Antitumor activity of NVP-BKM120--a selective pan class I PI3 kinase inhibitor showed differential forms of cell death based on p53 status of glioma cells.

PURPOSE: The aim of this study was to show preclinical efficacy and clinical development potential of NVP-BKM120, a selective pan class I phosphatidylinositol-3 kinase (PI3K) inhibitor in human glioblastoma (GBM) cells in vitro and in vivo. EXPERIMENTAL DESIGN: The effect of NVP-BKM120 on cellular growth was assessed by CellTiter-Blue assay. Flow cytometric analyses were carried out to measure the cell-cycle, apoptosis, and mitotic index. Mitotic catastrophe was detected by immunofluorescence. The efficacy of NVP-BKM120 was tested using intracranial U87 glioma model. RESULTS: We tested the biologic effects of a selective PI3K inhibitor NVP-BKM120 in a set of glioma cell lines. NVP-BKM120 treatment for 72 hours resulted in a dose-dependent growth inhibition and effectively blocked the PI3K/Akt signaling cascade. Although we found no obvious relationship between the cell line's sensitivity to NVP-BKM120 and the phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) statuses, we did observe a differential sensitivity pattern with respect to p53 status, with glioma cells containing wild-type p53 more sensitive than cells with mutated or deleted p53. NVP-BKM120 showed differential forms of cell death on the basis of p53 status of the cells with p53 wild-type cells undergoing apoptotic cell death and p53 mutant/deleted cells having a mitotic catastrophe cell death. NVP-BKM120 mediates mitotic catastrophe mainly through Aurora B kinase. Knockdown of p53 in p53 wild-type U87 glioma cells displayed microtubule misalignment, multiple centrosomes, and mitotic catastrophe cell death. Parallel to the assessment of the compound in in vitro settings, in vivo efficacy studies using an intracranial U87 tumor model showed an increased median survival from 26 days (control cohort) to 38 and 48 days (treated cohorts). CONCLUSION: Our present findings establish that NVP-BKM120 inhibits the PI3K signaling pathways, leading to different forms of cell death on the basis of p53 statuses. Further studies are warranted to determine if NVP-BKM120 has potential as a glioma treatment.

Heart rate correction models to detect QT interval prolongation in novel pharmaceutical development.

INTRODUCTION: The QT interval of the electrocardiogram (ECG) reflects the duration of ventricular depolarization and repolarization. A drug-induced prolongation of ventricular repolarization, and thereby QT prolongation, is recognized to be a marker for an enhanced risk for ventricular arrhythmia. The assessment of a drug's effect on the QT interval has therefore become routine within pharmaceutical research and development. However, the heart rate has a major influence on the QT interval; the QT interval shortens as heart rate increases such that one needs to account for such heart rate-dependent changes when evaluating possible drug-induced effects on the QT interval. The relationship between the QT interval and heart rate can be modeled mathematically and using this function a so-called "corrected" QT interval (QTc) can be generated to assess drug-induced effects independent from heart rate-dependent effects. In the past few years, a large number of mathematical relationship have been described that supposedly best describe the heart rate-QT relationship. In this paper we describe a novel approach for selecting the optimal mathematical function for this purpose for a given individual. METHODS: Mongrel, purpose-bred dogs (16, males and females) were instrumented with radiotelemetry transmitters (ITS) for measurement of aortic pressure (AP), left ventricular pressure (LVP), the lead II ECG and body temperature. ECGs were recorded continuously without drug treatment and include a range of HRs due to spontaneous, physiological changes over the 24h of data acquisition. Various mathematical models (>20) were then used to evaluate the HR-QT relationship and these were compared statistically to objectively select the model best fitting the data set of each individual animal. RESULTS: In this study a dynamic analysis algorithm was developed to find the optimal descriptor of the HR-QT relationship for a given individual animal under control conditions. The use of this optimal relationship provides the best possible approach for detecting drug-induced effects on the QT interval for compounds that also affect the heart rate. DISCUSSION: Several numerical methods to optimize the correction functions and statistical procedures to perform significance tests were discussed and implemented in a QT/RR relationship analysis system, named QTana. Given a sample data set, QTana searches the best correction model(s) from the integrated 11 QT/RR relationship modeling functions.

Establishment and characterization of clinically relevant models of ependymoma: a true challenge for targeted therapy.

