The Biological Role and Translational Implications of the Long Non-Coding RNA GAS5 in Breast Cancer.

The lncRNA GAS5 plays a significant role in tumorigenicity and progression of breast cancer (BC). In this review, we first summarize the role of GAS5 in cell biology, focusing on its expression data in human normal tissues. We present data on GAS5 expression in human BC tissues, highlighting its downregulation in all major BC classes. The main findings regarding the molecular mechanisms underlying GAS5 dysregulation are discussed, including DNA hypermethylation of the CpG island located in the promoter region of the gene. We focused on the action of GAS5 as a miRNA sponge, which is able to sequester microRNAs and modulate the expression levels of their mRNA targets, particularly those involved in cell invasion, apoptosis, and drug response. In the second part, we highlight the translational implications of GAS5 in BC. We discuss the current knowledge on the role of GAS5 as candidate prognostic factor, a responsive molecular therapeutic target, and a circulating biomarker in liquid biopsies with clinical importance in BC. The findings position GAS5 as a promising druggable biomolecule and stimulate the development of strategies to restore its expression levels for novel therapeutic approaches that could benefit BC patients in the future.

Longitudinal Circulating Levels of miR-23b-3p, miR-126-3p and lncRNA GAS5 in HCC Patients Treated with Sorafenib.

Human hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The multikinase inhibitor sorafenib is a systemic drug for unresectable HCC. The identification of molecular biomarkers for the early diagnosis of HCC and responsiveness to treatment are needed. In this work, we performed an exploratory study to investigate the longitudinal levels of cell-free long ncRNA GAS5 and microRNAs miR-126-3p and -23b-3p in a cohort of 7 patients during the period of treatment with sorafenib. We used qPCR to measure the amounts of GAS5 and miR-126-3p and droplet digital PCR (ddPCR) to measure the levels of miR-23b-3p. Patients treated with sorafenib displayed variable levels of GAS5, miR-126-3p and miR-23b-3p at different time-points of follow-up. miR-23b-3p was further measured by ddPCR in 37 healthy individuals and 25 untreated HCC patients. The amount of miR-23b-3p in the plasma of untreated HCC patients was significantly downregulated if compared to healthy individuals. The ROC curve analysis underlined its diagnostic relevance. In conclusion, our results highlight a potential clinical significance of circulating miR-23b-3p and an exploratory observation on the longitudinal plasmatic levels of GAS5, miR-126-3p and miR-23b-3p during sorafenib treatment.

DNA methylation variations in familial female and male breast cancer.

In total, ~25% of familial breast cancer (BC) is attributed to germline mutations of the BRCA1 and BRCA2 genes, while the rest of the cases are included in the BRCAX group. BC is also known to affect men, with a worldwide incidence of 1%. Epigenetic alterations, including DNA methylation, have been rarely studied in male breast cancer (MBC) on a genome-wide level. The aim of the present study was to examine the global DNA methylation profiles of patients with BC to identify differences between familial female breast cancer (FBC) and MBC, and according to BRCA1, BRCA2 or BRCAX mutation status. The genomic DNA of formalin-fixed paraffin-embedded tissues from 17 women and 7 men with BC was subjected to methylated DNA immunoprecipitation and hybridized on human promoter microarrays. The comparison between FBC and MBC revealed 2,846 significant differentially methylated regions corresponding to 2,486 annotated genes. Gene Ontology enrichment analysis revealed molecular function terms, such as the GTPase superfamily genes (particularly the GTPase Rho GAP/GEF and GTPase RAB), and cellular component terms associated with cytoskeletal architecture, such as 'cytoskeletal part', 'keratin filament' and 'intermediate filament'. When only FBC was considered, several cancer-associated pathways were among the most enriched KEGG pathways of differentially methylated genes when the BRCA2 group was compared with the BRCAX or BRCA1+BRCAX groups. The comparison between the BRCA1 and BRCA2+BRCAX groups comprised the molecular function term 'cytoskeletal protein binding'. Finally, the functional annotation of differentially methylated genes between the BRCAX and BRCA1+BRCA2 groups indicated that the most enriched molecular function terms were associated with GTPase activity. In conclusion, to the best of our knowledge, the present study was the first to compare the global DNA methylation profile of familial FBC and MBC. The results may provide useful insights into the epigenomic subtyping of BC and shed light on a possible novel molecular mechanism underlying BC carcinogenesis.

