CD151 Regulates Tumorigenesis by Modulating the Communication between Tumor Cells and Endothelium

The tetraspanin CD151 forms stoichiometric complexes with laminin-binding integrins (e.g., alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4) and regulates their ligand-binding and signaling functions. We have found that high expression of CD151 in breast cancers is associated with decreased overall survival (3.44-fold higher risk of death). Five-year estimated survival rates were 45.8% (95% confidence interval, 16.4-71.4%) for CD151-positive Patients and 79.9% (95% confidence interval, 62.2-90.0%) for CD151-negative Patients. Furthermore, CD151 was positively associated with axillary lymph node involvement. To study the biological significance of this observation, we investigated the contribution of CD151 in breast cancer tumorigenesis using MDA-MB-231 cells as a model system. Stable down-regulation of this tetraspanin by short-hairpin RNA decreased the tumorigenicity of these cells in mice. Detailed immunohistologic analysis of CD151 (+) and CD151(-) xenografts showed differences in tumor vascular pattern. Vascularization observed at the subcutaneous border of the CD151 (+) tumors was less pronounced or absent in the CD151(-) xenografts. In vitro experiments have established that depletion of CD151 did not affect the inherent proliferative capacity of breast cancer cells in three-dimensional extracellular matrices, but modified their responses to endothelial cells in coculture experiments. The modulatory activity of CD151 was dependent on its association with both alpha 3 beta 1 and alpha 6 beta 4 integrins. These data point to a new role of CD151 in tumorigenesis, whereby it functions as an important regulator of communication between tumor cells and endothelial cells. These results also identify CD151 as a potentially novel prognostic marker and target for therapy in breast cancer

R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage

How DNA damage leads to chronic inflammation and tissue degeneration with aging remains to be fully resolved. Here, we show that DNA damage leads to cellular senescence, fibrosis, loss-of-tissue architecture, and chronic pancreatitis in mice with an inborn defect in the excision repair cross complementation group 1 (Ercc1) gene. We find that DNA damage-driven R-loops causally contribute to the active release and buildup of single-stranded DNAs (ssDNAs) in the cytoplasm of cells triggering a viral-like immune response in progeroid and naturally aged pancreata. To reduce the proinflammatory load, we developed an extracellular vesicle (EV)-based strategy to deliver recombinant S1 or ribonuclease H nucleases in inflamed Ercc1(−/−) pancreatic cells. Treatment of Ercc1(−/−) animals with the EV-delivered nuclease cargo eliminates DNA damage-induced R-loops and cytoplasmic ssDNAs alleviating chronic inflammation. Thus, DNA damage-driven ssDNAs causally contribute to tissue degeneration, Ercc1(−/−) paving the way for novel rationalized intervention strategies against age-related chronic inflammation

Tissue-infiltrating macrophages mediate an exosome-based metabolic reprogramming upon DNA damage

DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1(F/-)) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1(F/-) animal sera and are secreted in macrophage media after DNA damage. The Er1(F/-) EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging

Μελέτη του ρόλου και της ρύθμισης της TPL2/COT (MAP3K8) στην καρκινογένεση του πνεύμονα: ένα πρωτο-ογκογονίδιο με ογκοκατασταλτική δράση

