Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM

Novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis

Purpose: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. Experimental Design: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II - diffuse astrocytoma, grade III - anaplastic astrocytoma, and grade IV - glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). Results: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage-inducible α (GADD45α) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45α and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45α conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. Conclusions: Our study reveals that GADD45α and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45α overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients

Die Rolle der Mikrogliazellen in Gliomentwicklung und Vorlauf

Malignant gliomas are aggressive tumors of the CNS and offer a poor prognosis. Glioma infiltration is mainly facilitated by brain immune cells or microglia. Soluble factor(s) released by glioma cells attract microglia in and around the tumor to aid glioma diffusion. Invasion by gliomas is further mediated by extracellular matrix degrading enzymes like matrix metalloproteases (MMPs). Metalloproteases are secreted as pro-forms that are activated by membrane anchored metalloproteases or by autocatalysis. Glioma cells produce pro-MMP2 which is catalytically activated by MT1-MMP on microglial cells. Thus, glioma- associated microglia play a pro-neoplastic role by regulating the activity and expression of MT1-MMP via recruitment of Toll-like-receptor adaptor molecule MyD88 and stimulation of p38 MAPK pathway. Project 1: The exact molecular mechanism by which gliomas induce MyD88 dependent-MT1-MMP over-expression in microglia via TLRs was unknown so far. Here for the first time, it is reported that glioma-released factor(s) induce MT1-MMP over-expression in microglia via Toll-like receptor 2 (TLR2), possibly in association with its heterodimeric partners TLR1 and/or TLR6. Receptor sub-type specific ligands of TLRs induced variable changes in MT1-MMP gene expression, with TLR1/2 specific ligand Pam3Csk4 inducing the highest upregulation compared to other agonists. Deletion of TLR2 gene caused inhibition in MT1-MMP gene expression in microglia exposed to glioma conditioned medium (GCM). Microglia derived from TLR1 and 6 knock-out mice also responded with a reduced MT1-MMP expression after stimulation with GCM. Knock-out of TLR7 and 9 gene loci did not decrease GCM-induced MT1-MMP expression. Most importantly, a significant abrogation in MT1-MMP expression and reduction in tumor expansion were also observed in TLR2 knock-out mice intra-cerebrally implanted with glioma cells. Further-more, ablation of the TLR2 gene locus also led to a significant improvement of survival in glioma-implanted TLR2 KO mice as compared to wild-type mice. These results highlight for the first time the role of TLR2, in association with TLRs 1 and/or 6 in glioma-induced MT1-MMP over-expression in microglial cells. Targeting TLR2 could thus offer new insights into the molecular basis of microglia-associated glioma development. Project 2: Pharmacological intervention of microglia-glioma cross-talk using the broad spectrum antibiotic Minocycline yielded encouraging results. Minocycline, a second- generation derivative of tetracycline, and a blocker of microglia activation and p38 MAPK signaling could effectively abrogate glioma-induced MT1-MMP over- expression and activity in microglia at the mRNA and protein expression levels. When Minocycline was administered ex vivo to organotypic brain slices inoculated with glioma cells, a significant decrease in tumor expansion was observed. An effect of Minocycline was not observed in microglia-depleted slices, emphasizing its specific effect on microglial cells. The in vivo oral administration of Minocycline not only led to a reduction in tumor expansion but also caused a decrease in MT1-MMP expression, suggesting its efficacy as a potential drug to target glioma-microglia interaction. This study highlighted the molecular mechanism of glioma-induced MT1-MMP over-expression in microglia via TLR2 and also the promise of Minocycline for treating malignant gliomas.Bösartige Gliome sind aggressive Tumore des zentralen Nervensystems (ZNS) und Patienten haben trotz bestmöglicher Behandlung nur eine durchschnittliche Überlebenszeit von 12 Monaten nach der Diagnose. Gliome