Roles for the merlin tumour suppressor have been characterized in many cell types and interaction of merlin with many other proteins regulates a diverse number of cell functions ranging from signalling at the cell membrane to controlling protein degradation in the nucleus (McClatchey and Giovannini 2005
; Ammoun et al., 2008
; McClatchey and Fehon 2009
; Li et al., 2010
; Cooper et al., 2011
). The hallmark feature of neurofibromatosis type 2 is the occurrence of Merlin-null vestibular schwannomas, often bilaterally, and currently there is no effective clinical treatment for these tumours (Hanemann, 2008
). Whereas a lot is now known about the transcription factors and signalling that control normal Schwann cell–axon interaction, cell cycle exit and differentiation, little is known about how merlin loss impacts on these processes and leads to tumour development. We have performed a number of experiments to examine the process of differentiation and cell cycle exit in human Merlin-null schwannoma cells. The zinc finger transcription factor KROX20 is a key regulator of Schwann cell differentiation, regulating both cell cycle exit and the gene expression that drives myelination (Topilko et al., 1994
; Zorick et al., 1999
; Parkinson et al., 2004
). The normal control by KROX20 of cell cycle exit and myelin gene induction, using enforced KROX20 expression, is unchanged in Merlin-null schwannoma cells, but the ability of these cells to induce endogenous markers of differentiation, such as KROX20 and OCT6, in response to a myelination cue is markedly reduced, implying a Merlin-dependent loss of correct signalling upstream of KROX20. The mouse SC4 immortalized Merlin-null schwannoma cell line has been widely used in understanding the biology of merlin (Morrison et al., 2007
; Hennigan et al., 2012
). Interestingly, in SC4 cells, we did not observe a significant increase in either periaxin expression or downregulation of c-Jun when KROX20 was expressed (data not shown). We presume that the additional genetic events in the immortalization of these cells preclude their normal differentiation in response to KROX20 in contrast to primary human Schwann or schwannoma cells.
The transcriptional control of OCT6 and KROX20 have been well characterized in Schwann cells, and both rely on the high mobility group transcription factor SOX10 for their expression in developing Schwann cells (Ghislain and Charnay 2006
; Finzsch et al., 2010
; Reiprich et al., 2010
; Bremer et al., 2011
). Correspondingly, we have found reduced levels of SOX10
messenger RNA and protein in all the human schwannoma tumours we have examined, and in further confirmation of the role of SOX10, re-expression of SOX10 in human schwannoma cells reverts many of the changes seen in these cells back to a normal phenotype, critically including restoring the capacity to express myelin proteins in vitro
and suppressing basal and PDGF-induced proliferation.
To separate the effects of merlin and SOX10 loss in Schwann cells, we have used small interfering RNA and mice with conditional alleles of these genes, and, both in vitro
and in vivo
, we see that loss of merlin has no apparent direct effect on SOX10 expression in Schwann cells. Analysis of the role of SOX10 in Schwann cells in vivo
has shown that it is required for normal Schwann cell–axon interaction, cell cycle withdrawal, expression of both OCT6 and KROX20 and the normal induction and maintenance of myelination in peripheral nerves. In addition, loss of SOX10 leads to an increase in other cell types within the nerve, such as endothelial cells and pericytes and an apparent decrease in S100-expressing Schwann cells (Finzsch et al., 2010
; Bremer et al., 2011
), although we, in agreement with other studies (Rosenbaum et al., 1998
; Kanaan et al., 2008
), find no decrease in S100 staining in the schwannoma tumours we have analysed; in accordance with this, we find no increase in S100β messenger RNA levels when SOX10 is re-expressed in schwannoma cells (data not shown).
