A novel program of infiltrative control in astrocytomas: ADAM23 depletion promotes cell invasion by activating γ-secretase complex.

Background: Infiltration is a life-threatening growth pattern in malignant astrocytomas and a significant cause of therapy resistance. It results in the tumor cell spreading deeply into the surrounding brain tissue, fostering tumor recurrence and making complete surgical resection impossible. We need to thoroughly understand the mechanisms underlying diffuse infiltration to develop effective therapies. Methods: We integrated in vitro and in vivo functional assays, RNA sequencing, clinical, and expression information from public data sets to investigate the role of ADAM23 expression coupling astrocytoma's growth and motility. Results: ADAM23 downregulation resulted in increased infiltration, reduced tumor growth, and improved overall survival in astrocytomas. Additionally, we show that ADAM23 deficiency induces γ-secretase (GS) complex activity, contributing to the production and deposition of the Amyloid-ß and release of NICD. Finally, GS ablation in ADAM23-low astrocytomas induced a significant inhibitory effect on the invasive programs. Conclusions: Our findings reveal a role for ADAM23 in regulating the balance between cell proliferation and invasiveness in astrocytoma cells, proposing GS inhibition as a therapeutic option in ADAM23 low-expressing astrocytomas.

Small Indels in the Androgen Receptor Gene: Phenotype Implications and Mechanisms of Mutagenesis.

CONTEXT: Despite high abundance of small indels in human genomes, their precise roles and underlying mechanisms of mutagenesis in Mendelian disorders require further investigation. OBJECTIVE: To profile the distribution, functional implications, and mechanisms of small indels in the androgen receptor (AR) gene in individuals with androgen insensitivity syndrome (AIS). METHODS: We conducted a systematic review of previously reported indels within the coding region of the AR gene, including 3 novel indels. Distribution throughout the AR coding region was examined and compared with genomic population data. Additionally, we assessed their impact on the AIS phenotype and investigated potential mechanisms driving their occurrence. RESULTS: A total of 82 indels in AIS were included. Notably, all frameshift indels exhibited complete AIS. The distribution of indels across the AR gene showed a predominance in the N-terminal domain, most leading to frameshift mutations. Small deletions accounted for 59.7%. Most indels occurred in nonrepetitive sequences, with 15.8% situated within triplet regions. Gene burden analysis demonstrated significant enrichment of frameshift indels in AIS compared with controls (P < .00001), and deletions were overrepresented in AIS (P < .00001). CONCLUSION: Our findings underscore a robust genotype-phenotype relationship regarding small indels in the AR gene in AIS, with a vast majority presenting complete AIS. Triplet regions and homopolymeric runs emerged as prone loci for small indels within the AR. Most were frameshift indels, with polymerase slippage potentially explaining half of AR indel occurrences. Complex frameshift indels exhibited association with palindromic runs. These discoveries advance understanding of the genetic basis of AIS and shed light on potential mechanisms underlying pathogenic small indel events.

Identification of NID1 as a novel candidate susceptibility gene for familial non-medullary thyroid carcinoma using whole-exome sequencing.

The genetics underlying non-syndromic familial non-medullary thyroid carcinoma (FNMTC) is still poorly understood. To identify susceptibility genes for FNMTC, we performed whole-exome sequencing (WES) in a Brazilian family affected by papillary thyroid carcinoma (PTC) in three consecutive generations. WES was performed in four affected and two unaffected family members. Manual inspection in over 100 previously reported susceptibility genes for FNMTC showed that no variants in known genes co-segregated with disease phenotype in this family. Novel candidate genes were investigated using PhenoDB and filtered using Genome Aggregation (gnomAD) and Online Archive of Brazilian Mutations (ABraOM) population databases. The missense variant p.Ile657Met in the NID1 gene was the only variant that co-segregated with the disease, while absent in unaffected family members and controls. The allele frequency for this variant was <0.0001 in the gnomAD and ABbraOM databases. In silico analysis predicted the variant to be deleterious or likely damaging to the protein function. Somatic mutations in NID1 gene were found in nearly 500 cases of different cancer subtypes in the intOGen platform. Immunohistochemistry analysis showed NID1 expression in PTC cells, while it was absent in normal thyroid tissue. Our findings were corroborated using data from the TCGA cohort. Moreover, higher expression of NID1 was associated with higher likelihood of relapse after treatment and N1b disease in PTCs from the TCGA cohort. Although replication studies are needed to better understand the role of this variant in the FNMTC susceptibility, the NID1 variant (c.1971T>G) identified in this study fulfills several criteria that suggest it as a new FNMTC predisposing gene.

