In this study, we defined a specific miRNA profile found in most sporadic benign human meningioma tumors. Among dysregulated miRNAs, miR-200a was one of the most downregulated in meningiomas compared to the arachnoidal tissues from which these tumors arise. Moreover, we found that miR-200a functions as a potential tumor suppressor by inhibiting Wnt/β-catenin signaling through two complementary mechanisms: direct targeting of the β-catenin mRNA, leading to reduced β-catenin levels, and targeting of the mRNAs for ZEB1 and SIP1 with consequent upregulation of E-cadherin levels and sequestration of β-catenin. Dysregulation of Wnt/β-catenin signaling and the E-cadherin cell adhesion system have been implicated as important events in the initiation and/or progression of several forms of cancer (3
). These results provide insight into the mechanism by which reduced levels of miR-200a promote meningioma tumor growth through elevation of β-catenin and Wnt signaling, as well as reduction of E-cadherin.
To our knowledge, this is the first report analyzing the miRNA profiles of human meningioma tumors. This study focused on miR-200a, the most downregulated miRNA (about 25-fold) in 10 out of 14 meningioma tumors (7 with NF2
deleted and 7 without deletion). Elevation of miR-200a in meningioma cells reduced their apparent growth rate in culture via induction of apoptosis and suppressed tumor growth in vivo in a xenograft model. Recent studies showed that the members of the miR-200 family, including miR-200a, inhibit EMT as the initial step in tumorigenesis by directly targeting mRNAs for the transcriptional repressors ZEB1 and SIP1 (4
). Reduction of miR-200 members leads to increased levels of ZEB1 and SIP1, which bind to an E box in the promoter of the E-cadherin gene, thereby downregulating E-cadherin expression. Consistent with these previous reports, our studies showed that increased miR-200a levels act to downregulate the expression of ZEB1 and SIP1 in meningioma cells, with a commensurate increase in E-cadherin expression.
With respect to the role of miR-200a in β-catenin signaling, we found that miR-200a directly targets the 3′ UTR of β-catenin mRNA. To our knowledge, miR-200a is the first miRNA shown to be involved in the regulation of β-catenin levels. Given the wide role of Wnt/β-catenin signaling in many cancers, including colon cancer, hepatocellular carcinoma, melanoma, and ovarian and prostate cancers (33
), we presume that reduction in miR-200a may contribute to increased β-catenin signaling in other forms of cancer.
To investigate the downstream effect of miR-200a levels on β-catenin-responsive promoters, we overexpressed miR-200a in meningioma cells and monitored the activity of a promoter containing T-cell factor/lymphoid enhancer factor binding sites driving luciferase (Lenti-TOPFLASH [26
]) in response to the Wnt ligand, mWnt3a. Overexpression of miR-200a strongly inhibited the response of this promoter to Wnt induction, and this suppression could be overcome with an antisense inhibitor of miR-200a. Moreover, we found that β-catenin overexpression via an expression vector, Ad-β-catenin, significantly overcame the growth inhibition caused by elevated miR-200a in meningioma cells. Taken together, these data suggest that low levels of miR-200a in meningioma cells activate Wnt signaling by increasing the levels of β-catenin mRNA and its availability to enter the nucleus and activate genes supporting the growth of meningioma tumors. Furthermore, our data suggest that miR-200a can act as a key “negative regulator” of the Wnt/β-catenin signaling pathway and hence a potential therapeutic agent, with elevation of miR-200a levels inhibiting the growth of meningioma tumors in a xenograft model. The stability of β-catenin in the cytoplasm is tightly regulated by a degradation complex containing adenomatous polyposis coli (APC), axin, casein kinase 1, and glycogen synthase kinase 3 that marks β-catenin for proteolytic destruction (16
). Mutations in the APC
gene lead to constant activation of Wnt/β-catenin signaling as one of the main players in cancer (13
). Interestingly, in a recent report, miR-135a and -b were shown to target the 3′ UTR of the APC transcript and to suppress its expression in colon carcinoma (30
). Thus, potentiation of β-catenin/Wnt signaling seems to be a critical step in cancer that can be regulated by miRNAs.
We also evaluated the role of miR-200a in the level and localization of β-catenin in normal primary human arachnoidal cells in culture as the cell of origin of meningioma tumors. When levels of miR-200a were decreased by transfection with a specific inhibitor, the levels of β-catenin increased, and its localization changed from primarily at the plasma membrane to a more cytoplasmic and nuclear location. This observation is consistent with loss of membranous β-catenin immunostaining in most human meningioma tumors (3
). Together, these data implicate the downregulation of miR-200a in activation of Wnt/β-catenin signaling in the initial steps of tumorigenesis in meningiomas.
qRT-PCR analysis of β-catenin mRNA in human meningioma tumor samples demonstrated a significant inverse correlation between the levels of miR-200a and β-catenin mRNA. This increase in β-catenin mRNA levels in meningiomas appeared to have functional consequences, as the levels of a downstream target gene of the β-catenin nuclear complex, the cyclin D1 gene, were significantly elevated in meningioma tissue with low levels of miR-200a. On the other hand, we did not find a correlation between upregulation of c-myc, encoded by another β-catenin target gene, and downregulation of miR-200a in meningioma tissues (data not shown), possibly due to other modes of c-myc regulation in tumors (35
Our findings also support a role for E-cadherin in miR-200a-mediated growth inhibition of meningioma cells, as an siRNA for the E-cadherin message substantially blocked growth inhibition of meningioma cells resulting from elevated miR-200a. Reduced expression of miR-200a increases the levels of the transcriptional repressors ZEB1 and SIP1, which in turn downregulate E-cadherin in meningiomas, as shown by immunohistochemistry of meningioma tumors (3
). E-cadherin levels were also low in arachnoidal tissue and meningioma tumors that had no decrease in miR-200a levels, so a deficiency in miR-200a expression appears to be only one of the factors responsible for the E-cadherin reduction in meningiomas.
The activity of the Wnt/β-catenin signaling pathway is mainly determined by the amount of β-catenin free in the cytoplasm versus that sequestered in the plasma membrane or APC degradation complex (28
). Decreased levels of E-cadherin in meningioma cells should thus make more β-catenin available in the cytoplasm/nucleus, at the same time possibly decreasing links between E-cadherin and the cytoskeleton. The latter effect should also reduce cell-to-cell adhesion of arachnoidal cells as part of the tumorigenesis process. Disruption of cell-cell communication through miR-200a downregulation may thus promote meningioma tumor growth and progression through both decreased E-cadherin and elevated β-catenin levels.
In conclusion, this study has defined an miRNA signature of benign meningioma tumors and demonstrated a key role for miR-200a downregulation in promoting the growth of these tumors. Reduced levels of miR-200a appear to contribute to tumorigenesis through two converging pathways mediated by upregulation of three mRNA targets. Those for ZEB1 and SIP1 lead to increased levels of these transcriptional repressors, which decreases the levels of E-cadherin in the tumors. Upregulation of a new β-catenin mRNA target for miR-200a, discovered in this study, results in more β-catenin being available to enter the nucleus and activate genes in the Wnt signaling pathway. These miR-200a-mediated tumorigenic processes are undoubtedly shared with other tumors and provide potential targets for therapeutic intervention.