Using quantitative real-time RT/PCR, we confirmed our previous microarray findings that showed that miR-21 is over-expressed in VSs. Consistent with our results, miR-21 has been shown to be over-expressed in a variety of solid tumors (25
). In addition, important links between miR-21 expression and cancer-related cellular processes such as proliferation, migration, apoptosis and tumor growth have been demonstrated in human breast and hepatocellular cancer cells (12
). Inhibition of miR-21 in hepatocellular carcinoma cell lines increased expression of tumor suppressor PTEN, and decreased cellular proliferation, migration and invasion; while enhancing miR-21 expression had the opposite effects (12
). Despite demonstrating the presence of PTEN mRNA in the four VSs tested, we observed very low PTEN protein levels in three of the four tumors tested. This may be due to translational suppression by miR-21. VS4 was an exception in that it displayed the highest level of PTEN protein; however, this tumor also exhibited the lowest relative level of miR-21 expression. Alternatively, PTEN mRNA in VS4 may be missing the miR-21 binding site in the 3′ untranslated region due to either gene mutations or alternative splicing and thereby escape post-transcriptional regulation. Since PTEN is a strong negative regulator of the AKT pathway, decreased expression of PTEN would enhance signaling through this pathway leading to increased cellular proliferation, decreased apoptosis, or both. Interestingly, the PI3 kinase/AKT pathway has recently been shown to be activated in human VSs (15
STAT3 is a critical regulator of gene expression in response to many growth factors and cytokines (17
). The neuropoietic cytokines IL-6, CNTF, and LIF use the common signal transducing gp130 subunit to mediate STAT1/3 signaling and play important roles in nerve regeneration and repair (18
). IL-6 has been shown to mediate the activation of STAT3 in adult Schwann cells and rat schwannoma cells (27
). Furthermore, activation of STAT3 by IL-6 induces miR-21 expression in multiple myeloma cells (16
). We postulated that VSs may be secreting neuropoietic cytokines and stimulating their own growth through an autocrine pathway mediated, in part, by the overexpression of miR-21. The detection of mRNAs encoding these cytokines and their receptor subunits in VS supports this possibility. That we were unable to detect the expression of IL-6, IL-6Rα, LIF and LIFR in normal vestibular nerve may reflect the absence or low abundance of these mRNAs in this quiescent, non-proliferative control tissue. Membrane tyrosine kinase receptors such as the platelet derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and erbB receptors have also been implicated in the activation of STATs (17
). The EGFR and neuregulin-1/erbB signaling pathways contribute to cell proliferation in NF2-deficient cells and VS primary cultures, respectively (22
). However, it is not clear to what extent STAT3 activation and miR-21 overexpression mediate this effect. A recently published case study demonstrating some effectiveness of the EGFR inhibitor erlotinib on VS progression in an NF2 patient suggests that targeted therapies for NF2-related tumors hold promise (30
). We demonstrated STAT3 activation using western blot analysis in one tumor (VS2) since its expression of miR-21 was the highest compared to the seven other tumors. Our objective was to verify that STAT3 was activated, i.e., phosphorylated (PSTAT3), in this tumor. Our finding suggest that STAT3 activation may be required for miR-21 expression in these VSs.
Recent studies shed additional light on the role of miR-21 in cancer-related processes. MCF-7 breast cancer cells transfected with anti-miR-21 oligonucleotides resulted in increased apoptosis and inhibition of cell growth in vitro
and tumor growth in a xenograft mouse model (13
). Using similar in vitro
approaches, we found that miR-21 contributes to the proliferative potential and survival of VS cells, confirming the functional significance of miR-21 over-expression in these cells. The tumor suppressor PDCD4 (programmed cell death 4) is an important functional target of miR-21 and the down-regulation of PDCD4 by miR-21 in colorectal cancer stimulated invasion, extravasation, and metastasis (31
). It would be interesting to know whether PDCD4 is a target of miR-21 in VSs. In addition to supporting VS cell proliferation and survival, there is some evidence suggesting that miR-21 may also contribute to the cystic phenotype that is occasionally observed in more aggressive tumors. MiR-21 increases matrix metalloprotease-2 (MMP-2) expression in cardiac fibroblasts via the PTEN pathway and MMP-2 has been implicated in cyst development in VSs (33
It is clear that miR-21 plays a significant role in the regulation of multiple pathways controlling cell proliferation in many cancers and this attribute makes it a very attractive target for the development of new therapies. Future experiments aimed at manipulating miR-21 expression in xenograft or genetic animal schwannoma models will allow us to test the relative importance of miR-21 on VS growth in vivo
. Furthermore, the application of proteomics strategies in these cells should help identify new molecular targets of miR-21 as has been demonstrated in the MCF-7 breast cancer cell line (20
). It has also been shown that miR-21 plays a role in non-neoplastic processes. Cai et al. outlined the potential role of miR-21 in the molecular management of heart disease (36
). The application of these methodologies and findings in other biologic systems may shed new light on the molecular pathways controlling tumor growth and help identify novel therapeutic targets for the treatment of NF2 or sporadic VS.
Should miR-21 be confirmed to be integral to the growth and regulation of VS, it is possible to clinically manipulate this biologic phenomenon. Using molecules that can bind and interfere with these microRNAs, i.e., RNA interference (RNAi), would be one strategy to down regulate this pathway. Early clinical trials using this approach have been used in patients with macular degeneration and respiratory syncytial virus infections (37
). A miR-21 specific RNAi knockdown could be achieved systemically by administering a viral gene expression vector; however, the side-effects on a patient may be undesirable because of off target effects. A more focused delivery via an endoscopic direct injection, or delivery during an intentionally incomplete resection of the VS could be an alternative method.