Experimental targeting of basal-like breast cancer in vitro

15% aller Brustkrebszellen sind Mammakarzinome vom Basaltyp (BLBC), ein aggressiver Subtyp mit schlechter Heilungsprognose. Diese Zellen sind dreifach negativ (Östrogen, Progesteron, Her2/neu), was eine konventionelle Behandlung nicht möglich macht. Die Inhibierung verschiedener Signalwege könnte eine alternative Therapie darstellen. Um herauszufinden welche Signalwege eine Schlüsselrolle für diese Zellen spielen, wurden Dosis-Wirkungskurven (MTT-Assay) mit verschiedenen Inhibitoren, Zytotoxinen und Kombinationen beider erstellt. Es konnte gezeigt werden, dass der NF-κB Signalweg eine wichtige Rolle für das Überleben der BLBC Zellen spielt (HCC-38, HCC-1937, MDA-MB-231). Immunoblotting des kanonischen NF-κB Signalweges zeigte, dass unbehandelte Basalzellen die Proteine NF-κBp65 und phospho-NF-κBp65 überexpremierten. Die Inhibitoren Violacein, MG-132, BAY 11-7082 und Akt Inhibitor IV waren imstande die Expression von NF-κBp65 zu inhibieren oder zu vermindern. Die Inhibitoren Violacein, MG-132 und Akt Inhibitor IV verringerten zusätzlich auch die Expression von IκB-α und phospho-IκB-α in fast allen untersuchten Zellen. Die IKK Proteine waren unterhalb des Detektionslimits des Western Blots. Diese Ergebnisse zeigten, dass das Überleben der BLBC vom NF-κB Signalweg abhängt. Alle verwendeten Inhibitoren dieses Signalweges, mit Ausnahme des Tpl2 Kinase Inhibitors (Wachstumsarrest), führten zu einer Eradikation der Zellen (IC50: 0,01 µM – 3,4 µM). Die Regulation dieses Signalweges scheint sehr kompliziert zu sein und es liegt die Vermutung nahe, dass es eine Kommunikation zwischen dem kanonischen NF-κB Signalweg und anderen Signalwegen gibt. Zusammenfassend kann gesagt werden, dass es vielversprechend scheint, alternative Therapien für das Mammakarzinom vom Basaltyp auf dem NF-κB Signalweg basieren zu lassen.15% of all breast cancers belong to the basal-like breast cancer type (BLBC), an aggressive subtype with poor prognosis. This subgroup is triple negative (hormone receptors of estrogen, progesterone and for Her2) impairing conventional treatment strategies. Thus, we investigated targeting of signaling pathways as option in BLBC. By using MTT-assays, we established dose-response curves with signaling inhibitors, cytotoxics and combinations of both. We discovered that the NF-κB pathway is of vital importance for BLBC cancer cells (HCC-38, HCC-1937 and MDA-MB-231). Immunoblotting of the canonical pathway showed that untreated BLBC were overexpressing phosphorylated and unphosphorylated NF-κBp65 protein. Further, the inhibitors violacein, MG-132, BAY11-7082 and Akt Inhibitor IV were able to supress or downregulate the expression of NF-κBp65. The inhibitors violacein, MG-132 and Akt Inhibitor IV led to a downregulation of the phosphorylated and unphosphorylated IκB-α protein in almost all investigated cells. In contrast, IKK proteins were below the detection limit of the Western blot. These experiments demonstrate that the survival of BLBC in vitro relies on the NF-κB pathway. All used inhibitors of this pathway, except Tpl2 Kinase Inhibitor (growth arrest), led to a complete eradication of BLBC (IC50: 0.01 µM – 3.4 µM). Moreover, the canonical NF-κB pathway is likely interacting with other signaling pathways emphasizing the aggressive behavior of this subtype. These data suggest that therapeutic strategies based on the NF-κB pathway may be a promising approach for the treatment of BLBC

Journal of Cellular and Molecular Medicine / Gliomasphere marker combinatorics: multidimensional flow cytometry detects CD 44+/CD 133+/ITGA 6+/CD 36+ signature