Cancer can be perceived as a disease of communication between and within cells. The aberrations are pleiotropic, but mitogen‐activated protein kinase (MAPK) pathways feature prominently. Tpl2/COT is a serine‐threonine MAPK kinase kinase (MAP3K8) at the crossroad of various signal transduction pathways that control fundamental cellular processes such as growth, proliferation, differentiation, mi‐gration and apoptosis. Despite early studies identifying Tpl2 as a proto‐oncogene activated by C‐terminal truncation in mouse and rat, a detailed evaluation of its expression and function in human malignancy is missing and the physiological role of Tpl2/COT in carcinogenesis remains enigmatic.One of the three major human tissues where Tpl2 is expressed at highest levels is the lung. Lung cancer is the leading cause of cancer related death worldwide, re‐flecting the need for a better understanding of the mechanisms that underlie the development of pulmonary carcinomas. The Ph.D. thesis presented here focuses on the characterization of the role and of the mechanisms regulating the expres‐sion of Tpl2/COT1 in lung tumor cells during the onset of lung cancer development and through its progression.In this regard, we were able to reveal an unprecedented role for Tpl2/COT as sup‐pressor of lung carcinogenesis. This conclusion was based on a number of novel findings obtained through the extensive analysis of human tissue and of ex vivo and in vivo experimental models. We showed that COT expression is significantly reduced in human lung cancer compared to normal lung tissue providing the first demonstration that COT can be differentially expressed in a human malignancy. Importantly, the downregulation of COT is found to be of clinical relevance as it correlates with a more aggressive tumor phenotype and with poor prognosis in pa‐tients with lung cancer.Our data disclose 3 mechanisms responsible for COT downregulation in primary human lung tumors: (i) allelic imbalance at the COT locus; (ii) global DNA hypomethylation‐associated upregulation of miR‐370 which targets COT transcripts and (iii) activated Ras signaling affecting both COT transcription and protein stability. The operation of multiple genetic and epigenetic aberrations leading to loss of wild‐type COT in primary lung tumors underlines a previously unappreciat‐ed broader role for this pathway in lung carcinogenesis. This is corroborated by our experimental data showing that ablation of the Tpl2 gene in mice dramatically ac‐celerates the onset and multiplicity of urethane‐induced lung tumorigenesis.Additionally, we provide mechanistic evidence to suggest that Tpl2/COT antago‐nizes oncogene‐induced cell transformation and survival through a pathway involv‐ing the tumor suppressor p53 downstream of JNK. In particular, our studies define Tpl2/COT as a novel physiological antagonist of oncogenic Ras, thus also expanding our understanding of signaling and function of this major human oncogene. An im‐portant functional interaction between Tpl2/COT, JNK and p53 which controls apoptosis is revealed, providing a telling example of how tumor cells usurp alter‐native pathways that lower intrinsic barriers to malignant transformation.Ο καρκίνος μπορεί να θεωρηθεί ως ασθένεια στην επικοινωνία μεταξύ και στο εσωτερικό των κυττάρων. Οι δυσλειτουργίες που προκύπτουν είναι πλειοτροπι‐κές, αλλά τα μονοπάτια των κινασών που ενεργοποιούνται από μιττογόνα (Mito‐gen Activated Protein Kinases) φαίνεται να παίζουν εξέχοντα ρόλο στη διαδικασία της καρκινογένεσης. Η Tpl2 είναι μία ΜΑΡ3 κινάση σερίνης/θρεονίνης στο σταυ‐ροδρόμι πολλών και διαφορετικών μονοπατιών σηματοδότησης που ελέγχουν πρωτεύουσας σημασίας κυτταρικές διεργασίες, όπως η κυτταρική αύξηση, πολ‐λαπλασιασμός διαφοροποίηση, μετανάστευση και απόπτωση. Παρά τις αρχικές που οδήγησαν στην ταυτοποίηση της Tpl2 ως πρωτο‐ογκογονίδιο που ενεργοποι‐είται μετά από καρβοξυτελική αποκοπή σε ποντικούς και αρουραίους, μία λεπτο‐μερής εκτίμηση της έκφρασης και της λειτουργίας της Tpl2/COT στην ανθρώπινη κακοήθεια λείπει ακόμη από τη βιβλιογραφεία. Ο φυσιολογικός ρόλος της Tpl2 στην καρκινογένεση παραμένει αινιγματικός.Ένα από τα 3 κυριότερα όργανα όπου εμφανίζονται από τα υψηλότερα επίπεδα της COT είναι οι πνεύμονες. Ο καρκίνος των πνευμόνων είναι η σημαντικότερη αιτία θανάτου καρκινοπαθών παγκοσμίως, αντανακλώντας την άμεση ανάγκη για καλύτερη κατανόηση των μηχανισμών που διέπουν την ανάπτυξη καρκινωμάτων του αναπνευστικού. Η διδακτορική διατριβή που παρουσιάζεται εδώ εστιάζει στον χαρακτηρισμό του ρόλου και των μηχανισμών που ελέγχουν την έκφραση της Tpl2/COT στα καρκινικά κύτταρα του πνεύμονα, κατά την έναρξη της ανάπτυ‐ξης του καρκίνου του πνεύμονα καθώς και κατά τη διάρκεια εξέλιξής του.Ερευνώντας υπό αυτό το πρίσμα, καταφέραμε να αποκαλύψουμε έναν απροσδό‐κητο ρόλο για την Tpl2/CΟT ως καταστολέα της καρκινογένεσης του πνεύμονα . Αυτό το συμπέρασμα βασίστηκε σε έναν αριθμό νέων ευρημάτων στα οποία κα‐ταλήξαμε μετά από ενδελεχή ανάλυση ανθρώπινου ιστού και χρήση πειραματό‐ζωων και ex vivo μοντέλων. Δείξαμε πως η έκφραση της COT είναι σημαντικά μειωμένη στον καρκίνο του πνεύμονα στον άνθρωπο συγκριτικά με τον φυσιολο‐γικό ιστό, παρέχοντας έτσι την πρώτη απόδειξη πως η COT μπορεί να παρουσιά‐σει διαφορική έκφραση σε μία ανθρώπινη κακοήθεια. Ανάλογα σημαντικό ήταν το εύρημα πως η μείωση της COT έχει κλινική προέκταση καθώς συσχετίζεται με πιο επιθετικό φαινότυπο του όγκου και κακή πρόγνωση στους ασθενείς με καρκί‐νο του πνεύμονα.Τα δεδομένα μας περικλείουν 3 μηχανισμούς υπεύθυνους για τη μείωση της έκ‐φρασης της COT σε πρωτεύοντες όγκους του πνεύμονα στον άνθρωπο: (i) αλληλι‐κή αστάθεια στον γενετικό τόπο της COT (ii) υπερέκφραση του mir370 που στο‐χεύει τα μετάγραφα της COT μετά από υπομεθυλίωση του γενετικού του τόπου, στα πλαίσια της γενικής υπομεθυλίωσης που συμβαίνει κατά την καρκινογένεση (iii) ενεργοποίηση σηματοδότησης Ras που επηρεάζει και την μεταγραφή και την πρωτεϊνική σταθερότητα της Tpl2/COT.Η ύπαρξη πολλαπλών γενετικών και επιγενετικών ανωμαλιών που οδηγούν σε απώλεια ή σημαντική μείωση της αγρίου τύπου Tpl2 / COT σε πρωτεύοντες όγκο‐υς στον πνεύμονα υπογραμμίζει έναν έως τώρα μη εκτιμημένο, ευρείας σημαντι‐κότητας ρόλο αυτού του μονοπατιού στην καρκινογένεση του πνεύμονα. Αυτό ενισχύεται και επιβεβαιώνεται και από τα πειραματικά μας δεδομένα που δείχ‐νουν πως η έλλειψη του γονιδίου στα πειραματόζωα επιταχύνει δραματικά την έναρξη και την πολλαπλότητα της ογκογένεσης στον πνεύμονα μετά από χορήγη‐ση ουρεθάνης.Επιπρόσθετα, παρουσιάζουμε μηχανιστικές ενδείξεις που υποδεικνύουν πως η Tpl2/COT ανταγωνίζεται τον επαγόμενο από ογκογονίδια κυτταρικό μετασχημα‐τισμό και επιβίωση, μέσω ενός μονοπατιού που περιλαμβάνει την ογκο‐κατασταλτική p53 καθοδικά της JNK. Πιο συγκεκριμένα, οι μελέτες ορίζουν την Tpl2/COT ως ένα νέο φυσικό ανταγωνιστή της ογκογόνου Ras, διευρύνοντας α‐κόμη περισσότερο την κατανόηση της λειτουργίας και της σηματοδότησης ενός τόσο σημαντικού ανθρώπινου ογκογονιδίου. Μία ενδιαφέρουσα λειτουργική αλ‐ληλεπίδραση μεταξύ των Tpl2/COT, JNK και p53 που ελέγχει την απόπτωση απο‐καλύπτεται, παρέχοντας ταυτόχρονα ένα ενδεικτικό παράδειγμα για το πώς τα καρκινικά κύτταρα εκμεταλλεύονται και καθοδηγούν εναλλακτικά μονοπάτια για να μειώσουν τους έμφυτους φραγμούς στον καρκινικό μετασχηματισμό

