PAX8 in ovarian carcinoma: identification of new downstream networks and target genes

PAX8 is a transcription factor involved in the tissue-specific expression of several genes during development, tissue homeostasis and cancer. Recently, PAX8 has been reported to be an important marker for the diagnosis of ovarian carcinoma with a pivotal function in the tumorigenic phenotype of ovarian cancer cells. PAX8 is normally expressed in Fallopian tube secretory cells but not in ovarian surface epithelial cells; however, its expression is detected in the majority of high-grade serous ovarian carcinoma (HGSC) supporting the tubal origin of this cancer. To determine whether PAX8 contributes to ovarian cancer development, we initially conducted a transcriptome analyses to determine the distinctive molecular profiles of the Fallopian tube epithelial secretory cell line (FT194) and the ovarian cancer cell line (SKOV3), before and after PAX8 silencing. The bioinformatics analysis revealed several GO categories enriched in both PAX8-silenced FT-194 and SKOV3 cells. Among those categories, the results showed that both “cell migration” and the “positive regulation of cell migration” bioprocess displayed transcriptional change of 5% in SKOV3 cells and the adhesion category shows change of about 16% in SKOV3 and 14% in FT-194 cells. With respect to specific pathways, the highest differential changes upon PAX8 silencing were found in angiogenesis, Wnt, cadherin and integrin signalling pathways, in both cell types. Since migration and adhesion are important biological processes in both physiological and pathological conditions, migration and adhesion assays were performed using a primary human fallopian tube secretory cells (Primary hFTSECs) and a panel of ovarian cancer cell lines (SKOV3, KURAMOCHI, OVSAHO and PEA1). Interestingly, our results show that inhibition of PAX8 expression in Primary hFTSEC and in epithelial ovarian cancer cell lines significantly reduces the ability of the cells to migrate and adhere on Fibronectin and/or Collagen I substrates. Integrins are reported to be the major regulators of cellular attachment with the extracellular matrix and are required for cellular migration. In our transcriptome analysis, Integrin β3 was significantly downregulated after PAX8 silencing in SKOV3 cells. Therefore, we performed qRT-PCR on Primary hFTSEC and our panel of ovarian cancer cell lines and the results show a strong reduction of Integrin β3 expression in all ovarian cancer cell lines after PAX8 silencing, respect to the control cells. In parallel, we also show that loss of PAX8 does not affect the expression of Integrin αv, the ligand of Integrin β3 involved in ovarian cancer tumorigenesis. The Immunofluorescences assays of the functional heterodimer αvβ3 was tested in Primary hFTSEC and KURAMOCHI cell lines and in PAX8 silenced cells the signal was decreased. In conclusion, we believe that it is of great relevance to further study and decipher the link between PAX8 and Integrin β3 because it could help uncover the role of PAX8 in HGSC development

Deepening the understanding of CNVs on chromosome 15q11–13 by using hiPSCs: An overview

The human α7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is widely expressed in the central and peripheral nervous systems. This receptor is implicated in both brain development and adult neurogenesis thanks to its ability to mediate acetylcholine stimulus (Ach). Copy number variations (CNVs) of CHRNA7 gene have been identified in humans and are genetically linked to cognitive impairments associated with multiple disorders, including schizophrenia, bipolar disorder, epilepsy, Alzheimer’s disease, and others. Currently, α7 receptor analysis has been commonly performed in animal models due to the impossibility of direct investigation of the living human brain. But the use of model systems has shown that there are very large differences between humans and mice when researchers must study the CNVs and, in particular, the CNV of chromosome 15q13.3 where the CHRNA7 gene is present. In fact, human beings present genomic alterations as well as the presence of genes of recent origin that are not present in other model systems as well as they show a very heterogeneous symptomatology that is associated with both their genetic background and the environment where they live. To date, the induced pluripotent stem cells, obtained from patients carrying CNV in CHRNA7 gene, are a good in vitro model for studying the association of the α7 receptor to human diseases. In this review, we will outline the current state of hiPSCs technology applications in neurological diseases caused by CNVs in CHRNA7 gene. Furthermore, we will discuss some weaknesses that emerge from the overall analysis of the published articles

Candidate genes and pathways downstream of PAX8 involved in ovarian high-grade serous carcinoma

Understanding the biology and molecular pathogenesis of ovarian epithelial cancer (EOC) is key to developing improved diagnostic and prognostic indicators and effective therapies. Although research has traditionally focused on the hypothesis that high-grade serous carcinoma (HGSC) arises from the ovarian surface epithelium (OSE), recent studies suggest that additional sites of origin exist and a substantial proportion of cases may arise from precursor lesions located in the Fallopian tubal epithelium (FTE). In FTE cells, the transcription factor PAX8 is a marker of the secretory cell lineage and its expression is retained in 96% of EOC. We have recently reported that PAX8 is involved in the tumorigenic phenotype of ovarian cancer cells. In this study, to uncover genes and pathways downstream of PAX8 involved in ovarian carcinoma we have determined the molecular profiles of ovarian cancer cells and in parallel of Fallopian tube epithelial cells by means of a silencing approach followed by an RNA-seq analysis. Interestingly, we highlighted the involvement of pathways like WNT signaling, epithelial-mesenchymal transition, p53 and apoptosis. We believe that our analysis has led to the identification of candidate genes and pathways regulated by PAX8 that could be additional targets for the therapy of ovarian carcinom