Our examination of SOX10-null mouse Schwann cells in vitro
shows that loss of SOX10 alone replicates phenotypic changes associated with human schwannoma cells, such as increased numbers of focal adhesions, increased proliferation and increased expression of and signalling through the PDGF receptor beta (PDGFRB). From these data, we would propose a model by which loss of Merlin and SOX10 contribute to the phenotype of human schwannoma cells. For the example of the PDGFRB, loss of Merlin in cells only causes a modest (1.3-fold) increase in PDGFRB messenger RNA levels but is involved in recycling of the receptor protein at the cell surface (Lallemand et al., 2009
), whereas our data show that SOX10 appears to transcriptionally repress the PDGFRB gene; thus, the combined effects of both merlin and SOX10 loss will lead to the large increases seen in PDGFRB protein levels in human schwannoma cells. Similarly, for CD44
messenger RNA, which encodes the hyaluronate receptor that transmits proliferative signalling in Merlin-null cells (Morrison et al., 2001
; Bai et al., 2007
), SOX10 transcriptionally represses CD44
messenger RNA levels when reintroduced into human schwannoma cells.
The mechanism by which SOX10 expression is lost in human schwannomas is unclear; the simplest explanation would be that loss of merlin leads to changes in cell signalling that repress SOX10
transcription, but our in vitro
and in vivo
analysis show this to not be the case. Analysis of both Merlin-null knockout cells and Merlin re-expression experiments in human schwannoma cells shows that the acute loss of Merlin has no effect on levels of SOX10. What regulates SOX10 in Schwann cells is presently unclear, but an upstream enhancer, known as U3, contributes to SOX10 expression and can be activated by a number of transcription factors such as SOX10, SOX9, AP2α (TFAP2A) and FOXD3 in vitro
(Wahlbuhl et al., 2012
). A role for activated Notch has also been shown in repressing SOX10 expression in Schwann cells (Li et al., 2004
), and Merlin loss in Drosophila
leads to increased Notch signalling (Maitra et al., 2006
), but inhibition of Notch signalling by the γ-secretase inhibitor N
-(3, 5-difluorophenacetyl)-lalanyl]-S-phenylglycine t-butyl ester; 25 µM of the γ-secretase SOX10 expression in schwannoma cells (data not shown).
The key to the loss of SOX10 in human schwannomas, we propose, may be the chromosomal location of SOX10 and Merlin on chromosome 22. Remarkably, the two genes are close to one another on the long arm of chromosome 22 (merlin 22q12.2, SOX10 22q13.1). The second ‘hit’ and loss of merlin function in neurofibromatosis type 2 commonly involves loss of heterozygosity or a large deletion of chromosome 22, and our meta-analysis of cytogenetic studies on schwannoma tumours indicates that the region encoding SOX10 would be lost in >50% of these tumours (Bruder et al., 1999
; Antinheimo et al., 2000
; Mantripragada et al., 2003
; Koutsimpelas et al., 2011
), although this is probably an underestimate as the chromosomal marker resolution of several of these studies is not particularly good. SOX10 has been shown to bind and activate its own promoter (Wahlbuhl et al., 2012
); therefore, it is possible that the loss of one allele could lead to the transcriptional silencing of the remaining intact allele in Schwann cells and the corresponding fall in both SOX10 messenger RNA and protein observed in schwannoma tumours. Another alternative, which has been proposed, given the differences observed between patients with neurofibromatosis type 2 and the phenotype of mice with Merlin-null Schwann cells, is that there is a third ‘hit’ at another locus in addition to the loss of merlin (Woods et al., 2003
). Such an additional genetic change could directly or indirectly affect SOX10 expression and contribute to the phenotype we observe in human schwannoma cells. The production and characterization of a conditional SOX10 allele, and the separation of merlin and SOX10 on different mouse chromosomes (chromosomes 11 and 15, respectively) would now allow the testing of the effects of SOX10 hemizygosity or complete loss on the biology of Merlin-null cells and may lead, perhaps, to the development of a mouse model that more closely matches the human disease.
In conclusion, we have identified consistent loss of SOX10 in human schwannoma cells, and our results identify that this loss strongly contributes to the phenotype of this tumour cell type. These results, for the first time, provide a direct link between the processes involved in normal Schwann cell differentiation and homeostasis in the peripheral nerve and the development and pathology of schwannoma tumours. Further characterization of the relative contributions of merlin and SOX10 to the biology of schwannoma cells may give important insights into how to treat these clinically important tumours.