Molecular Signature Expands the Landscape of Driver Negative Thyroid Cancers.

Thyroid cancer is the most common endocrine malignancy. However, the cytological diagnosis of follicular thyroid carcinoma (FTC), Hürthle cell carcinoma (HCC), and follicular variant of papillary thyroid carcinoma (FVPTC) and their benign counterparts is a challenge for preoperative diagnosis. Nearly 20-30% of biopsied thyroid nodules are classified as having indeterminate risk of malignancy and incur costs to the health care system. Based on that, 120 patients were screened for the main driver mutations previously described in thyroid cancer. Subsequently, 14 mutation-negative cases that are the main source of diagnostic errors (FTC, HCC, or FVPTC) underwent RNA-Sequencing analysis. Somatic variants in candidate driver genes (ECD, NUP98,LRP1B, NCOR1, ATM, SOS1, and SPOP) and fusions were described. NCOR1 and SPOP variants underwent validation. Moreover, expression profiling of driver-negative samples was compared to 16 BRAF V600E, RAS, or PAX8-PPARg positive samples. Negative samples were separated in two clusters, following the expression pattern of the RAS/PAX8-PPARg or BRAF V600E positive samples. Both negative groups showed distinct BRS, ERK, and TDS scores, tumor mutation burden, signaling pathways and immune cell profile. Altogether, here we report novel gene variants and describe cancer-related pathways that might impact preoperative diagnosis and provide insights into thyroid tumor biology.

Evidence of Cooperation between Hippo Pathway and RAS Mutation in Thyroid Carcinomas.

Thyroid cancer incidences have been steadily increasing worldwide and are projected to become the fourth leading cancer diagnosis by 2030. Improved diagnosis and prognosis predictions for this type of cancer depend on understanding its genetic bases and disease biology. RAS mutations have been found in a wide range of thyroid tumors, from benign to aggressive thyroid carcinomas. Based on that and in vivo studies, it has been suggested that RAS cooperates with other driver mutations to induce tumorigenesis. This study aims to identify genetic alterations or pathways that cooperate with the RAS mutation in the pathogenesis of thyroid cancer. From a cohort of 120 thyroid carcinomas, 11 RAS-mutated samples were identified. The samples were subjected to RNA-Sequencing analyses. The mutation analysis in our eleven RAS-positive cases uncovered that four genes that belong to the Hippo pathway were mutated. The gene expression analysis revealed that this pathway was dysregulated in the RAS-positive samples. We additionally explored the mutational status and expression profiling of 60 RAS-positive papillary thyroid carcinomas (PTC) from The Cancer Genome Atlas (TCGA) cohort. Altogether, the mutational landscape and pathway enrichment analysis (gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genome (KEGG)) detected the Hippo pathway as dysregulated in RAS-positive thyroid carcinomas. Finally, we suggest a crosstalk between the Hippo and other signaling pathways, such as Wnt and BMP.

Global changes in nitration levels and DNA binding profile of Trypanosoma cruzi histones induced by incubation with host extracellular matrix.

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.

Histopathological Characterization and Whole Exome Sequencing of Ectopic Thyroid: Fetal Architecture in a Functional Ectopic Gland from Adult Patient.