Differential expression profiling of long non-coding RNA GAS5 and miR-126-3p in human cancer cells in response to sorafenib.

Long non-coding RNAs (lncRNAs) and microRNAs are involved in numerous physio-pathological conditions included cancer. To better understand the molecular mechanism of the oral antitumor multikinase inhibitor sorafenib, we profiled the expression of a panel of lncRNAs and miRNAs by qPCR array in a sorafenib-treated hepatocellular carcinoma (HCC) cell line. Among the most affected ncRNAs, we found that sorafenib mediated the dysregulation of the lncRNAs GAS5, HOTTIP and HOXA-AS2 and the miR-126-3p, in a panel of human cancer cell lines (HCC, renal and breast carcinomas). By luciferase gene reporter assay, we discovered that GAS5 may act as a sponge for miR-126-3p in HCC cells. The expression level of GAS5 and miR-126-3p was verified in human liquid and/or solid biopsies from HCC patients. miR-126-3p expression in HCC tissues was decreased respect to their correspondent peritumoral tissues. The levels of plasmatic circulating miR-126-3p and GAS5 were significantly higher and lower in HCC patients compared to healthy subjects, respectively. This study highlighted the capability of sorafenib to modulate the expression of a wide range of ncRNAs and specifically, GAS5 and miR-126-3p were involved in the response to sorafenib of different cancer cell types.

Biological Function of MicroRNA193a-3p in Health and Disease.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that act mainly as negative regulators of gene expression. Several studies demonstrated that miRNAs take part in numerous biological processes, such as proliferation, apoptosis, and migration. The dysregulation of miRNAs has been frequently observed in different types of disease, including cancer. Here, we provide a comprehensive review on the human miR-193a-3p by considering its role in both physiological and pathological contexts. Different mechanisms involved in regulating miR-193a-3p expression have been reported, including epigenetic modifications and transcription factors. In physiological contexts, miR-193a-3p seemed able to limit proliferation and cell cycle progression in normal cells. Remarkably, several publications demonstrated that miR-193a-3p acted as a tumor suppressor miRNA in cancer by targeting different genes involved in proliferation, apoptosis, migration, invasion, and metastasis. Furthermore, the downregulation of miR-193a-3p has been observed in many primary tumors and altered levels of circulating miR-193a-3p have been identified in serum or plasma of cancer patients and subjects affected by Parkinson's disease or by schizophrenia. In a clinical perspective, further studies are needed to explore the antitumor effects of the miR-193a-3p mimics delivery and the relevance of this miRNA detection as a possible diagnostic and prognostic biomarker.

Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells.

Sorafenib is currently used to treat advanced and/or unresectable hepatocellular carcinoma (HCC), but the increase of the median survival was only 3 months. Moreover, sorafenib has severe side effects and patients develop resistance quickly. Epigenetic alterations such as DNA methylation play a decisive role in the development and progression of HCC. To our knowledge, there are no studies that analysed the global DNA methylation changes in HCC cells treated with sorafenib. Using MeDip-chip technologies, we found 1230 differentially methylated genes in HA22T/VGH cells treated with sorafenib compared to untreated cells. Gene ontology and pathway analysis allowed identifying several enriched signaling pathways involved in tumorigenesis and cancer progression. Among the genes differentially methylated we found genes related to apoptosis, angiogenesis and invasion, and genes belonging to pathways known to be deregulated in HCC such as RAF/MEK/ERK, JAK-STAT, PI3K/AKT/mTOR and NF-κB. Generally, we found that oncogenes tended to be hypermethylated and the tumor suppressor genes tended to be hypomethylated after sorafenib treatment. Finally, we validated MeDip-chip results for several genes found differentially methylated such as BIRC3, FOXO3, MAPK3, SMAD2 and TSC2, using both COBRA assay and direct bisulfite sequencing and we evaluated their mRNA expression. Our findings suggest that sorafenib could affect the methylation level of genes associated to cancer-related processes and pathways in HCC cells, some of which have been previously described to be directly targeted by sorafenib.

A novel mitochondrial tRNA(Ala) gene variant causes chronic progressive external ophthalmoplegia in a patient with Huntington disease.