sind sehr resistent gegen programmierten Zelltod, entziehen sich der Immunabwehr und dringen ins gesunde Hirnparenchym ein, was eine chirurgische Entfernung erschwert. Die Ausbreitung der Gliomzellen wird begünstigt durch die Immunzellen des ZNS – die Mikroglia. Von den Gliomzellen abgesonderte, lösliche Moleküle rekrutieren Mikroglia innerhalb oder in der Nähe des Tumors, welche ihrerseits die Invasion der Gliomzellen in das umliegende Parenchym unterstützen. Die Invasion der Gliomzellen in das Hirnparenchym wird durch die Produktion von Enzymen, welche die extrazelluläre Matrix degradieren, z.B. die Matrix- Metalloproteasen (MMPs), ermöglicht. Sie werden als inaktive Form sezerniert und anschließend aktiviert, entweder durch autokatalytische Spaltung oder via membranverankerte Metalloproteasen. Die von den Gliomzellen abgesonderten pro- MMP2-Moleküle werden durch die Mikroglia-produzierte, membrangebundene MT1-MMP katalytisch prozessiert und aktiviert. Folglich spielen gliom-assoziierte Mikroglia durch die Regulation der Aktivität von MMP2, vermittelt durch die Produktion von MT1-MMP - welche durch die Rekrutierung des Toll-like-Rezeptor Adaptormoleküls MyD88 und die Stimulation des p38 Signalwegs erfolgt, eine tumorfördernde Rolle. Projekt 1: Der genaue molekulare Mechanismus, durch welchen Gliome die MyD88-abhängige MT1-MMP-Überexpression in Mikroglia durch die TLRs hervorrufen, war bis jetzt nicht bekannt. In dieser Arbeit wird erstmalig gezeigt, dass ein (oder mehrere) von Gliomen produzierter löslicher Faktor die Überexpression von MT1-MMP in Mikroglia über den Toll-like-Rezeptor 2 (TLR2) auslöst, möglicherweise durch die Heterodimerisierung mit TLR1 und/oder TLR6. Liganden, die spezifisch für die Toll-like-Rezeptor-Untertypen sind, induzierten Änderungen der Genexpression von MT1-MMP, wobei der TLR1/2 spezifische Agonist Pam3Csk4 die größte Überexpression bewirkte. Die Deletion des TLR2 Gens verhinderte die MT1-MMP Überexpression in Mikroglia, welche in vitro mit gliomkondizioniertem Medium (GKM) behandelt wurden. Mikroglia, welche aus TLR1 und -6-Knockout-Mäusen isoliert wurden, zeigten ebenfalls eine verringerte Expression von MT1-MMP nach Behandlung mit GKM. Dieser Effekt wurde bei Zellen mit einer Deletion von TLR7 und TLR9 nicht beobachtet. Ausserdem wurde eine signifikante Reduktion der MT1-MMP Expression und dem Tumorwachstum in TLR2-Knockout-Mäusen festgestellt, die intrazerebral mit Tumoren implantiert wurden. Hinzu kommt, dass die Deletion des TLR2-Gens zu einer geringfügigen, aber signifikanten Verlängerung des Überlebens von tumorimplantierten Knockout-Mäusen im Vergleich zu den Wildtyp-Mäusen führte. Diese Ergebnisse demonstrieren zum ersten Mal die Rolle von TLR2, in Verbindung mit TLR1 und TLR6, in der gliom-induzierten MT1-MMP Überexpression in Mikroglia. Der Fokus auf den TLR2-Signalweg könnte also neue Einblicke in die molekularen Grundlagen der mikroglia-unterstützten Gliomentwicklung und –evolution liefern. Projekt 2: Pharmakologische Eingriffe in das Zusammenspiel von Mikroglia und Gliome durch den Einsatz von Minocyclin (Breitbandantibiotikum mit antiinflammatorischer Wirkung) lieferten ermutigende Ergebnisse, die für eine Entwicklung von therapeutischen Strategien gegen Gliominvasion und Wachstum anregend sein könnten. Minocyclin, ein Derivat des Antibiotikums Tetracyclin, das die Aktivierung von Mikroglia und spezifisch vom p38 MAPK Signalweg inhibiert, verhinderte die gliominduzierte Überexpression von MT1-MMP in Mikroglia, sowohl auf der mRNA- als auch auf der Proteinebene. Minocyclinbehandlung von ex vivo organotypischen Hirnschnitten, in die Gliomzellen injiziert wurden, führte zu einer signifikanten Reduktion der Tumorausbreitung. Dieser Effekt konnte jedoch nicht in Schnitten, die frei von Mikroglia waren, beobachtet werden, was auf einen mikrogliaspezifischen Effekt schließen lässt. Die Minocyclinbehandlung in einem experimentellen Gliommausmodells führte zu einer verringerten Tumorgröße, begleitet durch eine reduzierte MT1-MMP-Expression. Daraus lässt sich schließen, dass Minocyclin als potentielles Medikament eingreifend in das Zusammenspiel von Mikroglia und Gliome fungieren könnte. Zusammenfassend hebt diese Studie den molekularen Mechanismus der gliominduzierten MT1-MMP Überexpression in Mikroglia mittels den MyD88-abhängigen TLR2 Signalweg hervor und wirft neues Licht auf den möglichen Einsatz von Minocyclin als Begleitung der existierenden Therapieansätze von bösartigen Gliomen