The development of new therapies for ependymoma is dramatically limited by the absence of optimal in vivo and in vitro models. Successful ependymoma treatment requires a profound understanding of the disease's biological characteristics. This study focuses on the establishment and characterization of in vivo and in vitro models of ependymoma to study the molecular pathways necessary for growth and progression in ependymoma. In addition, this study also emphasizes the use of these models for therapeutic intervention of ependymomas. We established optimal conditions for the long-term growth of 2 tumor xenografts and cultures of 2 human ependymoma cell lines. This study also describes the establishment of in vivo models. Histopathologic features of tumors from both intracranial and subcutaneous sites in mice revealed perivascular pseudorosettes and ependymal rosettes, which are typical morphologic features of ependymoma similar to those observed in human specimens. The in vitro models revealed glial fibrillary acidic protein and vimentin expression, and ultrastructural studies demonstrated numerous microvilli, caveolae, and microfilaments commonly seen in human ependymoma. To study signaling pathway alterations in ependymoma, we profiled established ependymoma models with Western blot analysis that demonstrated aberrant activation mainly of the phosphoinositide 3-kinase and epidermal growth factor receptor signaling pathways. Targeting phosphoinositide 3-kinase and epidermal growth factor receptor signaling pathways with small molecule inhibitors showed growth inhibitory effects. These models can also be used to study the standard therapies used for ependymomas, as shown by some of the drugs used in this study. Therefore, the models developed will assist in the biological studies and preclinical drug screening for ependymomas.

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

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.

PTEN down regulates AP-1 and targets c-fos in human glioma cells via PI3-kinase/Akt pathway.

The continual activation of signaling cascades results in dramatic consequences that include loss of cellular growth control and neoplastic transformation. We show here that phosphoinositide 3-kinase and its mediator Akt was constitutively activated in glioma and that this might be due to the aberrant expression of their natural antagonist PTEN. The PTEN (phosphatase and tensin homologue deleted on chromosome ten) tumor suppressor gene modulates cell growth and survival through mechanisms that are incompletely understood. In this study, we investigated the possibility that PTEN mediates its effects through modulation of transcription factor AP-1, which is in part due to decrease in c-fos expression which was dependent on PI3kinase activity. Consistent with a reduction in the c-fos levels, an AP-1 dependent reporter gene was poorly induced in the PTEN expressing cell lines. In contrast to its effect on c-fos, PTEN did not affect the expression of c-Jun and other fos family members. We also show that the effect of PTEN on c-fos expression was due to its ability to antagonize PI3-kinase and could be mimicked by the expression of dominant negative Akt mutant. Taken together, these data indicate that the aberrant expression of PTEN contributes to the activation of the PI3kinase/Akt pathway and its transcription factor mediators in glioma. We conclude that the ectopic expression of PTEN down regulates the proliferation of glioma cells through the suppression of AP-1 and that this target might be essential for its central role in the growth and survival of glioma cancer cells.

PTEN enhances TNF-induced apoptosis through modulation of nuclear factor-kappaB signaling pathway in human glioma cells.

The PTEN tumor suppressor gene modulates cell growth and survival known to be regulated by the activation of the transcription factor NFkappaB, suggesting PTEN might affect the NFkappaB activation pathway. We found that PTEN inhibited NFkappaB activation induced by TNF. The suppression of NFkappaB activation correlated with sequential inhibition of the tumor necrosis factor-induced expression of NFkappaB-regulated anti-apoptotic (IAP1, IAP2, Bcl-2, Bcl-xL, cFLIP, Bfl-1/A1, and survivin) gene products. Downregulation of the antiapoptotic genes by PTEN increased TNF-induced apoptosis, as indicated by caspase activation, TUNEL, annexin staining, and esterase assay. We conclude that the ectopic expression of PTEN enhances TNF-induced apoptosis and downregulates the proliferation of glioma cells through the suppression of various molecules including NFkappaB, and various mediators of cellular survival and proliferation, and that this targets might be essential for its central role in the growth and survival of glioma cancer cells.

Inhibition of Akt survival pathway by a small-molecule inhibitor in human glioblastoma.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Akt are important regulators of the phosphatidylinositol 3-kinase (PI3K) pathway and thus are important to the regulation of a wide spectrum of tumor-related biological processes. Akt regulates several critical cellular functions, including cell cycle progression; cell migration, invasion, and survival; and angiogenesis. Decreased expression of PTEN and overexpression of the Akt proto-oncogene, which is located downstream of PI3K, have been shown in a variety of cancers, including glioblastoma. Novel small-molecule inhibitors of receptors and signaling pathways, including inhibitors of the PI3K pathway, have shown antitumor activity, but inhibitors of Akt have not been examined. In this study, we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We showed that two newly developed Akt inhibitors, KP-372-1 and KP-372-2 (herein called KP-1 and KP-2), effectively inhibited the PI3K/Akt signaling cascade. KP-1 and KP-2 blocked both the basal and epidermal growth factor-induced phosphorylation of Akt Ser473 at 125 and 250 nmol/L, which, in turn, reduced the activation of intracellular downstream targets of Akt, including GSK-3beta and p70s6k. Furthermore, the treatment of U87 and U251 glioma cells with 125 to 250 nmol/L KP-1 and KP2 for 48 hours inhibited cell growth by approximately 50%. This decrease in cell growth stemmed from the induction of apoptosis. Collectively, these results provide a strong rationale for the pharmacologic targeting of Akt for the treatment of gliomas.

Targeting integrin-linked kinase inhibits Akt signaling pathways and decreases tumor progression of human glioblastoma.