Chronic progressive external ophthalmoplegia is a mitochondrial disorder usually caused by single or multiple mitochondrial DNA (mtDNA) deletions and, more rarely, by maternally inherited mtDNA point mutations, most frequently in tRNA genes (MTT). We report on a patient presenting with a progressive eyelid ptosis with bilateral ophthalmoparesis, dysphagia, dysphonia and mild proximal limb weakness associate with a mild movement disorder characterized by abnormal involuntary movements involving head and limbs, imbalance and gait instability. Muscle biopsy demonstrated the presence of ragged red fibers and several cytochrome-C-oxidase negative fibers. Molecular analysis showed the novel m.5613T > C heteroplasmic mutation in the mitochondrial tRNA(Ala) gene (MTTA) which disrupts a conserved site and fulfills the accepted criteria of pathogenicity. Moreover, a 38 CAG trinucleotide repeat expansion was found on the huntingtin gene, thus configuring a singular CPEO/"reduced penetrance" Huntington disease "double trouble". With this novel MTTA point mutation, we extend the spectrum of provisional pathogenic changes in this gene, which is a very rare site of pathogenic mutation, and confirm that clinical expression of these mutations is hardly ever heterogeneous, including myopathy and CPEO. Mitochondrial involvement is an emerging key determinant in the pathogenesis of Huntington disease and it is well known that mutant huntingtin influences the mitochondrial respiratory complexes II and III. A synergist effect of the HTT and MTTA mutations on respiratory chain function may be hypothesized in our patient and should be regarded as a spur for further studies on the mtDNA/HTT reciprocal interactions.

Is Having Clonal Cytogenetic Abnormalities the Same as Having Leukaemia.

A finding of cytogenetic abnormalities, even when these are clonal and even when the abnormalities are typically associated with leukaemia, is not the same as a person having leukaemia. We describe a person who had acute myeloid leukaemia (AML) and achieved a complete haematological remission and who then had persistent and transient clonal cytogenetic abnormalities for 22 years but no recurrence of leukaemia. These data suggest that clones of myeloid cells with mutations and capable of expanding to levels detectable by routine cytogenetic analyses do not all eventuate in leukaemia, even after a prolonged observation interval. The possibility of incorrectly diagnosing a person as having leukaemia becomes even greater when employing more sensitive techniques to detect mutations such as by polymerase chain reaction and whole-exome or whole-genome sequencing. Caution is needed when interpreting clonal abnormalities in AML patients with normal blood and bone marrow parameters.

Lynch syndrome from a surgeon perspective: retrospective study of clinical impact of mismatch repair protein expression analysis in colorectal cancer patients less than 50 years old.

BACKGROUND: In clinical practice, unexpected diagnosis of colorectal cancer in young patients requires prompt surgery, thus genetic testing for Lynch Syndrome is frequently missed, and clinical management may result incorrect. METHODS: Patients younger than 50 years old undergoing colorectal resection for cancer in the period 1994-2007 were identified (Group A, 49 cases), and compared to a group of randomly selected patients more than 50 (Group B, 85 cases). In 31 group A patients, immunohistochemical expression analysis of MLH1, MSH2 and MSH6 was performed; personal and familial history of patients with defective MMR proteins expression was further investigated, searching for synchronous and metachronous tumors in probands and their families. RESULTS: Fifty-one percent of patients did not express one or more MMR proteins (MMR-) and should be considered Lynch Syndrome carriers (16 patients, group A1); while only 31.2% of them were positive for Amsterdam criteria, 50% had almost another tumor, 37.5% had another colorectal tumor and 68% had relatives with colorectal tumor. This group of patients, compared with A2 group (< 50 years old, MMR+) and B group, showed typical characteristics of HNPCC, such as proximal location, mucinous histotype, poor differentiation, high stage and shorter survival. CONCLUSIONS: The present study confirms that preoperative knowledge of MMR proteins expression in colorectal cancer patients would allow correct staging, more extended colonic resection, specific follow-up and familial screening.

Gene expression profile in fibroblasts of Huntington's disease patients and controls.

