particularly those that involve non-loss-of-function mutations or mutations in essential genes. We previously identified a strong positive genetic interaction between specific mutations in KEL1 (P344T) and HSL7 (A695fs) that arose in an experimentally evolved Saccharomyces cerevisiae population. Because this genetic interaction is not phenocopied by gene deletion, it was previously unknown. Using “evolutionary replay” experiments, we identified additional mutations that have positive genetic interactions with the kel1-P344T mutation. We replayed the evolution of this population 672 times from six timepoints. We identified 30 populations where the kel1-P344T mutation reached high frequency. We performed whole-genome sequencing on these populations to identify genes in which mutations arose specifically in the kel1-P344T background. We reconstructed mutations in the ancestral and kel1-P344T backgrounds to validate positive genetic interactions. We identify several genetic interactors with KEL1, we validate these interactions by reconstruction experiments, and we show these interactions are not recapitulated by loss-of-function mutations. Our results demonstrate the power of experimental evolution to identify genetic interactions that are positive, allele specific, and not readily detected by other methods, shedding light on an underexplored region of the yeast genetic interaction network.     

Differential expression of CD45 isoforms is controlled by the combined activity of basal and inducible splicing-regulatory elements in each of the variable exons

The human CD45 gene encodes five isoforms of a transmembrane tyrosine phosphatase that differ in their extracellular domains as a result of alternative splicing of exons 4-6. Expression of the CD45 isoforms is tightly regulated in peripheral T cells such that resting cells predominantly express the larger CD45 isoforms, encoded by mRNAs containing two or three variable exons. In contrast, activated T cells express CD45 isoforms encoded by mRNAs lacking most or all of the variable exons. We have previously identified the sequences within CD45 variable exon 4 that control its level of inclusion into spliced mRNAs. Here we map the splicingregulatory sequences within CD45 variable exons 5 and 6. We show that, like exon 4, exons 5 and 6 each contain an exonic splicing silencer (ESS) and an exonic splicing enhancer (ESE), which together determine the level of exon inclusion in na?ve cells. We further demonstrate that the primary activation-responsive silencing motif in exons 5 and 6 is homologous to that in exon 4 and, as in exon 4, binds specifically to the protein heterogeneous nuclear ribonucleoprotein L. Together these studies reveal common themes in the regulation of the CD45 variable exons and provide a mechanistic explanation for the observed physiological expression of CD45 isoforms.     

MECP2 genomic structure and function: insights from ENCODE

MECP2, a relatively small gene located in the human X chromosome, was initially described with three exons transcribing RNA from which the protein MeCP2 was translated. It is now known to have four exons from which two isoforms are translated; however, there is also evidence of additional functional genomic structures within MECP2, including exons potentially transcribing non-coding RNAs. Accompanying the recognition of a higher level of intricacy within MECP2 has been a recent surge of knowledge about the structure and function of human genes more generally, to the extent that the definition of a gene is being revisited. It is timely now to review the published and novel functional elements within MECP2, which is proving to have a complexity far greater than was previously thought.