Microarray analysis of gene expression in vestibular schwannomas reveals SPP1/MET signaling pathway and androgen receptor deregulation

Abstract

Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (Nrg1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan low density arrays. We performed a mutational analysis of NF2 by PCR/denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)/B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatment.

Figure 1

Three-dimensional representation of principal component analysis. Grey points represent 31 vestibular schwannomas, while the 9 controls are shown in white. The more remote control corresponds to human Schwann cell culture. Schwannomas appear tight together, contrary to the controls, which are less uniform.

Figure 2

Cluster of samples. Hierarchical cluster of Euclidean distances of NM set of probes from RefSeq annotation. Controls and tumors are clearly grouped, whereas schwannomas show a similar pattern.

Figure 3

Volcano plot resulting from the comparison of schwannomas to controls. Dotted lines represent 2-fold (vertical) and p<0.05 cut-off (horizontal). Only grey points matched these criteria.

Figure 4

Microarray and qRT-PCR comparison. Fold change of 33 genes obtained by both microarray (black lines) and qRT-PCR analysis (grey lines). Values more than or equal to 1 represent upregulation and more than 1 downregulation in schwannomas. By the qRT-PCR method, gene deregulation was usually higher, due to the wider dynamic range of this technique compared to the microarray analysis.