We investigated the role of the PI3K inhibitor PX-866 in a set of well-characterized glioblastoma cell lines and found that PX-866 treatment for 72 hours resulted in dose-dependent growth inhibition. Consistent with its role as a PI3K antagonist, PX-866 inhibited the ability of EGF to induce Akt phosphorylation and also inhibited other PI3K-mediated downstream targets. We suggest here a dual mechanism of PX-866-induced inhibition of PI3K signaling involving (i) downregulation of cell cycle-regulating protein cyclin D1, the synthesis of which is regulated by p70S6K and 4E-BP-1 and involves cell cycle arrest in G1 phase, and (ii) induction of autophagy. This dual mechanism of action of PX-866 may contribute to the efficacy of PX-866 observed in our studies. The antiproliferative activity of PX-866 occurs partly through inhibition of cell-cycle progression in glioma cells. Regardless of their PTEN status, glioma cells treated with PX-866 showed G1
cell-cycle arrest with no apparent induction of apoptosis. Thus, apoptosis could not have accounted for the potent effect of PX-866 on glioma cell growth. Recently, several lines of evidence have demonstrated that small-molecular mass inhibitors such as imatinib,26
induce a type II programmed cell death, autophagy, which suggests that autophagy may be a response of tumor cells to the blockage of signals for survival. Therefore, cell-cycle arrest in G1 may directly contribute to autophagy as PX-866 increased LC3 cleavage, and this may represent a highly conserved mitotic or postmitotic checkpoint control of autophagy.28
It has recently been reported that cooperativity between blockade of PI3K and mTOR signaling impacts autophagy in glioma cells.29
A similar observation of G1
cell-cycle arrest has been reported for other PI3K inhibitors.30
The PI3K/Akt signaling pathway mediates invasion, angiogenesis, and the expression of VEGF in cells.31–33
This stimulation of VEGF in cancer cells can be mediated by autocrine or chronic stimulation by growth factors such as insulin-like growth factor-1, constitutive activation of PI3K, or constitutive activation of Akt due to inactivation of the tumor suppressor PTEN.32,33
Here, we report that inhibition of PI3K by a potent PI3K inhibitor resulted in statistically significant suppression of VEGF secretion. A similar antiangiogenic activity was previously observed for another PI3K inhibitor, SF1326.34
Importantly, PX-866 also inhibited the invasive potential of both U87 and LN229 cells. Thus, we conclude that PX-866 is directly involved in the inhibition of invasion and angiogenic potential of glioblastoma cells. In addition, in the present study, it was clear that PI3K triggers the invasion and angiogenesis of glioblastoma cells and that the pharmacologic inhibition of this pathway significantly abolishes invasiveness and angiogenesis. Recent studies have shown that the PI3K pathway is an attractive therapeutic target because of its integral role in the regulation of tumor proliferation, invasiveness, and survival.35
Multiple studies with small interfering RNAs and pharmacologic inhibitors have shown the importance of PI3K blockade, and several agents that target this pathway are already undergoing clinical testing; some have already shown promise in clinical trials.36,37
For example, LY294002 has been tested in an ectopic skin and orthotopic brain tumor model and has been shown to inhibit glioma tumor growth.38
LY294002 has also shown efficacy against ovarian carcinoma.39
Consistent with these results, we also found therapeutic activity of PX-866 in human tumor xenograft models, where immunohistochemical and RPPA analyses showed that p-Akt and pS6 inhibition were linked to an 84% growth inhibition in a subcutaneous tumor model and an increase in the median survival of mice with intracranial U87 tumors from 32 to 39 days. Also, MRI findings indicated that tumor growth was inhibited approximately 75% after PX-866 treatment. This was similar to the growth inhibition observed in the subcutaneous tumors. Although the increase in survival was only 7 days (30%) more in PX-866-treated animals, this increase in 30% is statistically significant (P < .001) in a nude mouse model. Although we have shown adequate targeting of PX-866 to the tumor by showing treatment with PX-866 effectively decreased p-Akt and pS6 in the treated animals using Western blots and the novel RPPA technique, the effectiveness of the treatment does not seem to be so robust due to the activation of a collateral pathway for tumor survival. We believe that tumor heterogeneity and complex compensatory or collateral pathways negate the therapeutic efficacy of suppressing a single node. The logical next step is to identify the compensatory pathways responsible for maintaining cell survival when PI3K is inactivated and suggests that additional refinement of this overall approach is necessary.
As expected, 1
H spectroscopy showed a significant difference in the Cho/NAA ratio between tumors and normal contralateral brain tissue, consistent with previous findings, such as those reported by Workman et al.40
More importantly, PX-866 treatment led to significant alterations in the spectra of treated tumors compared with those of untreated tumors. Specifically, spectra from treated tumors had significantly lower Cho/NAA. This observation is consistent with our previous finding that a drop in phosphocholine, the main component of the choline peak, is associated with PI3K inhibition in cells treated with wortmannin, an analog of PX-866.41
In addition, our observation that relatively small tumors were characterized by an increase in the lactate–lipid peak typically associated with necrosis in brain tumors42
is consistent with our histological findings indicating that PX-866 can lead to necrosis. Thus, the drop in Cho/NAA in PX-866 tumors is likely indicative of PI3K inhibition, tumor cell death, and the presence of a larger proportion of cells with normal Cho/NAA. This more normal appearing metabolic signature could predict better long-term response.
In summary, our results affirm that PX-866 is an effective specific inhibitor of PI3K; that the PI3K/Akt pathway is a highly specific molecular target for the development of molecular therapeutics for glioblastoma and other cancers with aberrant PTEN/PI3K expression; and that PX-866 treatment results in MRS-detectable metabolic changes that indicate a partial normalization of the metabolic profile of treated tumors. This illustrates the potential of MRS to noninvasively monitor the molecular effects of PX-866 in glioblastomas in vivo. Further studies monitoring the longitudinal effect of PX-866 treatment need to be performed to confirm the value of MRS in monitoring early molecular response to this PI3K inhibitor in patients.