Cellular and in vivo activity of a novel PI3K inhibitor, PX-866, against human glioblastoma

Department of Neuro-Oncology, Unit 100, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Neuro-Oncology (Impact Factor: 5.56). 02/2010; 12(6):559-69. DOI: 10.1093/neuonc/nop058
Source: PubMed


The phosphatidylinositol-3-kinase (PI3K)/Akt oncogenic pathway is critical in glioblastomas. Loss of PTEN, a negative regulator
of the PI3K pathway or activated PI3K/Akt pathway that drive increased proliferation, survival, neovascularization, glycolysis,
and invasion is found in 70%–80% of malignant gliomas. Thus, PI3K is an attractive therapeutic target for malignant glioma.
We report that a new irreversible PI3K inhibitor, PX-866, shows potent inhibitory effects on the PI3K/Akt signaling pathway
in glioblastoma. PX-866 did not induce any apoptosis in glioma cells; however, an increase in autophagy was observed. PX-866
inhibited the invasive and angiogenic capabilities of cultured glioblastoma cells. In vivo, PX-866 inhibited subcutaneous
tumor growth and increased the median survival time of animals with intracranial tumors. We also assessed the potential of
proton magnetic resonance spectroscopy (MRS) as a noninvasive method to monitor response to PX-866. Our findings show that
PX-866 treatment causes a drop in the MRS-detectable choline-to-NAA, ratio and identify this partial normalization of the
tumor metabolic profile as a biomarker of molecular drug action. Our studies affirm that the PI3K pathway is a highly specific
molecular target for therapies for glioblastoma and other cancers with aberrant PI3K/PTEN expression.

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Available from: Yong-Wan Kim, Oct 04, 2015
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    • "We have previously used MRS to develop and mechanistically validate biomarkers of response to emerging targeted therapies [15]–[21]. In particular, we have shown that treatment with the HDAC inhibitor SAHA is associated with increased PC levels [22]. "
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    ABSTRACT: Histone deacetylase (HDAC) inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS)-detectable phosphocholine (PC) is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using (13)C MRS to monitor [1,2-(13)C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by (31)P MRS (129±7% of control). Furthermore, cellular choline kinase activity was elevated (177±31%), while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively) relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC) increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA2) isoforms, resulting in increased PLA2 activity (162±18%) in SAHA-treated cells. Importantly, the levels of total choline (tCho)-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using (1)H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow-up diagnostics of HDAC inhibitor treatment.
    PLoS ONE 04/2013; 8(4):e62610. DOI:10.1371/journal.pone.0062610 · 3.23 Impact Factor
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    • "In this study, both PCho and GPC increased in basal-like xenografts after blockade of the PI3K signaling. Previous in vitro studies of PI3K inhibitors in prostate cancer, colon cancer and breast cancer cell lines have suggested a reduced PCho concentration and an increased GPC concentration, whereas in vivo studies in glioblastoma xenografts have suggested a decrease in tCho [40,62,63]. However, we anticipate that the metabolic changes depend on the oncogenic signaling abnormalities seen in different cancer subtypes. "
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    ABSTRACT: Introduction The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in cancer cells through numerous mutations and epigenetic changes. The recent development of inhibitors targeting different components of the PI3K pathway may represent a valuable treatment alternative. However, predicting efficacy of these drugs is challenging, and methods for therapy monitoring are needed. Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype, frequently associated with PI3K pathway activation. The objectives of this study were to quantify the PI3K pathway activity in tissue sections from xenografts representing basal-like and luminal-like breast cancer before and immediately after treatment with PI3K inhibitors, and to identify metabolic biomarkers for treatment response. Methods Tumor-bearing animals (n = 8 per treatment group) received MK-2206 (120 mg/kg/day) or BEZ235 (50 mg/kg/day) for 3 days. Activity in the PI3K/Akt/mammalian target of rapamycin pathway in xenografts and human biopsies was evaluated using a novel method for semiquantitative assessment of Aktser473 phosphorylation. Metabolic changes were assessed by ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy. Results Using a novel dual near-infrared immunofluorescent imaging method, basal-like xenografts had a 4.5-fold higher baseline level of pAktser473 than luminal-like xenografts. Following treatment, basal-like xenografts demonstrated reduced levels of pAktser473 and decreased proliferation. This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors. BEZ235 also caused increased glucose and glycerophosphocholine concentrations. No response to treatment or change in metabolic profile was seen in luminal-like xenografts. Analyzing tumor sections from five patients with BLBC demonstrated that two of these patients had an elevated pAktser473 level. Conclusion The activity of the PI3K pathway can be determined in tissue sections by quantitative imaging using an antibody towards pAktser473. Long-term treatment with MK-2206 or BEZ235 resulted in significant growth inhibition in basal-like, but not luminal-like, xenografts. This indicates that PI3K inhibitors may have selective efficacy in basal-like breast cancer with increased PI3K signaling, and identifies lactate, phosphocholine and glycerophosphocholine as potential metabolic biomarkers for early therapy monitoring. In human biopsies, variable pAktser473 levels were observed, suggesting heterogeneous PI3K signaling activity in BLBC.
    Breast cancer research: BCR 02/2013; 15(1):R16. DOI:10.1186/bcr3391 · 5.49 Impact Factor
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    • "Our observation of enhanced autophagy in anti-miR-21-treated cells after irradiation can also be explained partly by inactivation of the PI3K/AKT pathway. As shown earlier, the mammalian target of rapamycin, a potent inhibitor of autophagy, is a down-stream target of Akt, and chemical inhibition of the PI3K/Akt pathway in PTEN-deficit glioma cell lines increases autophagy [47]. However, decrease of autophagy by miR-21 over-expression in LN428 was not associated with concomitant increase of phospho-Akt and anti-miR-21 showed additive effect on autophagy with LY294002 after irradiation in U373 (Supplementary Data). "
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    ABSTRACT: Radiation is a core part of therapy for malignant glioma and is often provided following debulking surgery. However, resistance to radiation occurs in most patients, and the underlying molecular mechanisms of radio-resistance are not fully understood. Here, we demonstrated that microRNA 21 (miR-21), a well-known onco-microRNA in malignant glioma, is one of the major players in radio-resistance. Radio-resistance in different malignant glioma cell lines measured by cytotoxic cell survival assay was closely associated with miR-21 expression level. Blocking miR-21 with anti-miR-21 resulted in radio-sensitization of U373 and U87 cells, whereas overexpression of miR-21 lead to a decrease in radio-sensitivity of LN18 and LN428 cells. Anti-miR-21 sustained γ-H2AX DNA foci formation, which is an indicator of double-strand DNA damage, up to 24 hours and suppressed phospho-Akt (ser473) expression after exposure to γ-irradiation. In a cell cycle analysis, a significant increase in the G(2)/M phase transition by anti-miR-21 was observed at 48 hours after irradiation. Interestingly, our results showed that anti-miR-21 increased factors associated with autophagosome formation and autophagy activity, which was measured by acid vesicular organelles, LC3 protein expression, and the percentage of GFP-LC3 positive cells. Furthermore, augmented autophagy by anti-miR-21 resulted in an increase in the apoptotic population after irradiation. Our results show that miR-21 is a pivotal molecule for circumventing radiation-induced cell death in malignant glioma cells through the regulation of autophagy and provide a novel phenomenon for the acquisition of radio-resistance.
    PLoS ONE 10/2012; 7(10):e47449. DOI:10.1371/journal.pone.0047449 · 3.23 Impact Factor
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