The phosphatidylinositol 3-kinase pathway is an important regulator of a wide spectrum of tumor-related biological processes, including cell proliferation, survival, and motility, as well as neovascularization. Protein kinase B/Akt is activated in a complex manner through the phosphorylation of protein kinase B/Akt on Thr308 and Ser473. Although protein-dependent kinase-1 has been shown to phosphorylate Akt at Thr308, it is not clear whether there is a distinct kinase that exclusively phosphorylates Akt at Ser473. A possible candidate is integrin-linked kinase (ILK), which has been shown to phosphorylate Akt at Ser473 in vitro. ILK is a multidomain focal adhesion protein that is believed to be involved in signal transmission from integrin and growth factor receptors. Further, ILK is implicated in the regulation of anchorage-dependent cell growth/survival, cell cycle progression, invasion and migration, and tumor angiogenesis. In this study, we tested the hypothesis that ILK inhibition would inhibit these processes in gliomas in which it is constitutively expressed. We found that a newly developed small-molecule compound (QLT0267) effectively inhibited signaling through the ILK/Akt cascade in glioma cells by blocking the phosphorylation of Akt and downstream targets, including mammalian target of rapamycin and glycogen synthase kinase-3beta. Treatment of glioma cells with 12.5 micromol/L QLT0267 inhibited cell growth by 50% at 48 hours. An anchorage-dependent cell growth assay confirmed the cell growth-inhibitory effect of QLT0267. Further, the decrease in cell growth was associated with a dramatic accumulation of cells in the G2-M phase of the cell cycle. Although the cell growth-inhibitory effects of the ILK inhibitor were achieved only at a high concentration, the QLT0267 was able to reduce cellular invasion and angiogenesis at much lower concentrations as shown by in vitro invasion assays and vascular endothelial growth factor secretion. Thus, blocking the ILK/Akt pathway is a potential strategy for molecular targeted therapy for gliomas.

MMAC/PTEN tumor suppressor gene regulates vascular endothelial growth factor-mediated angiogenesis in prostate cancer.

Prostate cancer presents with a broad spectrum of biologic behavior, ranging from being an indolent, incidental finding to an aggressively invasive and metastatic disease. An improved understanding of the events involved in prostate cancer progression is critically important to its diagnosis and staging, as well as to the development of new therapies. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of an angiogenic, gene-driven switch. In prostate cancer, one of the most powerful stimulators of angiogenesis is the vascular endothelial growth factor (VEGF). VEGF transcription can be induced by hypoxia through activation of the PI3 kinase pathway and hypoxia-inducible factor alpha. MMAC/PTEN (henceforth referred to as PTEN) is a recently identified tumor suppressor gene residing on chromosome 10q23, which is frequently inactivated in a wide range of human tumors, including advanced prostate cancer. The goal of this study was to determine whether PTEN inhibits angiogenesis by modulating VEGF activity. Our results showed that reintroduction of the PTEN gene into human prostate PC-3 and LNCaP cells decreased VEGF secretion, which was accompanied by various biologic activities, including inhibited endothelial cell growth and migration. PTEN expression also down-regulated VEGF mRNA levels, as detected by RT-PCR analysis. Concomitant with lessened VEGF expression was the reduction of VEGF promoter activity in PTEN-expressing cells. Our findings suggest that PTEN modulates angiogenesis by regulating VEGF expression.

Motif analysis of the tumor suppressor gene MMAC/PTEN identifies tyrosines critical for tumor suppression and lipid phosphatase activity.

The tumor suppressor gene, MMAC/PTEN, has phosphatase, C2, and PDZ-binding domains as well as potential sites of regulation by phosphorylation, including tyrosine phosphorylation, which may contribute to its ability to modulate cell growth and viability. Several obvious and significant motifs were found in MMAC/PTEN, including most notably, a catalytic domain of tyrosine phosphatase (IHCxxGxxRS/T) and several potential tyrosine phosphorylation sites. To examine the functional significance of tyrosine phosphorylation of MMAC/PTEN, retroviral constructs were generated with mutations at two putative tyrosine phosphorylation sites (Y240A/Y240F and Y315A/Y315F). Stable expression of wild-type MMAC/PTEN in U251 human glioma cells (which do not normally produce a functional MMAC/PTEN gene product) resulted in a significant reduction of tumor growth in nude mice, decreased growth rate, saturation density, and colony formation in vitro, as well as dephosphorylation of D3-phosphorylated phosphatidylinositols (PtdIns) in vitro. Mutation of Y240 or Y315 to either alanine or phenylalanine abrogated the ability of MMAC/PTEN to alter growth rate, saturation density, and colony formation in vitro. The ability of MMAC/PTEN to limit tumor growth in nude mice was markedly decreased but not abrogated by mutation of Y240 or Y315 to alanine. Thus, Y240 and Y315 are required for MMAC/PTEN to decrease tumor growth in vitro and in vivo. In contrast to wild-type MMAC/PTEN, mutant MMAC/PTEN containing Y240A or Y315A was unable to dephosphorylate D3-phosphorylated PtdIns in vitro. Thus, Y240A and Y315A are involved in the ability of MMAC/PTEN to dephosphorylate PtdIns and regulate tumor cell growth in vitro and in vivo.