Preclinical therapeutic efficacy of a novel blood-brain barrier-penetrant dual PI3K/mTOR inhibitor with preferential response in PI3K/PTEN mutant glioma.
A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer.
In this study we compared the intratumoral and circulating EPCs of glioma patients for a set of common glioma genotypical aberrations (amplification of EGFR; deletion of PTEN and aneusomy of chromosomes 7 and 10).
Because PTEN negatively regulates on the downstream effects of phosphatidylinositol-3-kinase (PI3-K), we hypothesized that the loss of PTEN/MMAC1 and the activation of RAS may be largely equivalent because RAS is a known positive upstream regulator of PI3-K. We expanded our previous survey of PTEN/MMAC1 mutations and analyzed the RAS status of 53 cutaneous melanoma cell lines, 18 glioma cell lines, and 17 uncultured cutaneous melanoma metastasis.
Forty-five LGG tumor specimens from newly diagnosed patients were analyzed for methylation of the putative 5'-promoter region of PTEN using methylation-specific PCR as well as phosphorylation of S6 and PRAS40 and expression of PTEN protein using immunohistochemistry.
Using the C6 glioma cell line, which does not express measurable amounts of PTEN protein and in which in vitro invasiveness is MMP dependent, the authors determined that in vitro glioma cell invasiveness was significantly reduced when cells were preincubated overnight with LY294002 or wortmannin, two specific inhibitors of PI 3-K signaling.
The authors introduced wild-type PTEN complementary DNA into four human glioma cell lines (T98G, U-251MG, U-87MG, and A172) containing endogenous aberrant PTEN alleles.
Introduction of PTEN gene in glioma cell lines markedly reduced the levels of Rac-GTP and Cdc42-GTP, activated forms of these small GTP-binding proteins, and decreased the phosphorylation levels of focal adhesion kinase.
Through comparative analyses of 286 cases of IDH-wildtype and IDH-mutant lower-grade glioma from a TCGA data set, we report that IDH-mutant gliomas have increased expression of tumor-suppressor genes (NF1, PTEN, and PIK3R1) and decreased expression of oncogenes(AKT2, ARAF, ERBB2, FGFR3, and PDGFRB) and glioma progression genes (FOXM1, IGFBP2, and WWTR1) compared with IDH-wildtype gliomas.
In contrast, ectopic expression of wild type PTEN, but not the PTEN(G129R) mutant, in PTEN-mutant gliomas markedly sensitizes these cells to irradiation and to CD95-ligand (CD95L)-induced apoptosis.
The present study suggests that PTEN mutations are late events in the malignant progression of glioma and the occurrence of PTEN mutations are significantly correlated to patients' short-term survival.
Six constitutional missense mutations of the p53 gene were identified (13.6%), but no mutations of the p16 and PTEN genes were found, suggesting that (1) germline p53 mutations contribute to a small portion of astrocytic tumors, (2) inherited mutations of the p16 and PTEN gene do not predispose to the development of gliomas, and (3) other genes are involved in glioma predisposition.
miR-21 may regulate the proliferation and apoptosis of human glioma cells by downregulating the expression of the PTEN protein, and miR-21 may represent a potential therapeutic target for the treatment of glioma.
We used a combination of Northern blot analysis, protein truncation assays, and sequence analysis to determine the types and frequency of PTEN mutations in glioma cell lines so that we could define appropriate recipients to assess the growth suppressive function of PTEN by gene transfer.
These results suggest that mutation of the PTEN gene is a later event than that of the p53 gene in glioma progression and is associated with both the genetic pathways.
Consistent with this idea, several integrin alpha(V)beta(3)-expressing glioma cell lines with PTEN mutations and high levels of phospho-Akt (pAkt) were unaffected by exposure to an active fragment of tumstatin (T3), whereas alpha(V)beta(3)-expressing glioma cell lines with a functional PTEN/low levels of pAkt exhibited T3-induced growth suppression that could be bypassed by small interfering RNA-mediated suppression of PTEN, introduction of a constitutively expressed Akt, or introduction of the Akt and mTOR target eukaryotic translation initiation factor 4E.
We conclude that methylation of the PTEN promoter may represent an alternate mechanism by which PI3K signaling is increased in grade II and III gliomas as well as secondary GBMs, a finding that offers new therapeutic approaches in these patients.