KDM7A-DT induces genotoxic stress, tumorigenesis, and progression of p53 missense mutation-associated invasive breast cancer

Stress-induced promoter-associated and antisense lncRNAs (si-paancRNAs) originate from a reservoir of oxidative stress (OS)-specific promoters via RNAPII pausing-mediated divergent antisense transcription. Several studies have shown that the KDM7A divergent transcript gene (KDM7A-DT), which encodes a si-paancRNA, is overexpressed in some cancer types. However, the mechanisms of this overexpression and its corresponding roles in oncogenesis and cancer progression are poorly understood. We found that KDM7A-DT expression is correlated with highly aggressive cancer types and specific inherently determined subtypes (such as ductal invasive breast carcinoma (BRCA) basal subtype). Its regulation is determined by missense TP53 mutations in a subtype-specific context. KDM7A-DT transcribes several intermediate-sized ncRNAs and a full-length transcript, exhibiting distinct expression and localization patterns. Overexpression of KDM7A-DT upregulates TP53 protein expression and H2AX phosphorylation in nonmalignant fibroblasts, while in semi-transformed fibroblasts, OS superinduces KDM7A-DT expression in a TP53-dependent manner. KDM7A-DT knockdown and gene expression profiling in TP53-missense mutated luminal A BRCA variant, where it is abundantly expressed, indicate its significant role in cancer pathways. Endogenous over-expression of KDM7A-DT inhibits DNA damage response/repair (DDR/R) via the TP53BP1-mediated pathway, reducing apoptosis and promoting G2/M checkpoint arrest. Higher KDM7A-DT expression in BRCA is associated with KDM7A-DT locus gain/amplification, higher histologic grade, aneuploidy, hypoxia, immune modulation scores, and activation of the c-myc pathway. Higher KDM7A-DT expression is associated with relatively poor survival outcomes in patients with luminal A or Basal subtypes. In contrast, it is associated with favorable outcomes in patients with HER2+ER- or luminal B subtypes. KDM7A-DT levels are coregulated with critical transcripts and proteins aberrantly expressed in BRCA, including those involved in DNA repair via non-homologous end joining and epithelial-to-mesenchymal transition pathway. In summary, KDM7A-DT and its si-lncRNA exhibit several intrinsic biological and clinical characteristics that suggest important roles in invasive BRCA and its subtypes. KDM7A-DT-defined mRNA and protein subnetworks offer resources for identifying clinically relevant RNA-based signatures and prospective targets for therapeutic intervention

Transcriptional Regulation of PIK3CA Oncogene by NF-kappa B in Ovarian Cancer Microenvironment

Peer reviewe

Functional genomic analysis: identification of genes up-regulated in ovarian tumours: study of their importance in cancer diagnosis and treatment

Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. Like other solid tumors, the progression of ovarian cancer strongly depends on the establishment of a supportive tumor microenvironment that promotes growth and expansion of cancer cells. In this study, we assessed global gene expression changes of tumor cells as a result of their adaptation process to two parameters of tumor microenvironment: lymphocyte infiltration and hypoxia. The development of a sensitive and reproducible fluorescent differential display methodology (ADDER) allowed the identification of rare mRNA genes that are over- or under-expressed when lymphocytes infiltrate ovarian cancer at an early stage. Among them, ttf1 was further validated in a significant number of samples (n=96) as a gene over-expressed in tumors positive to lymphocyte presence. Complementary to this approach, we analyzed data from microarray experiments retrieved from free-accessed databases and identified genes whose expression shows a strong positive correlation with the lymphocyte marker CD8A in 11 types of cancer. Ten of these genes were further validated by qPCR in 57 ovarian cancer samples. During the course of this task various reports revealed that non-coding RNA molecules, like miRNAs, play an essential role in tumorigenesis. So, we expanded the scope of our study on differential gene expression in ovarian cancer in order to analyze changes in these genes as well. Utilizing a miRNA-specific Taqman qPCR approach we defined the expression signature of 156 miRNAs in several (n=18) ovarian cancer cell lines compared to HOSE cell lines. We then used this technique to analyze the miRNA expression profile of two ovarian cancer lines and a HeLa cell line in hypoxic conditions or under physiological oxygen atmosphere (normoxia). Only miR-210 was significantly up-regulated under hypoxia in all three cell lines. MiR-210 promoter dissection as well as the analysis of its expression in cell lines with constitutive HIF expression yielded evidence consistent with a HIF-mediated mechanism of miR-210 induction in hypoxia. Gene dosage aberrations in the miR-210 locus was also studied. We found an unexpectedly high frequency of deletions of the miR-210 gene in ovarian cancer. Finally, using bioinformatics and in vitro approaches we identified 6 mRNAs as putative targets of miR-210. One of miR-210 target genes was E2F3, involved in cell cycle regulation. We showed that induction of miR-210 in cell causes very effective suppression of E2F3 protein levels, linking for the first time an miRNA gene with hypoxia and cell cycle regulation.Ο καρκίνος των ωοθηκών είναι η κύρια αιτία θανάτου από γυναικολογικές νεοπλασίες παγκοσμίως. Όπως όλοι οι συμπαγείς όγκοι, η πρόοδος του καρκίνου των ωοθηκών εξαρτάται από την καθιέρωση του κατάλληλου υποστηρικτικού μικροπεριβάλλοντος, το οποίο προωθεί την επιβίωση και την αύξηση των καρκινικών κυττάρων. Σε αυτή τη μελέτη, αξιολογήσαμε τις αλλαγές της γονιδιακής έκφρασης των καρκινικών κυττάρων ως αποτέλεσμα της διαδικασίας προσαρμογής τους σε δύο παραμέτρους του μικροπεριβάλλοντος: τη διήθηση λεμφοκυττάρων και την υποξία. Η ανάπτυξη μιας ευαίσθητης και επαναλήψιμης μεθοδολογίας διαφορικής έκθεσης με φθορισμό (ADDER) επέτρεψε τον προσδιορισμό σπάνιων γονιδίων mRNA που υπέρ- ή υπό-εκφράζονται όταν τα λεμφοκύτταρα διηθούν τον όγκο των ωοθηκών σε πρώιμο στάδιο. Από αυτά, το γονίδιο ttf1 επιβεβαιώθηκε περαιτέρω σε ένα σημαντικό αριθμό δειγμάτων (n=96) ως γονίδιο που υπερεκφράζεται στους θετικούς, ως προς την παρουσία λεμφοκυττάρων, όγκους ωοθηκών. Συμπληρωματικά αναλύσαμε δεδομένα από πειράματα με μικροσυστοιχίες που ανακτήθηκαν από ελεύθερα προσβάσιμες βάσεις δεδομένων στο διαδίκτυο και προσδιορίσαμε τα γονίδια των οποίων η έκφραση συσχετίζεται θετικά με το δείκτη λεμφοκυττάρων CD8A σε 11 τύπους καρκίνων. Δέκα από αυτά τα γονίδια επικυρώθηκαν περαιτέρω με qPCR σε 57 δείγματα καρκίνου ωοθήκης. Κατά τη διάρκεια της παρούσας εργασίας, έγινε εμφανές από διάφορες δημοσιεύσεις ότι τα μη κωδικά μόρια RNA, όπως τα miRNA, διαδραματίζουν ουσιαστικό ρόλο στη διαδικασία της καρκινογένεσης. Έτσι, επεκτείναμε το πεδίο της μελέτης μας για τη διαφορική έκφραση γονιδίων στον καρκίνο των ωοθηκών προκειμένου να αναλυθούν οι αλλαγές της έκφρασης και αυτών των γονιδίων. Χρησιμοποιώντας μια miRNA-ειδική Taqman μέθοδο qPCR καθορίσαμε το πρότυπο έκφρασης 156 miRNA σε διάφορες (n=18) καρκινικές κυτταρικές σειρές ωοθηκών σε σύγκριση με κυτταρικές σειρές φυσιολογικού επιθηλίου ωοθηκών. Στη συνέχεια, χρησιμοποιήσαμε αυτήν την τεχνική για να αναλύσουμε το πρότυπο έκφρασης miRNA δύο καρκινικών κυτταρικών σειρών ωοθηκών και μιας καρκινικής κυτταρικής σειράς HeLa σε συνθήκες υποξίας ή σε φυσιολογικά επίπεδα οξυγόνου. Μόνο το miR-210 υπερεκφραζόταν σημαντικά στην υποξία και στις τρεις κυτταρικές σειρές. Δεδομένα από την ανάλυση του υποκινητή για το miR-210 καθώς επίσης και από την ανάλυση της έκφρασης του σε κυτταρικές σειρές με συστατική έκφραση για το μεταγραφικό παράγοντα HIF υποδεικνύουν ότι ο HIF πιθανώς ρυθμίζει την επαγωγή του miR-210 στην υποξία. Επίσης μελετήθηκαν οι αλλαγές στον αριθμό γονιδιωματικών αντιγράφων του γενετικού τόπου του miR-210. Βρήκαμε μια απροσδόκητα υψηλή συχνότητα ελλειμμάτων για το miR-210 στον καρκίνο των ωοθηκών. Τέλος, χρησιμοποιώντας τη βιοπληροφορική και in vitro προσεγγίσεις προσδιορίσαμε 6 mRNA ως υποθετικούς στόχους του miR-210. Ένα από τα γονίδια-στόχους του miR-210 ήταν ο μεταγραφικός παράγοντας E2F3 που αποτελεί σημαντικό ρυθμιστή του κυτταρικού κύκλου. Δείξαμε ότι η υπερέφκραση του miR-210 έχει ως αποτέλεσμα την πολύ αποτελεσματική καταστολή του E2F3 σε πρωτεϊνικό επίπεδο, γεγονός που συνδέει για πρώτη φορά ένα γονίδιο miRNA με την υποξία και τη ρύθμιση του κυτταρικού κύκλου

Network-based Bayesian inference revealed critical ncRNAs signal drivers from transcriptomics in CLL

The purpose of this study is to create a novel workflow based on NetBID tool (Network-based Bayesian inference of signal drivers), in order to unveil ncRNA-gene associations with biological merit in a type of multi-factorial disease like B cell lymphomas (Chronic Lymphocytic Leukemia). Preliminary analysis of BloodCancerMultiOmics2017 database reveals central ncRNAs drivers in CLL patients in comparison with samples that differentiate in drug response with Ibrutinib. These initial findings hopefully pave the way to discover through a Systems-approach more ncRNAs that participate in CLL pathobiology and could be of therapeutic interest. </p

Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p=0.0067) and 0.861 (p<0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p=0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling

Distinctive molecular signature and activated signaling pathways in aortic smooth muscle cells of patients with myocardial infarction

Background and aims: We aim to identify significant transcriptome alterations of vascular smooth muscle cells (VSMCs) in the aortic wall of myocardial infarction (MI) patients. Providing a robust transcriptomic signature, we aim to highlight the most likely aberrant pathway(s) in MI VSMCs. Methods and results: Laser-captured microdissection (LCM) was used to obtain VSMCs from aortic wall tissues harvested during coronary artery bypass surgery. Microarray gene analysis was applied to analyse VSMCs from 17 MI and 19 non-MI patients. Prediction Analysis of Microarray (PAM) identified 370 genes that significantly discriminated MI and non-MI samples and were enriched in genes responsible for muscle development, differentiation and phenotype regulation. Incorporation of gene ontology (GO) led to the identification of a 21-gene VSMCs-associated classifier that discriminated between MI and non-MI patients with 92% accuracy. The mass spectrometry-based iTRAQ analysis of the MI and non-MI samples revealed 94 proteins significantly differentiating these tissues. Ingenuity Pathway Analysis (IPA) of 370 genes revealed top pathways associated with hypoxia signaling in the cardiovascular system. Enrichment analysis of these proteins suggested an activation of the superoxide radical degradation pathway. An integrated transcriptome-proteome pathway analysis revealed that superoxide radical degradation pathway remained the most implicated pathway. The intersection of the top candidate molecules from the transcriptome and proteome highlighted superoxide dismutase (SOD1) overexpression. Conclusions: We provided a novel 21-gene VSMCs-associated MI classifier in reference to significant VSMCs transcriptome alterations that, in combination with proteomics data, suggests the activation of superoxide radical degradation pathway in VSMCs of MI patients.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

lncRNA NORAD is consistently detected in breastmilk exosomes and its expression is downregulated in mothers of preterm infants

Breast milk is the ideal food for infants and undoubtedly has immediate and long-term benefits. Breast milk contains extracellular vesicles (EVs) i.e., exosomes secreted by maternal breast cells. Exosomes carry genetic material, such as long non-coding RNAs (lncRNAs), which possibly participate in cell-to-cell communications, as they are known to regulate critical gene pathways. The aim of the present study was to screen human breastmilk exosomes for their lncRNA cargo and to examine exosomal lncRNA levels associated with milk obtained from mothers that gave birth at term or prematurely (&lt;37 weeks of gestation). Samples were collected at 3 weeks postpartum from 20 healthy, breastfeeding mothers; 10 mothers had given birth at full-term and 10 mothers preterm. Exosomal RNA was extracted from all samples and the expression of 88 distinct lncRNAs was determined using reverse transcription-quantitative PCR. A total of 13 lncRNAs were detected in &gt;= 85% of the samples, while 31 were detected in &gt;= 50% of the samples. Differential expression analysis of the lncRNAs between the two groups revealed &gt;= 2-fold differences, with generally higher lncRNA concentrations found in the milk of the mothers that gave birth at term compared with those that gave birth preterm. Among these, the non-coding RNA activated at DNA damage (NORAD) was prominently detected in both groups, and its expression was significantly downregulated in the breast milk exosomes of mothers who delivered preterm. On the whole, the present study demonstrates that breast milk lncRNAs may be important factors of normal early human development. Collectively, the presence of lncRNAs in human breast milk may explain the consistent inability of researchers to fully ‘humanize’ animal milk