Suppression of Integrin alpha 3 beta 1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Integrin receptors for cell adhesion to extracellular matrix have important roles in promoting tumor growth and progression. Integrin α3β1 is highly expressed in breast cancer cells where it is thought to promote invasion and metastasis; however, its roles in regulating malignant tumor cell behavior remain unclear. In the current study, we used short-hairpin RNA (shRNA) to show that suppression of α3β1 in a human breast cancer cell line, MDA-MB-231, leads to decreased tumorigenicity, reduced invasiveness, and decreased production of factors that stimulate endothelial cell migration. Real-time PCR revealed that suppression of α3β1 caused a dramatic reduction in expression of the cyclooxygenase-2 (COX-2) gene, which is frequently over-expressed in breast cancers and has been exploited as a therapeutic target. Decreased COX-2 was accompanied by reduced prostaglandin E2 (PGE(2)), a major prostanoid produced downstream of COX-2 and an important effector of COX-2 signaling. shRNA-mediated suppression of COX-2 showed that it has a role in tumor cell invasion and crosstalk to endothelial cells. Furthermore, treatment with PGE(2) restored these functions in α3β1-deficient MDA-MB-231 cells. These findings identify a role for α3β1 in regulating two properties of tumor cells that facilitate cancer progression: invasiveness and ability to stimulate endothelial cells. They also reveal a novel role for COX-2 as a downstream effector of α3β1 in tumor cells, thereby identifying α3β1 as a potential therapeutic target to inhibit breast cancer