Ectopic thyroid results from a migration defect of the developing gland during embryogenesis causing congenital hypothyroidism. But it has also been detected in asymptomatic individuals. This study aimed to investigate the histopathological, functional, and genetic features of human ectopic thyroids. Six samples were histologically examined, and the expression of the specific thyroid proteins was assessed by immunohistochemistry. Two samples were submitted to whole exome sequencing. An oropharynx sample showed immature fetal architecture tissue with clusters or cords of oval thyrocytes and small follicles; one sample exhibited a normal thyroid pattern while four showed colloid goiter. All ectopic thyroids expressed the specific thyroid genes and T4 at similar locations to those observed in normal thyroid. No somatic mutations associated with ectopic thyroid were found. This is the first immature thyroid fetal tissue observed in an ectopic thyroid due to the arrest of structural differentiation early in the colloid stage of development that proved able to synthesize thyroid hormone but not to respond to TSH. Despite the ability of all ectopic thyroids to synthetize specific thyroid proteins and T4, at some point in life, it may be insufficient to support body growth leading to hypothyroidism, as observed in some of the patients.

A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology.

Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin-fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G>T:A and C:G>A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct.

Measuring plasma levels of three microRNAs can improve the accuracy for identification of malignant breast lesions in women with BI-RADS 4 mammography.

A BI-RADS category of 4 from a mammogram indicates suspicious breast lesions, which require core biopsies for diagnosis and have an approximately one third chance of being malignant. Human plasma contains many circulating microRNAs, and variations in their circulating levels have been associated with pathologies, including cancer. Here, we present a novel methodology to identify malignant breast lesions in women with BI-RADS 4 mammography. First, we used the miRNome array and qRT-PCR to define circulating microRNAs that were differentially represented in blood samples from women with breast tumor (BI-RADS 5 or 6) in comparison to controls (BI-RADS 1 or 2). Next, we used qRT-PCR to quantify the level of this circulating microRNAs in patients with mammograms presenting with BI-RADS category 4. Finally, we developed a machine learning method (Artificial Neural Network - ANN) that receives circulating microRNA levels and automatically classifies BI-RADS 4 breast lesions as malignant or benign. We identified a minimum set of three circulating miRNAs (miR-15a, miR-101 and miR-144) with altered levels in patients with breast cancer. These three miRNAs were quantified in plasma from 60 patients presenting biopsy-proven BI-RADS 4 lesions. Finally, we constructed a very efficient ANN that could correctly classify BI-RADS 4 lesions as malignant or benign with approximately 92.5% accuracy, 95% specificity and 88% sensibility. We believe that our strategy of using circulating microRNA and a machine learning method to classify BI-RADS 4 breast lesions is a non-invasive, non-stressful and valuable complementary approach to core biopsy in women with BI-RADS 4 lesions.

MicroRNA Expression Signature Is Altered in the Cardiac Remodeling Induced by High Fat Diets.

Recent studies have revealed the involvement of microRNAs (miRNAs) in the control of cardiac hypertrophy and myocardial function. In addition, several reports have demonstrated that high fat (HF) diet induces cardiac hypertrophy and remodeling. In the current study, we investigated the effect of diets containing different percentages of fat on the cardiac miRNA expression signature. To address this question, male C57Bl/6 mice were fed with a low fat (LF) diet or two HF diets, containing 45 kcal% fat (HF45%) and 60 kcal% fat (HF60%) for 10 and 20 weeks. HF60% diet promoted an increase on body weight, fasting glycemia, insulin, leptin, total cholesterol, triglycerides, and induced glucose intolerance. HF feeding promoted cardiac remodeling, as evidenced by increased cardiomyocyte transverse diameter and interstitial fibrosis. RNA sequencing analysis demonstrated that HF feeding induced distinct miRNA expression patterns in the heart. HF45% diet for 10 and 20 weeks changed the abundance of 64 and 26 miRNAs in the heart, respectively. On the other hand, HF60% diet for 10 and 20 weeks altered the abundance of 27 and 88 miRNAs in the heart, respectively. Bioinformatics analysis indicated that insulin signaling pathway was overrepresented in response to HF diet. An inverse correlation was observed between cardiac levels of GLUT4 and miRNA-29c. Similarly, we found an inverse correlation between expression of GSK3ß and the expression of miRNA-21a-3p, miRNA-29c-3p, miRNA-144-3p, and miRNA-195a-3p. In addition, miRNA-1 overexpression prevented cardiomyocyte hypertrophy. Taken together, our results revealed differentially expressed miRNA signatures in the heart in response to different HF diets. J. Cell. Physiol. 231: 1771-1783, 2016. © 2015 Wiley Periodicals, Inc.