Glioblastoma is the most dangerous brain cancer. One reason for glioblastoma's aggressiveness are glioblastoma stemlike cells. To target them, a number of markers have been proposed (CD 133, CD 44, CD 15, A2B5, CD 36, CXCR 4, IL 6R, L1CAM , and ITGA 6). A comprehensive study of coexpression patterns of them has, however, not been performed so far. Here, we mapped the multidimensional coexpression profile of these stemnessassociated molecules. Gliomaspheres an established model of glioblastoma stemlike cells were used. Seven different gliomasphere systems were subjected to multicolor flow cytometry measuring the nine markers CD 133, CD 44, CD 15, A2B5, CD 36, CXCR 4, IL 6R, L1CAM , and ITGA 6 all simultaneously based on a novel 9marker multicolor panel developed for this study. The viSNE dimensionality reduction algorithm was applied for analysis. All gliomaspheres were found to express at least five different glioblastoma stemlike cell markers. Multidimensional analysis showed that all studied gliomaspheres consistently harbored a cell population positive for the molecular signature CD 44+/CD 133+/ITGA 6+/CD 36+. Glioblastoma patients with an enrichment of this combination had a significantly worse survival outcome when analyzing the two largest available The Cancer Genome Atlas datasets (MIT /Harvard Affymetrix: P = 0.0015, University of North Carolina Agilent: P = 0.0322). In sum, we detected a previously unknown marker combination demonstrating feasibility, usefulness, and importance of highdimensional gliomasphere marker combinatorics.(VLID)510252

Immunological analysis of phase II glioblastoma dendritic cell vaccine (Audencel) trial: immune system characteristics influence outcome and Audencel up-regulates Th1-related immunovariables

Abstract Audencel is a dendritic cell (DC)-based cellular cancer immunotherapy against glioblastoma multiforme (GBM). It is characterized by loading of DCs with autologous whole tumor lysate and in vitro maturation via “danger signals”. The recent phase II “GBM-Vax” trial showed no clinical efficacy for Audencel as assessed with progression-free and overall survival in all patients. Here we present immunological research accompanying the trial with a focus on immune system factors related to outcome and Audencel’s effect on the immune system. Methodologically, peripheral blood samples (from apheresis before Audencel or venipuncture during Audencel) were subjected to functional characterization via enzyme-linked immunospot (ELISPOT) assays connected with cytokine bead assays (CBAs) as well as phenotypical characterization via flow cytometry and mRNA quantification. GBM tissue samples (from surgery) were subjected to T cell receptor sequencing and immunohistochemistry. As results we found: Patients with favorable pre-existing anti-tumor characteristics lived longer under Audencel than Audencel patients without them. Pre-vaccination blood CD8+ T cell count and ELISPOT Granzyme B production capacity in vitro upon tumor antigen exposure were significantly correlated with overall survival. Despite Audencel’s general failure to induce a significant clinical response, it nevertheless seemed to have an effect on the immune system. For instance, Audencel led to a significant up-regulation of the Th1-related immunovariables ELISPOT IFNγ, the transcription factor T-bet in the blood and ELISPOT IL-2 in a dose-dependent manner upon vaccination. Post-vaccination levels of ELISPOT IFNγ and CD8+ cells in the blood were indicative of a significantly better survival. In summary, Audencel failed to reach an improvement of survival in the recent phase II clinical trial. No clinical efficacy was registered. Our concomitant immunological work presented here indicates that outcome under Audencel was influenced by the state of the immune system. On the other hand, Audencel also seemed to have stimulated the immune system. Overall, these immunological considerations suggest that DC immunotherapy against glioblastoma should be studied further – with the goal of translating an apparent immunological response into a clinical response. Future research should concentrate on investigating augmentation of immune reactions through combination therapies or on developing meaningful biomarkers

EVI1 promotes tumor growth via transcriptional repression of MS4A3

Background The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells. Methods U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice. Results Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis. Conclusions Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.(VLID)486732