Huntington's disease is an inherited disorder caused by expanded stretch of consecutive trinucleotides (cytosine-adenosine-guanine, CAG) within the first exon of the huntingtin (HTT) gene on chromosome 4 (p16.3). The mutated huntingtin (mHTT) gains toxic function, probably through mechanisms that involve aberrant interactions in several pathways, causing cytotoxicity. Pathophysiology of disease involves several tissues; indeed it has been shown that there is a broad toxic effect of mHTT in the peripheral tissue of patients with HD, not only in the central nervous system. In this study we compared gene expression profiles (GEP) of HD fibroblasts and matched controls using microarray technology. We used RT-PCR to test the consistency of the microarray data and we found four genes up-regulated in HD patients with respect to control individuals. The genes appear to be involved in different pathways that have been shown to be perturbed even in HD models and patients. Although our study is preliminary and has to be extended to a larger cohort of HD patients and controls, nevertheless it shows that gene expression profiles seem to be altered in the fibroblasts of HD patients. Validation of the differential expressions at the protein level is required to ascertain if this cell type can be considered a suitable model for the identification of HD biomarkers.

BRCA1 and BRCA2 genetic test in high risk patients and families: counselling and management.

Hereditary breast cancer accounts for 5-10% of all cases of breast cancer and 10-15% of ovarian cancer and is characterised by dominant inheritance, early onset, the severity of the disease and bilaterality. About 30% of cases with hereditary breast and ovarian cancer have mutations in the BRCA1 and BRCA2 genes. Women with a mutation in the BRCA1 gene have a 80-90% lifetime risk of developing breast cancer, and 40-65% chance of developing ovarian cancer. Most studies carried out throughout the world indicate that the prevalence of BRCA1 and BRCA2 mutation is lower than originally suggested by early studies on large families with several affected members. Studies performed in Italy have reported different prevalence of BRCA1 and BRCA2 mutations, probably due to different selection criteria and to the variability of the techniques used. In this study, we performed a screening of BRCA1 and BRCA2 in families from northern Italy with familial recurrence of breast cancer or ovarian cancer in which the individual risk of patients of being carriers of BRCA1 and BRCA2 mutation was evaluated using BRCAPRO (CAGene) software. We enrolled 27 patients of 101 unrelated families selected when they fulfilled the inclusion criteria of the American Society of Clinical Oncology (ASCO). Specific risk evaluation, genetic test administration if needed, and discussion of the results were offered during multi-disciplinary genetic, surgical and psychological counselling. Seven probands (35%) found BRCA1/2 sequence variation carriers; no BRCA1 and BRCA2 mutations were detected in the remaining 13 probands. Two (15%) patients had BRCA1 mutations and 5 (25%) patients had BRCA2 mutations. In the latter case, BRCA2 delA 9158fs+29stop mutation in exon 22, never previously described and a new sequence variation (T703N) in exon 11 were identified.

CFTR mutation in an Arab patient: clinical and functional features of 875+1G-->A/875+1G-->A genotype.

Cystic fibrosis (CF; MIM# 219700) is the most frequent recessive disease in Caucasian patients. However, immigration from the Middle East and Africa to Europe is revealing different CFTR mutations. Here, we have described an 875+1G-->A mutation, found for the first time in a homozygous state in an 8 yr old boy. He was the child of a couple of Egyptian first level cousins, both carriers of the mutation. The functional test revealed the 875+1G-->A to be a severe mutation, leading to defective protein function as detected by nasal potential difference (NPD) measurements.

Germline and somatic c-met mutations in multifocal/bilateral and sporadic papillary renal carcinomas of selected patients.

Papillary renal carcinoma (PRC) comprises about 10% of all kidney epithelial tumors. Familiar/hereditary papillary renal carcinomas (HPRCs) have been described, but the majority of cases seem to be sporadic. HPRC is characterized by the predisposition to develop bilateral, multifocal renal tumors. Activating mutations in the tyrosine kinase domain (TK) of the hepatocyte growth factor (HGF) receptor, c-met, have been identified in both hereditary and sporadic PRC. The main aim of this study was to examine a family with no history of PRC in which the proband was a female patient affected by multiple and bilateral PRC at early onset. DNA mutation analysis has been performed by direct sequencing of exons 14-21 of c-met gene which include the TK domain. The proband displayed the germline c-met missense mutation g.3522G--> A in exon 16. Two other family members were found to carry the same mutation. The mutation analysis extended to 15 selected patients, allowed to identify the first case of an Italian patient affected by PRC displaying the somatic missense mutation g.3997 T-->C curved arrow C located in exon 19 of c-met. The mutation frequency of the selected-based population of PRC patients in this report was 12.5%. Furthermore, the phosphorylated c-met expression detected by immunohistochemistry in PRCs with germline/somatic or no c-met mutation, supports the concept that c-met activation may occur in PRC oncogenesis by c-met mutations and/or c-met over-expression.