IFNα Impairs Autophagic Degradation of mtDNA Promoting Autoreactivity of SLE Monocytes in a STING-Dependent Fashion

Interferon α (IFNα) is a prompt and efficient orchestrator of host defense against nucleic acids but upon chronicity becomes a potent mediator of autoimmunity. Sustained IFNα signaling is linked to pathogenesis of systemic lupus erythematosus (SLE), an incurable autoimmune disease characterized by aberrant self-DNA sensing that culminates in anti-DNA autoantibody-mediated pathology. IFNα instructs monocytes differentiation into autoinflammatory dendritic cells (DCs) than potentiates the survival and expansion of autoreactive lymphocytes, but the molecular mechanism bridging sterile IFNα-danger alarm with adaptive response against self-DNA remains elusive. Herein, we demonstrate IFNα-mediated deregulation of mitochondrial metabolism and impairment of autophagic degradation, leading to cytosolic accumulation of mtDNA that is sensed via stimulator of interferon genes (STING) to promote induction of autoinflammatory DCs. Identification of mtDNA as a cell-autonomous enhancer of IFNα signaling underlines the significance of efficient mitochondrial recycling in the maintenance of peripheral tolerance. Antioxidant treatment and metabolic rescue of autolysosomal degradation emerge as drug targets in SLE and other IFNα-related pathologies. In lupus, sustained IFNα signaling leads to anti-DNA autoimmunity. Gkirtzimanaki et al. propose that excessive IFNα damages mitochondrial respiration, leading to oxidative stress that impairs lysosomal degradation and obstructs autophagic clearance. Undegraded mtDNA escapes in the cytoplasm and is sensed, priming monocytes cell-autonomously to initiate an anti-viral-like response against self-DNA. © 2018 The Author

IFNα Impairs Autophagic Degradation of mtDNA Promoting Autoreactivity of SLE Monocytes in a STING-Dependent Fashion

Summary: Interferon α (IFNα) is a prompt and efficient orchestrator of host defense against nucleic acids but upon chronicity becomes a potent mediator of autoimmunity. Sustained IFNα signaling is linked to pathogenesis of systemic lupus erythematosus (SLE), an incurable autoimmune disease characterized by aberrant self-DNA sensing that culminates in anti-DNA autoantibody-mediated pathology. IFNα instructs monocytes differentiation into autoinflammatory dendritic cells (DCs) than potentiates the survival and expansion of autoreactive lymphocytes, but the molecular mechanism bridging sterile IFNα-danger alarm with adaptive response against self-DNA remains elusive. Herein, we demonstrate IFNα-mediated deregulation of mitochondrial metabolism and impairment of autophagic degradation, leading to cytosolic accumulation of mtDNA that is sensed via stimulator of interferon genes (STING) to promote induction of autoinflammatory DCs. Identification of mtDNA as a cell-autonomous enhancer of IFNα signaling underlines the significance of efficient mitochondrial recycling in the maintenance of peripheral tolerance. Antioxidant treatment and metabolic rescue of autolysosomal degradation emerge as drug targets in SLE and other IFNα-related pathologies. : In lupus, sustained IFNα signaling leads to anti-DNA autoimmunity. Gkirtzimanaki et al. propose that excessive IFNα damages mitochondrial respiration, leading to oxidative stress that impairs lysosomal degradation and obstructs autophagic clearance. Undegraded mtDNA escapes in the cytoplasm and is sensed, priming monocytes cell-autonomously to initiate an anti-viral-like response against self-DNA. Keywords: autoimmunity, SLE, lysosome, metabolis