Long Non-Coding RNA MAGI2-AS3 is a New Player with a Tumor Suppressive Role in High Grade Serous Ovarian Carcinoma

High-Grade Serous Ovarian Carcinoma (HGSC) is the most incidental and lethal subtype of epithelial ovarian cancer (EOC) with a high mortality rate of nearly 65%. Recent findings aimed at understanding the pathogenesis of HGSC have attributed its principal source as the Fallopian Tube (FT). To further comprehend the exact mechanism of carcinogenesis, which is still less known, we performed a transcriptome analysis comparing FT and HGSC. Our study aims at exploring new players involved in the development of HGSC from FT, along with their signaling network, and we chose to focus on non-coding RNAs. Non-coding RNAs (ncRNAs) are increasingly observed to be the major regulators of several cellular processes and could have key functions as biological markers, as well as even a therapeutic approach. The most physiologically relevant and significantly dysregulated non-coding RNAs were identified bioinformatically. After analyzing the trend in HGSC and other cancers, MAGI2-AS3 was observed to be an important player in EOC. We assessed its tumor-suppressive role in EOC by means of various assays. Further, we mapped its signaling pathway using its role as a miRNA sponge to predict the miRNAs binding to MAGI2AS3 and showed it experimentally. We conclude that MAGI2-AS3 acts as a tumor suppressor in EOC, specifically in HGSC by sponging miR-15-5p, miR-374a-5p and miR-374b-5p, and altering downstream signaling of certain mRNAs through a ceRNA network

Isolation of Enterobacter aerogenes carrying blaTEM-1 and blaKPC-3 genes recovered from a hospital Intensive Care Unit.

Enterobacter aerogenes has recently emerged as an important hospital pathogen. In this study, we showed the emergence of E. aerogenes isolates carrying the blaKPC gene in patients colonized by carbapenem-resistant Klebsiella pneumoniae strains. Two multiresistant E. aerogenes isolates were recovered from bronchial aspirates of two patients hospitalized in the Intensive Care Unit at the "Santa Maria della Scaletta" Hospital, Imola. The antimicrobial susceptibility test showed the high resistance to carbapenems and double-disk synergy test confirmed the phenotype of KPC and AmpC production. Other investigation revealed that ESBL and blaKPC genes were carried on the conjugative pKpQIL plasmid. This is a relevant report in Italy that describes a nosocomial infection due to the production of KPC beta-lactamases by an E. aerogenes isolate in patients previously colonized by K. pneumoniae carbapenem-resistant. In conclusion, it's necessary a continuous monitoring of multidrug-resistant strains for the detection of any KPC-producing bacteria that could expand the circulation of carbapenem-resistant pathogens

Mitochondrial malfunctioning, proteasome arrest and apoptosis in cancer cells by focused intracellular generation of oxygen radicals

Photofrin/photodynamic therapy (PDT) at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53+/+) and H1299 (p53−/−) cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones) with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis

Photodynamic and antibiotic therapy in combination to fight biofilms and resistant surface bacterial infections

Although photodynamic therapy (PDT), a therapeutic approach that involves a photosensitizer, light and O₂, has been principally considered for the treatment of specific types of cancers, other applications exist, including the treatment of infections. Unfortunately, PDT does not always guarantee full success since it exerts lethal effects only in cells that have taken up a sufficient amount of photosensitizer and have been exposed to adequate light doses, conditions that are not always achieved. Based on our previous experience on the combination PDT/chemotherapy, we have explored the possibility of fighting bacteria that commonly crowd infected surfaces by combining PDT with an antibiotic, which normally does not harm the strain at low concentrations. To this purpose, we employed 5-aminolevulinic acid (5-ALA), a pro-drug that, once absorbed by proliferating bacteria, is converted into the natural photosensitizer Protoporphyrin IX (PpIX), followed by Gentamicin. Photoactivation generates reactive oxygen species (ROS) which damage or kill the cell, while Gentamicin, even at low doses, ends the work. Our experiments, in combination, have been highly successful against biofilms produced by several Gram positive bacteria (i.e., Staphylococcus aureus, Staphylococcus epidermidis, etc.). This original approach points to potentially new and wide applications in the therapy of infections of superficial wounds and sores

In vitro interaction of Stenotrophomonas maltophilia with human monocyte-derived dendritic cells

Stenotrophomonas maltophilia is increasingly identified as an opportunistic pathogen in immunocompromised, cancer and cystic fibrosis (CF) patients. Knowledge on innate immune responses to S. maltophilia and its potential modulation is poor. The present work investigated the ability of 12 clinical S. maltophilia strains (five from CF patients, seven from non-CF patients) and one environmental strain to survive inside human monocyte-derived dendritic cells (DCs). The effects of the bacteria on maturation of and cytokine secretion by DCs were also measured. S. maltophilia strains presented a high degree of heterogeneity in internalization and intracellular replication efficiencies as well as in the ability of S. maltophilia to interfere with normal DCs maturation. By contrast, all S. maltophilia strains were able to activate DCs, as measured by increase in the expression of surface maturation markers and proinflammatory cytokines secretion