Expression of vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 in primary and recurrent WHO grade III meningiomas

WHO grade III meningiomas are malignant neoplasms for which new and more targeted treatment strategies are urgently needed. Although clinical trials investigating anti-angiogenic vascular endothelial growth factor (VEGF) targeted therapies are currently recruiting, knowledge about the expression of VEGF and VEGF receptors remains to be determined. Methods: We investigated the expression of VEGF and its receptors VEGFR1 and VEGFR2 in 32 WHO grade III meningioma samples by immunohistochemistry. Furthermore, we performed in-situ hybridisation for VEGF. Results: We found low VEGF expression in tumor and endothelial cells. Highest VEGF expression levels were seen in peri-necrotic tumor cells potentially suffering from hypoxia. VEGFR1 and 2 were virtually absent on tumor cells, although endothelial cells displayed significantly higher levels reaching stronger expression for VEGFR2 than VEGFR1. Conclusions: Our findings showing constant expression levels of VEGFR2 in endothelial cells serve as a first indication that the use of small tyrosine kinase inhibitors such as Sunitinib directly targeting the VEGF-receptors might be worth testing, also in the clincial context in cases of therapy-refractory meningiomas. Further investigations are needed to study the response to drugs targeting the VEGF pathway in relation to the expression profile of VEGF and its receptors in high grade meningiomas

Pericytes/vessel-associated mural cells (VAMCs) are the major source of key epithelial-mesenchymal transition (EMT) factors SLUG and TWIST in human glioma

Epithelial-to-mesenchymal transition (EMT) is supposed to be responsible for increased invasion and metastases in epithelial cancer cells. The activation of EMT genes has further been proposed to be important in the process of malignant transformation of primary CNS tumors. Since the cellular source and clinical impact of EMT factors in primary CNS tumors still remain unclear, we aimed at deciphering their distribution in vivo and clinico-pathological relevance in human gliomas. We investigated 350 glioma patients for the expression of the key EMT factors SLUG and TWIST by immunohistochemistry and immunofluorescence related to morpho-genetic alterations such as EGFR-amplification, IDH-1 (R132H) mutation and 1p/19q LOH. Furthermore, transcriptional cluster and survival analyses were performed. Our data illustrate that SLUG and TWIST are overexpressed in gliomas showing vascular proliferation such as pilocytic astrocytomas and glioblastomas. EMT factors are exclusively expressed by non-neoplastic pericytes/vessel-associated mural cells (VAMCs). They are not associated with patient survival but correlate with pericytic/VAMC genes in glioblastoma cluster analysis. In summary, the upregulation of EMT genes in pilocytic astrocytomas and glioblastomas reflects the level of activation of pericytes/VAMCs in newly formed blood vessels. Our results underscore that the negative prognostic potential of the EMT signature in the group of diffuse gliomas of WHO grade II-IV does most likely not derive from glioma cells but rather reflects the degree of proliferating mural cells thereby constituting a potential target for future alternative treatment approaches

Differential expression of the tumor suppressor A-kinase anchor protein 12 in human diffuse and pilocytic astrocytomas is regulated by promoter methylation

The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY® of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12α promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12α promoter methylation and markedly increased AKAP12α mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined

Nuclear localization of netrin-1 in brain metastases.

<p>(A) Box plots showing different expression profiles of nuclear netrin-1 in brain metastases of the tumor types indicated (see also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092311#pone.0092311.s004" target="_blank">Table S1</a></b>). (B, upper) Example of NSCLC with strong cytoplasmic and nuclear netrin-1 expression. Dotted line shows tumor to mesenchymal stroma border with a regionally heterogenous netrin-1 expression pattern. While tumor cells adjacent to the stroma show strong nuclear staining, tumor cells inside the tumor nodule exhibit weaker netrin-1 expression without showing a nuclear localization. (B, lower) Brain metastases of breast carcinoma exclusively showing nuclear netrin-1 expression (scale bar 50 μm). (C) Comparison of netrin-1 protein expression and mRNA levels in an exemplaric case of melanoma brain metastasis (scale bar 50 μm). While netrin-1 protein can be detected in cytoplasms but stronger in nuclei, in-situ hybridization reveals a mild cytoplasmic staining compared to stronger signals within the nucleus.</p

Univariate survival analysis for total netrin-1 and nuclear netrin-1 expression.

<p>Univariate Kaplan-Meier survival analysis showing poor prognosis for high netrin-1 tumor cell scores in patients with brain metastases. Survival probability of (A) all brain metastases (entire cohort) and (B) the subentity of NSCLC. (C, D) Kaplan-Meier survival analysis showing worse survival in brain metastases patients when netrin-1 locates to the nucleus in (C) the total cohort and (D) subgroups comparing patients without nuclear netrin-1 to the cohort exhibiting highest nuclear netrin-1 levels. (Low netrin-1 score4; low nuclear netrin-1< or = 5% or high nuclear netrin-1>5%.).</p

Netrin-1 is strongly expressed in tumor cells of human brain metastases.

<p>Arrowheads in (A) indicate different subcellular localisation. Brain metastases of NSCLC and SCLC show strong cytoplasmic staining, while the depicted breast carcinoma shows an additional membraneous signal (see blow up and arrowheads). Nuclear localization is exemplarily shown in melanoma (see blow up and arrowhead) and colon cancer metastases samples (scale bar 100 μm). Box plots of netrin-1 tumor cell scores are shown in (B) (see also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092311#pone.0092311.s004" target="_blank">Table S1</a></b>). In-situ hybridization reveals tumor cells and endothelial cells (arrowheads) as source for netrin-1 expression (C: example of a SCLC, scale bars 50 μm).</p

Multivariate survival analysis including all brain metastasis patients.

<p>Cox hazard ratios of nuclear netrin-1, Ki67, patient age and number of brain metastases are depicted (single comparisons of diagnoses and edema scores are not shown. n/a not applicable). n/a: not assessed.</p

Netrin-1 expression is an independent prognostic factor for poor patient survival in brain metastases

The multifunctional molecule netrin-1 is upregulated in various malignancies and has recently been presented as a major general player in tumorigenesis leading to tumor progression and maintenance in various animal models. However, there is still a lack of clinico-epidemiological data related to netrin-1 expression. Therefore, the aim of our study was to elucidate the association of netrin-1 expression and patient survival in brain metastases since those constitute one of the most limiting factors for patient prognosis. We investigated 104 brain metastases cases for netrin-1 expression using in-situ hybridization and immunohistochemistry with regard to clinical parameters such as patient survival and MRI data. Our data show that netrin-1 is strongly upregulated in most cancer subtypes. Univariate analyses revealed netrin-1 expression as a significant factor associated with poor patient survival in the total cohort of brain metastasis patients and in sub-entities such as non-small cell lung carcinomas. Interestingly, many cancer samples showed a strong nuclear netrin-1 signal which was recently linked to a truncated netrin-1 variant that enhances tumor growth. Nuclear netrin-1 expression was associated with poor patient survival in univariate as well as in multivariate analyses. Our data indicate both total and nuclear netrin-1 expression as prognostic factors in brain metastases patients in contrast to other prognostic markers in oncology such as patient age, number of brain metastases or Ki67 proliferation index. Therefore, nuclear netrin-1 expression constitutes one of the first reported molecular biomarkers for patient survival in brain metastases. Furthermore, netrin-1 may constitute a promising target for future anti-cancer treatment approaches in brain metastases