Although our previous studies had indicated that aberrant FoxM1B expression is critical to the regulation of tumorigenicity, invasion, and angiogenesis of human glioma cells, it was not known whether FoxM1B plays an important role in the transformation of human astrocytes into glioma cells. In this study, we found that overexpression of FoxM1 in immortalized human astrocytes not only transforms the cells but also causes them to progress into GBM cells in the brain. We also provided evidence that FoxM1 up-regulated the expression of NEDD-4, which resulted in increased PTEN protein degradation. Furthermore, the increased PTEN protein degradation in turn led to the up-regulation of activation of Akt pathway, a potential mechanism for the development of GBM.
Glioma formation and progression are widely regarded as multistep processes resulting from complex interplay between multiple genetic and epigenetic events (3
). Studies have shown that the loss of p53 and pRb pathways is not sufficient to induce glioma and that additional molecular events are needed such as Ras activation and PTEN loss (40
). Our results support this finding; specifically, we found that a combinationof FoxM1 overexpression, loss of p53 and pRb pathways, and human telomerase reverse transcriptase overexpression is sufficient to induce NHA cells to transform into glioma.
Moreover, our results support the involvement of PTEN underexpression in transformation and in GBM formation. Brain-specific inactivation of PTEN in a mouse model caused increased astrocyte proliferation that may render these cells susceptible to neoplastic transformation (42
). PTEN heterozygous knockout accelerated astrocytoma development in mice in which the Rb family proteins were inactivated (44
). Moreover, it has been shown that successive loss of each PTEN allele may contribute to de novo formation of high-grade astrocytoma and progression into glioblastoma, respectively (45
Numerous studies have suggested that PTEN can be inactivated not only by gene deletion and mutation but also by defects in posttranslational regulation during tumorigenesis (12
). Recent studies postulated that PTEN is subject to ubiquitination mediated by NEDD4-1, an E3 ligase, leading to PTEN degradation (12
). Moreover, the overexpression of NEDD4-1 was shown to induce PTEN degradation and promote K-Ras–mediated transformation (12
). In bladder cancers, the PTEN level was inversely correlated with the level of NEDD4-1 (12
). In breast cancer, NEDD4-1 was shown to be critical for targeting PTEN for degradation (47
). Therefore, these studies suggest that NEDD4-1 shows oncogenic activity that is PTEN-dependent.
In the current study, we found that knockdown of NEDD4-1 in NHA-E6/E7/hTERT/FoxM1B cells caused an increase in endogenous PTEN, suggesting the role of NEDD4-1 in PTEN regulation in glioma cells. Furthermore, we demonstrated that the overexpression of FoxM1 down-regulated PTEN expression in NHA-E6/E7/hTERT, Hs683, and COS-1 cells (all with wild-type PTEN). The mechanism for the overexpression of FoxM1 that accelerates PTEN degradation is as follows: FoxM1 regulates the expression of NEDD4-1, which promotes PTEN ubiquitination and degradation in these cells.
PTEN inactivation could result in uncontrolled PI3K/Akt signaling activation, a mechanism of tumor formation. We have observed that increased expression of FoxM1 in NHA-E6/E7/hTERT caused a significant increase in the level of p-Akt and induced tumor colony formation in a soft agar assay and tumor formation in a mouse brain tumor model. Moreover, using the PI3K-specific inhibitors LY294002 and wortmannin can inhibit FoxM1-induced colony formation in vitro. Therefore, the results indicate that PI3K/Akt activation plays an important role in the glioma formation.
Several studies have reported that cyclin D1 and cyclin E expression correlate significantly with the degree of malignancy in astrocytomas (49
). Survivin expression is also increased in GBMs and correlates with patient survival (50
). In this study, we found that FoxM1B overexpression in glioma cells significantly increased survivin, cyclin D1, and cyclin E expression, consistent with findings in other types of cells (5
). Thus, the molecular mechanisms by which FoxM1 regulates the growth of glioma cells are associated with increasing the expression of these molecules that function as cell-cycle regulators and/or inhibitors of tumor cell apoptosis and are critical for tumor cell proliferation.
In summary, our findings demonstrated that FoxM1 plays an important role in promoting astrocyte transformation and GBM formation by the regulation of multiple factors. Of more importance, we have provided a new mechanism of FoxM1-induced transformation and GBM formation involving the overexpression of FoxM1 that up-regulates the expression of NEDD4-1 and hence destabilizes PTEN and activates the Akt pathway. Our findings strongly suggest that FoxM1 plays an important role in glioma transformation and may be a therapeutic target for glioma.