We reveal here that constitutively active EGFRvIII cooperates with the loss of Pten to synergistically induce expression of miR-146a in Ink4a/Arf−/− astrocytes. Counterintuitively, upregulation of miR-146a inhibits tumor growth and the formation and migration of glioma stem-like cells by both malignant murine Ink4a/Arf−/− Pten−/− EgfrvIII astrocytes and human glioblastoma cells. We further show for the first time that miR-146a directly downregulates Notch1 and potentiates differentiation of normal NSCs. Knocking down miR-146a function enhances glioma stem-like cell formation and exacerbates tumor burden. These data suggest that miR-146a integrates oncogenic cues to restrict tumor development as a feedback mechanism (H).
Previously, miR-146a was shown to be induced by endotoxin (lipopolysaccharide) through two consensus NF-κB binding sites in the promoter region (9
). This region is highly homologous between human and mouse, suggesting an evolutionarily conserved regulatory mechanism for controlling miR-146a expression. NF-κB is constitutively activated in glioma and many other cancer cells, in which it promotes survival and metastatic potential of these cells as well as tumorigenesis (28
). One of the key pathways that controls NF-κB activity in gliomas is the phosphatidylinositol 3-kinase (PI3K) pathway (16
), which is a converging point of EGFR- and PTEN-dependent signal transduction. Activation of receptor tyrosine kinase EGFR leads to recruitment and activation of PI-3 kinase, which phosphorylates phosphoinositol lipids to generate phosphatidylinositol-3-phosphates. These lipids in turn recruit and activate AKT in the plasma membrane through PDK1. The function of PI3K is antagonized by PTEN, a lipid phosphatase that dephosphorylates phosphatidylinositol 3,4,5-trisphosphate. Activated AKT further phosphorylates IKK, thus promoting NF-κB activation. The synergistic induction of miR-146a by constitutively active EGFRvIII
and the inactivation of Pten
astrocytes may reflect a convergence of these two signaling pathways on NF-κB activity. Activated AKT also promotes Notch1 expression, which subsequently modulates transcription of Egfr
through p53 (44
). These data indicate that miR-146a belongs to an integrated genetic circuit consisting of the PTEN, EGFR, NF-κB, and Notch pathways. The results from our present study reveal that miR-146a regulates the activity of this circuit by targeting Notch1 expression.
Recent studies indicate that certain miRNAs (such as miR-124, miR-137, miR-128, and miR-7) function as tumor suppressors (19
). These miRNAs are rarely expressed in gliomas; however, their overexpression restrains proliferation and self-renewal of glioma stem-like cells by promoting neural differentiation (20
). miR-146a is unique in that it is significantly enriched in the human tissues of skin (melanoma), cervical, breast, pancreas and prostate cancers compared to the same noncancerous tissues (42
). Similarly, miR-146a was also upregulated in human glioblastoma tissues and in both human and mouse primary glioma cell lines (32
). In this regard, increased expression of miR-146a can be viewed as a biomarker for cancers. Unexpectedly, our study reveals that, instead of promoting gliomagenesis through its upregulation, miR-146a rather plays an inhibitory role in restricting the formation of glioma stem-like cells and tumor burden. This result is consistent with a demonstrated role of miR-146a in other cancers, such as pancreatic and breast cancers, where it inhibits cancer progression and invasion (8
). A recent report also showed a positive correlation of miR-146a expression with the survival time of gastric cancer patients (25
). Furthermore, the overexpression of miR-146a inhibits the proliferation and survival of breast, prostate, and pancreatic cancer cells through the downregulation of other targets in these cells, including ROCK1, EGFR, and MTA-2 (8
). Our study adds Notch1 as a major target of miR-146a in glioma cells. Supporting these cell culture models of tumorigenesis, it was recently reported that mice with a deletion of miR-146a spontaneously developed subcutaneous flank tumors (37
). These data clearly indicate that miR-146a serves as a native molecular brake for oncogenesis (2
In summary, our current results and other emerging data indicate that miR-146a constitutes an endogenous feedback system to counteract the oncogenic potential of dysregulated signaling pathways, such as activation of EGFR and inactivation of Pten in gliomas. By regulating multiple targets, including key neural stem cell factor Notch1, a miR-146a-mediated innate regulatory mechanism provides an opportunity to devise novel therapeutic strategies against aggressive and deadly brain tumors.