Microarray gene expression analysis revealed differential expression of members of the RD transcriptional network, PAX6, EYA4, SIX1, and DACH1, in MPNSTs relative to normal human Schwann cells. The NF1-GRD induced DACH1 expression, implicating Ras signaling in the decreased DACH1 expression seen in MPNSTs. Expression of EYA4 was dramatically upregulated in MPNST cells and primary tumors, and inhibition of EYA4 expression using shRNA reduced MPNST cell adhesion and migration and induced cell necrosis. Furthermore, tumorigenesis was profoundly inhibited when MPNST cells stably expressing shEYA4 RNA were injected into athymic nude mice, again correlating with necrosis. Our previous study (Miller et al., 2009) provided evidence that overexpression of SOX9 is sufficient to induce EYA4 expression. We propose a model in which overexpression of SOX9 and loss of NF1 functions independently lead to dysregulation of the EYA4-SIX1-DACH1 transcription in MPNST.
The most dramatic differential expression was observed for EYA4
, which was robustly overexpressed in all but one MPNST cell line and all primary MPNSTs, suggesting it and/or its family members, may represent an MPNST oncogene. Its activity may be context-specific, given other reports implicating EYA4
as a tumor suppressor gene in gastrointestinal tumors (Zou et al. 2005
; Osborn et al. 2006
). The expression pattern of PAX6, SIX1, EYA4
, and DACH1
in MPNST cells suggests a scenario whereby expression of multiple members of a potential transcriptional network is dysregulated in a manner that promotes tumorigenesis. High levels of PAX6, EYA4
, and SIX1
expression in combination with low levels of DACH1
expression are consistent with previous studies supporting a tumor promoting role for PAX (Maris et al. 1997
; Muratovska et al. 2003
), EYA (Ramdas et al. 2001
), and SIX (Christensen et al. 2008
) family members, and a tumor suppressor role for DACH1 (Wu et al. 2006
; Wu et al. 2008
; Wu et al. 2009
). However, the existence of a transcriptional network involving these genes has not been demonstrated here and has not been definitively identified in mammalian cells.
This study and our previous studies used expression differences between cultured Schwann cells and MPNST cells to identify genes relevant to tumorigenesis (Miller et al., 2006
, 2009). Gene expression changes are induced by cell isolation and tissue culture, and can interfere with the identification of relevant genes. In spite of these potential problems, our approach is supported by in vivo
validation of the microarray data in solid tumors for EYA4, coupled with functional validation in xenografts for EYA4.
Replacement of the NF1-GRD in MPNST cells rescued DACH1
expression but did not alter transcription of EYA
at 18 – 32 hours, although we cannot exclude the possibility that at other time points or levels of GRD expression transcription of EYA
might be changed. It is possible that a primary effect of loss of NF1 on DACH1
secondarily affects transcription of the other members of the complex. Indeed, several lines of evidence support feedback activation of the network members on each other (O'Neill et al. 1994
). Furthermore, while only some neurofibroma samples have elevated SIX1
expression was low in many neurofibroma Schwann cell cultures and primary neurofibromas. Thus, our data support the idea that decreased DACH1
expression is an early event downstream of NF1 loss.
EYA4 was up-regulated in NF1-related and in sporadic MPNST cell lines, although Ras-GTP is not elevated in the same sporadic MPNST cells (Mahller et al. 2006
). In addition, EYA4
expression was not affected by replacing the NF1-GRD in NF1-deficient MPNST cells, suggesting NF1-Ras signaling does not regulate EYA4
expression. In Drosophila it has been shown that Ras signaling phosphorylates EYA proteins, increasing its transcriptional activity (Hsiao et al. 2001
; Silver et al. 2003
). Thus, post-transcriptional regulation of EYA4 by the NF1-Ras pathway warrants future investigation. The mechanism(s) underlying high EYA4 expression in MPNST remain to be definitively determined but is likely to involve elevated SOX9 expression. SOX9
expression is elevated in both NF1-related and sporadic MPNST cell lines, and increasing SOX9 in wild type Schwann cells is sufficient to increase EYA4
mRNA expression, while decreasing SOX9
mRNA levels in MPNST cells decreases EYA4
expression (Miller and Jessen et al.
Our results provide several lines of evidence supporting a role for EYA4 in cell survival, specifically in inhibition of cell death by necrosis. Cell death is generally classified as either apoptosis or necrosis, a major distinction being the early breakdown of the plasma membrane in necrotic cells (Kroemer et al. 2009
). Molecular mechanisms regulating necrosis are not well understood, however overactivation of poly (ADP-ribose) polymerase (PARP) in response to extensive DNA damage results in depletion of ATP which can convert apoptosis to necrosis (Ha et al. 1999
). The absence of changes in proliferation and apoptosis along with an increase in propidium iodide uptake suggested that necrosis may be the cause of decreased cell numbers in MPNST cells with reduced EYA4 expression. Further investigation in vitro
supported necrotic cell death as increased side scatter was observed in FACS analysis. In addition, tumors that developed in xenografts of MPNST cells with low levels of EYA4 showed significant areas of necrosis. In other systems, loss of EYA induces apoptosis (Cook et al. 2009
), suggesting the mechanism of cell death resulting from decreased EYA may depend upon co-regulatory factors. Our data also support a role for EYA4 in MPNST cell adhesion and migration both of which are inhibited in response to reducing EYA4 expression. A reduction in cell adhesion can prompt anoikis, however, anoikis is usually associated with apoptosis rather than necrosis. Cellular changes downstream of EYA4, including cell adhesion, migration, and survival, may reflect the metastatic behavior of MPNSTs (Klymkowsky et al. 2009
) and provoke further investigation.
We conclude that the NF1-Ras pathway can regulate expression of DACH1, directly or indirectly, and EYA4 expression controls adhesion, migration, and blocks necrosis in MPNST cells. Inhibition of EYA4 reduced tumorigenic properties of MPNST cells in vitro and resulted in MPNST cell death ex vivo, suggesting that developing therapeutics aimed at diminishing EYA4 expression or EYA4 transcriptional targets represents a strategy for killing MPNST cells.