CD44 Modulates Cell Migration and Invasion in Ewing Sarcoma Cells.

The chimeric EWSR1::FLI1 transcription factor is the main oncogenic event in Ewing sarcoma. Recently, it has been proposed that EWSR1::FLI1 levels can fluctuate in Ewing sarcoma cells, giving rise to two cell populations. EWSR1::FLI1low cells present a migratory and invasive phenotype, while EWSR1::FLI1high cells are more proliferative. In this work, we described how the CD44 standard isoform (CD44s), a transmembrane protein involved in cell adhesion and migration, is overexpressed in the EWSR1::FLI1low phenotype. The functional characterization of CD44s (proliferation, clonogenicity, migration, and invasion ability) was performed in three doxycycline-inducible Ewing sarcoma cell models (A673, MHH-ES1, and CADO-ES1). As a result, CD44s expression reduced cell proliferation in all the cell lines tested without affecting clonogenicity. Additionally, CD44s increased cell migration in A673 and MHH-ES1, without effects in CADO-ES1. As hyaluronan is the main ligand of CD44s, its effect on migration ability was also assessed, showing that high molecular weight hyaluronic acid (HMW-HA) blocked cell migration while low molecular weight hyaluronic acid (LMW-HA) increased it. Invasion ability was correlated with CD44 expression in A673 and MHH-ES1 cell lines. CD44s, upregulated upon EWSR1::FLI1 knockdown, regulates cell migration and invasion in Ewing sarcoma cells.

De novo small deletion affecting transcription start site of short isoform of AUTS2 gene in a patient with syndromic neurodevelopmental defects.

Disruption of the autism susceptibility candidate 2 (AUTS2) gene through genomic rearrangements, copy number variations (CNVs), and intragenic deletions and mutations, has been recurrently involved in syndromic forms of developmental delay and intellectual disability, known as AUTS2 syndrome. The AUTS2 gene plays an important role in regulation of neuronal migration, and when altered, associates with a variable phenotype from severely to mildly affected patients. The more severe phenotypes significantly correlate with the presence of defects affecting the C-terminus part of the gene. This article reports a new patient with a syndromic neurodevelopmental disorder, who presents a deletion of 30 nucleotides in the exon 9 of the AUTS2 gene. Importantly, this deletion includes the transcription start site for the AUTS2 short transcript isoform, which has an important role in brain development. Gene expression analysis of AUTS2 full-length and short isoforms revealed that the deletion found in this patient causes a remarkable reduction in the expression level, not only of the short isoform, but also of the full AUTS2 transcripts. This report adds more evidence for the role of mutated AUTS2 short transcripts in the development of a severe phenotype in the AUTS2 syndrome.

Frequency of low-level and high-level mosaicism in sporadic retinoblastoma: genotype-phenotype relationships.

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene.

Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.

Molecular Approaches to Diagnosis in Ewing Sarcoma: RT-PCR.

Ewing sarcoma is a rare and aggressive tumor that affects children and young adults. Ewing sarcomas are characterized by specific chromosomal translocations that give rise to fusion transcripts that codify for aberrant transcription factors. More than 95% of Ewing sarcoma harbor translocations that produce the fusion of the EWSR1 gene with the transcription factors FLI1 or ERG. This feature can be used to diagnose this entity unambiguously.In this chapter we describe a RT-PCR method that allows for the detection of the most frequent alterations with elevated specificity and sensitivity which is able to distinguish among the different types of fusions. The method is fast and economical, and can be carried out with the conventional equipment available in any molecular biology laboratory.

The Transcription Factor FEZF1, a Direct Target of EWSR1-FLI1 in Ewing Sarcoma Cells, Regulates the Expression of Neural-Specific Genes.

Ewing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 promotes a specific cellular transcriptional program. Therefore, the study of EWSR1-FLI1 target genes is important to identify critical pathways involved in Ewing sarcoma tumorigenesis. In this work, we focused on the transcription factors regulated by EWSR1-FLI1 in Ewing sarcoma. Transcriptomic analysis of the Ewing sarcoma cell line A673 indicated that one of the genes more strongly upregulated by EWSR1-FLI1 was FEZF1 (FEZ family zinc finger protein 1), a transcriptional repressor involved in neural cell identity. The functional characterization of FEZF1 was performed in three Ewing sarcoma cell lines (A673, SK-N-MC, SK-ES-1) through an shRNA-directed silencing approach. FEZF1 knockdown inhibited clonogenicity and cell proliferation. Finally, the analysis of the FEZF1-dependent expression profile in A673 cells showed several neural genes regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1. In summary, FEZF1 is transcriptionally regulated by EWSR1-FLI1 in Ewing sarcoma cells and is involved in the regulation of neural-specific genes, which could explain the neural-like phenotype observed in several Ewing sarcoma tumors and cell lines.

Therapeutic Potential of EWSR1-FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma.

Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1-FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1-FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1-FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1-FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1-FLI1 at the cell level should be considered a therapeutic approach to develop in the future.

Role of global and local topology in the regulation of gene expression in Streptococcus pneumoniae.

The most basic level of transcription regulation in Streptococcus pneumoniae is the organization of its chromosome in topological domains. In response to drugs that caused DNA-relaxation, a global transcriptional response was observed. Several chromosomal domains were identified based on the transcriptional response of their genes: up-regulated (U), down-regulated (D), non-regulated (N), and flanking (F). We show that these distinct domains have different expression and conservation characteristics. Microarray fluorescence units under non-relaxation conditions were used as a measure of gene transcriptional level. Fluorescence units were significantly lower in F genes than in the other domains with a similar AT content. The transcriptional level of the domains categorized them was D>U>F. In addition, a comparison of 12 S. pneumoniae genome sequences showed a conservation of gene composition within U and D domains, and an extensive gene interchange in F domains. We tested the organization of chromosomal domains by measuring the relaxation-mediated transcription of eight insertions of a heterologous Ptccat cassette, two in each type of domain, showing that transcription depended on their chromosomal location. Moreover, transcription from the four promoters directing the five genes involved in supercoiling homeostasis, located either in U (gyrB), D (topA), or N (gyrA and parEC) domains was analyzed both in their chromosomal locations and in a replicating plasmid. Although expression from the chromosomal PgyrB and PtopA showed the expected domain regulation, their expression was down-regulated in the plasmid, which behaved as a D domain. However, both PparE and PgyrA carried their own regulatory signals, their topology-dependent expression being equivalent in the plasmid or in the chromosome. In PgyrA a DNA bend acted as a DNA supercoiling sensor. These results revealed that DNA topology functions as a general transcriptional regulator, superimposed upon other more specific regulatory mechanisms.