Cysteinyl leukotrienes and their receptors: Bridging inflammation and colorectal cancer.

Long-standing inflammation has emerged as a hallmark of neoplastic transformation of epithelial cells and may be a limiting factor of successful conventional tumor therapies. A complex milieu composed of distinct stromal and immune cells, soluble factors and inflammatory mediators plays a crucial role in supporting and promoting various types of cancers. An augmented inflammatory response can predispose a patient to colorectal cancer (CRC). Common risk factors associated with CRC development include diet and lifestyle, altered intestinal microbiota and commensals, and chronic inflammatory bowel diseases. Cysteinyl leukotrienes are potent inflammatory metabolites synthesized from arachidonic acid and have a broad range of functions involved in the etiology of various pathologies. This review discusses the important role of cysteinyl leukotriene signaling in linking inflammation and CRC

M2-like macrophages induce colon cancer cell invasion via matrix metalloproteinases

The inflammatory milieu plays an important role in colon cancer development and progression. Previously, we have shown that tumor-associated macrophages (TAMs), an important component of the tumor microenvironment, are enriched in tumors compared with normal tissue and confer a poorer prognosis. In the present study, we found that matrix metallopeptidase-9 (MMP-9), which degrades extracellular matrix proteins, was increased in biopsies from colon cancer patients and in mouse xenografts with SW480 cell-derived tumors. SW480 colon cancer cells exposed to M2-like macrophage-conditioned medium (M2-medium) exhibited increased MMP-9 mRNA, protein expression and gelatinase activity. A similar effect was obtained by the addition of tumor necrosis factor-α (TNFα) and leukotriene D4 (LTD4). MMP-9 expression and activity were reduced by a TNFα blocking antibody adalimumab and a cysteinyl leukotriene receptor 1 (CysLTR1, the receptor for LTD4) antagonist montelukast. M2-medium also induced changes in the epithelial-mesenchymal transition (EMT) markers E-cadherin, β-catenin, vimentin, and snail in SW480 cells. We also found that both M2-medium and TNFα and LTD4 induced stabilization/nuclear translocation of β-catenin. Furthermore, we also observed an elongated phenotype that may indicate increased invasiveness, as confirmed in a collagen I invasion assay. M2-medium increased the invasive ability, and a similar effect was also obtained by the addition of TNFα and LTD4. The specific MMP inhibitor I or adalimumab and montelukast reduced the number of invasive cells. In conclusion, our findings show that M2-medium enriched in TNFα and LTD4 promote colon cancer cell invasion via MMP-9 expression and activation and the induction of EMT

Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like1(Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is over expressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 up regulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM.Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM