Inhibition of the transcriptional repressor complex Bcl-6/BCoR induces endothelial sprouting but does not promote tumor growth.

The oncogenic potential of the transcriptional repressor Bcl-6 (B-cell lymphoma 6) was originally discovered in non-Hodgkin patients and the soluble Bcl-6 inhibitor 79-6 was developed to treat diffuse large B-cell lymphomas with aberrant Bcl-6 expression. Since we found Bcl-6 and its co-repressor BCoR (Bcl-6 interacting co-repressor) to be regulated in human microvascular endothelium by colorectal cancer cells, we investigated their function in sprouting angiogenesis which is central to tumor growth. Based on Bcl-6/BCoR gene silencing we found that the transcriptional repressor complex in fact constitutes an endogenous inhibitor of vascular sprouting by supporting the stalk cell phenotype: control of Notch target genes (HES1, HEY1, DLL4) and cell cycle regulators (cyclin A and B1). Thus, when endothelial cells were transiently transfected with Bcl-6 and/or BCoR siRNA, vascular sprouting was prominently induced. Comparably, when the soluble Bcl-6 inhibitor 79-6 was applied in the mouse retina model of physiological angiogenesis, endothelial sprouting and branching were significantly enhanced. To address the question whether clinical treatment with 79-6 might therefore have detrimental therapeutic effects by promoting tumor angiogenesis, mouse xenograft models of colorectal cancer and diffuse large B-cell lymphoma were tested. Despite a tendency to increased tumor vessel density, 79-6 therapy did not enhance tumor expansion. In contrast, growth of colorectal carcinomas was significantly reduced which is likely due to a combined 79-6 effect on cancer cells and tumor stroma. These findings may provide valuable information regarding the future clinical development of Bcl-6 inhibitors.

Overexpression of the transcriptional repressor complex BCL-6/BCoR leads to nuclear aggregates distinct from classical aggresomes.

Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed "nuclear aggresomes" and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical "nuclear aggresomes": Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones.

The differential activity of interferon-α subtypes is consistent among distinct target genes and cell types.

IFN-α proteins have been described to originate from 14 individual genes and allelic variants. However, the exceptional diversity of IFN-α and its functional impact are still poorly understood. To characterize the biological activity of IFN-α subtypes in relation to the cellular background, we investigated the effect of IFN-α treatment in primary fibroblasts and endothelial cells of vascular or lymphatic origin. The cellular response was evaluated for 13 distinct IFN-α proteins with respect to transcript regulation of the IFN-stimulated genes (ISGs) IFIT1, ISG15, CXCL10, CXCL11 and CCL8. The IFN-α proteins displayed a remarkably consistent potency in gene induction irrespective of target gene and cellular background which led to the classification of IFN-α subtypes with low (IFN-α1), intermediate (IFN-α2a, -4a, -4b, -5, -16, -21) and high (IFN-α2b, -6, -7, -8, -10, -14) activity. The differential potency of IFN-α classes was confirmed at the ISG protein level and the functional protection of cells against influenza virus infection. Differences in IFN activity were only observed at subsaturating levels of IFN-α proteins and did not affect the time course of ISG regulation. Cell-type specific responses were apparent for distinct target genes independent of IFN-α subtype and were based on different levels of basal versus inducible gene expression. While fibroblasts presented with a high constitutive level of IFIT1, the expression in endothelial cells was strongly induced by IFN-α. In contrast, CXCL10 and CXCL11 transcript levels were generally higher in endothelial cells despite a pronounced induction by IFN-α in fibroblasts. In summary, the divergent potency of IFN-α proteins and the cell-type specific regulation of individual IFN target genes may allow for the fine tuning of cellular responses to pathogen defense.

Neutralizing type I IFN antibodies trigger an IFN-like response in endothelial cells.

Neutralizing Abs to type I IFNs are of therapeutic significance, i.e., are currently evaluated for the treatment of autoimmune diseases with pathogenic IFN-alpha production such as for systemic lupus erythematosus. Unexpectedly, we observed that several neutralizing Abs reportedly known to counteract IFN-alpha or IFN-beta activity triggered an "IFN-like" response in quiescent primary human endothelial cells leading to activation of the transcription factor IFN-stimulated gene factor 3 and the expression of IFN-responsive genes. Furthermore, these Abs were found to enhance rather than inhibit type I IFN signals, and the effect was also detectable for distinct other cell types such as PBMCs. The stimulatory capacity of anti-IFN-alpha/beta Abs was mediated by the constitutive autocrine production of "subthreshold" IFN levels, involved the type I IFNR and was dependent on the Fc Ab domain, as Fab or F(ab')(2) fragments potently inhibited IFN activity. We thus propose that a combined effect of IFN recognition by the Ab paratope and the concomitant engagement of the Fc domain may trigger an IFN signal via the respective type I IFNR, which accounts for the observed IFN-like response to the neutralizing Abs. With respect to clinical applications, the finding may be of importance for the design of recombinant Abs vs Fab or F(ab')(2) fragments to efficiently counteract IFN activity without undesirable activating effects.

Retroviral targeting of proliferating endothelial cells.

Tumor growth requires the formation of new blood vessels by endothelial cells. Thus, surface molecules -- such as angiogenin receptors -- that are selectively expressed on growing endothelium represent an attractive target for directed delivery of compounds to tumor tissue. We attempted to obtain genetically engineered retroviral vectors targeted to the endothelium by inserting the human angiogenin sequence into Moloney murine leukemia virus envelope glycoprotein. Abundant expression of the chimeric protein could be verified. However, while being selective for proliferating human endothelial cells, the recombinant retroviral particles displayed low transduction efficiencies and thus have to be further improved.

Monitoring of circulating angiogenic factors in dendritic cell-based cancer immunotherapy.

BACKGROUND: A promising treatment approach for patients with malignant disease that recently has found its way into clinical trials is based on vaccination with autologous dendritic cells loaded with tumor antigens. However, adequate assays for monitoring clinical and immunologic responses still are under debate. In recent years, the determination of angiogenic markers has shown considerable potential in the diagnosis and prognosis of patients with malignant disease, because tumor growth and spread are promoted by angiogenesis, the formation of new blood vessels. METHODS: The authors established a method for measuring the plasma levels of three modulators of angiogenesis: vascular endothelial growth factor, platelet-derived endothelial cell growth factor, and thrombospondin-1. The angiogenic blood profile of a healthy control group was characterized and compared with a group of patients with malignant disease. Ultimately, levels of circulating angiogenic factors were monitored in the course of dendritic cell-based cancer immunotherapy. RESULTS: Baseline levels of angiogenic mediators varied substantially among healthy individuals but showed consistent values for each individual. Blood levels of circulating angiogenic factors were elevated significantly in patients with advanced disease and were highly sensitive to dendritic cell-based immunotherapy. CONCLUSIONS: To our knowledge, the current report was the first to analyze circulating levels of angiogenic factors during dendritic cell-based immunotherapy. The authors observed a noteworthy change in the angiogenic blood profile with treatment, and this change was correlated with the induction of an immunologic response.