Mesenchymal cells from human amniotic fluid survive and migrate after transplantation into adult rat brain.

Amniotic fluid has been recently suggested as an alternative source of mesenchymal stem cells. However, the fate of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) after in vivo transplantation has yet to be determined. In the present study we explored whether human AF-MSCs could survive and migrate following transplantation into the striatum of normal and ischemic rat. We found that the grafted cells could survive and migrate towards multiple brain regions in the normal animals, while they moved towards the injured region in the ischemic rat. Double-immunostaining analyses showed that the implanted human AF-MSCs express markers for immature neurons (Doublecortin) at 10 days, and for astrocytes (GFAP) at 10, 30 and 90 after transplantation. This study provides the first evidence that human amniotic fluid contains cells having the potential to survive and integrate into adult rat brain tissue and, therefore, to function as effective stem cells for therapeutic strategies.

Glutamate receptor RNA editing: a molecular analysis of GluR2, GluR5 and GluR6 in human brain tissues and in NT2 cells following in vitro neural differentiation.

The properties of some glutamate receptors are modified by RNA editing. This post-transcriptional mechanism involves the enzymatic deamination of specific adenosines in the pre-mRNA of the glutamate receptors, performed by specific RNA adenosine deaminases (ADARs). This event gives rise to the substitution of a gene-encoded amino acid with a different one that modifies the physiological properties of the ion channel. Here we report an analysis of the editing levels of AMPA GluR2, and kainate GluR5 and GluR6 in a human teratocarcinoma cell line (NT2) during in vitro neural differentiation, in conjunction with an analysis of the expression levels of GluR and ADAR genes. The editing levels were analysed using a specific standardised assay based on sequence analysis. This assay can be performed on all editing sites with a high level of sensitivity and reproducibility. Whereas GluR gene expression increased during NT2 neural differentiation, the expression of ADAR genes may be detected at comparable levels even in undifferentiated NT2 cells, remaining relatively stable during the differentiation process. Furthermore, most of the glutamate receptor editing sites increased their editing levels during NT2 neural differentiation, suggesting that the level of ADAR mRNAs is not closely related to the variable editing levels detected in the GluRs analysed. In human brain tissues, the editing levels appeared finely modulated in the different areas, indicating the possible formation of ion channels with different functional properties, thus generating a complex tissue-specific regulation of receptors and modulation of excitatory stimuli.

Matrix assembly induction and cell migration and invasion inhibition by a 13-amino acid fibronectin peptide.

Fibronectin (FN) is an extracellular matrix (ECM) protein involved in tumor growth and metastasis. Five human FN cDNA segments encoding for FN fragments, all starting with the II1 repeat and ending with different C-terminal extensions, have been stably expressed in chick embryo fibroblasts (CEF). These FN cDNAs induce the formation of an organized ECM in CEF as long as they retain a sequence coding for a 13-amino acid stretch (FN13), with collagen binding activity, localized between type II2 and I7 repeats. An FN13 synthetic peptide induces in control CEF the assembly of an FN-ECM comparable with that observed in CEF-expressing FN fragments. The activity of FN13 is specific for its amino acid sequence, although the cysteine present in the 6th position can be substituted with a polar serine without affecting the induction of a fibrillar FN-ECM. A less fibrillar matrix is induced by FN13-modified peptides in which the cysteine is methylated or substituted by a non-polar alanine. FN13 induces the assembly of an FN-ECM also in Rous sarcoma virus-transformed CEF lacking the ECM and in hepatoma (SK-Hep1) and fibrosarcoma (HT-1080) human cell lines. FN13 also promotes the adhesion of CEF and Rous sarcoma virus-CEF at levels comparable with those obtained with purified intact FN. Finally, FN13 inhibits the migratory and invasive properties of tumorigenic cells, whereas intact FN favors their migration. All FN13-modified peptides show similar effects, although with reduced efficiency. None of these activities is supported by a scrambled peptide. These data suggest a possible role of FN13 in tumor growth and metastasis inhibition and its possible use as anti-tumorigenic agent.