The human cell surfaceome of breast tumors.

INTRODUCTION: Cell surface proteins are ideal targets for cancer therapy and diagnosis. We have identified a set of more than 3700 genes that code for transmembrane proteins believed to be at human cell surface. METHODS: We used a high-throuput qPCR system for the analysis of 573 cell surface protein-coding genes in 12 primary breast tumors, 8 breast cell lines, and 21 normal human tissues including breast. To better understand the role of these genes in breast tumors, we used a series of bioinformatics strategies to integrates different type, of the datasets, such as KEGG, protein-protein interaction databases, ONCOMINE, and data from, literature. RESULTS: We found that at least 77 genes are overexpressed in breast primary tumors while at least 2 of them have also a restricted expression pattern in normal tissues. We found common signaling pathways that may be regulated in breast tumors through the overexpression of these cell surface protein-coding genes. Furthermore, a comparison was made between the genes found in this report and other genes associated with features clinically relevant for breast tumorigenesis. CONCLUSIONS: The expression profiling generated in this study, together with an integrative bioinformatics analysis, allowed us to identify putative targets for breast tumors.

SurfaceomeDB: a cancer-orientated database for genes encoding cell surface proteins.

Cell surface proteins (CSPs) are excellent targets for the development of diagnostic and therapeutic reagents, and it is estimated that 10-20% of all genes in the human genome encode CSPs. In an effort to integrate all data publicly available for genes encoding cell surface proteins, a database (SurfaceomeDB) was developed. SurfaceomeDB is a gene-centered portal containing different types of information, including annotation for gene expression, protein domains, somatic mutations in cancer, and protein-protein interactions for all human genes encoding CSPs. SurfaceomeDB was implemented as an integrative and relational database in a user-friendly web interface, where users can search for gene name, gene annotation, or keywords. There is also a streamlined graphical representation of all data provided and links to the most important data repositories and databases, such as NCBI, UCSC Genome Browser, and EBI.

Analysis of allelic differential expression in the human genome using allele-specific serial analysis of gene expression tags.

Recent reports have demonstrated that a significant proportion of human genes display allelic differential expression (ADE). ADE is associated with phenotypic variability and may contribute to complex genetic diseases. Here, we present a computational analysis of ADE using allele-specific serial analysis of gene expression (SAGE) tags representing 1295 human genes. We identified 472 genes for which unequal representation (>3-fold) of allele-specific SAGE tags was observed in at least one SAGE library, suggesting the occurrence of ADE. For 235 out of these 472 genes, the difference in the expression level between both allele-specific SAGE tags was statistically significant (p < 0.05). Eleven candidate genes were then subjected to experimental validation and ADE was confirmed for 8 out of these 11 genes. Our results suggest that at least 25% of the human genes display ADE and that allele-specific SAGE tags can be efficiently used for the identification of such genes.

Detection and evaluation of intron retention events in the human transcriptome.

Alternative splicing is a very frequent phenomenon in the human transcriptome. There are four major types of alternative splicing: exon skipping, alternative 3' splice site, alternative 5' splice site, and intron retention. Here we present a large-scale analysis of intron retention in a set of 21,106 known human genes. We observed that 14.8% of these genes showed evidence of at least one intron retention event. Most of the events are located within the untranslated regions (UTRs) of human transcripts. For those retained introns interrupting the coding region, the GC content, codon usage, and the frequency of stop codons suggest that these sequences are under selection for coding potential. Furthermore, 26% of the introns within the coding region participate in the coding of a protein domain. A comparison with mouse shows that at least 22% of all informative examples of retained introns in human are also present in the mouse transcriptome. We discuss that the data we present suggest that a significant fraction of the observed events is not spurious and might reflect biological significance. The analyses also allowed us to generate a reliable set of intron retention events that can be used for the identification of splicing regulatory elements.