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ABSTRACT: The IL-6/JAK/STAT pathway is a key signal transduction pathway implicated in the pathogenesis of many human cancers, suggesting that kinase inhibitors targeting JAK/STAT3 may have a broad spectrum of antitumor activity. AZD1480, a pharmacological JAK1/2 inhibitor, exhibits anti-tumor potency in multiple adult malignancies. To evaluate the efficacy of inhibition of JAK/STAT3 signal transduction pathway we assessed the activity of AZD1480 in pediatric malignancies using preclinical models of three highly malignant pediatric solid tumors: neuroblastoma (NB), rhabdomyosarcoma (RMS) and the Ewing Sarcoma Family Tumors (ESFT). In this study, we employed panels of biomedical and biological experiments to evaluate the in vitro and in vivo activity of AZD1480 in NB, RMS and ESFT. Our data indicate that AZD1480 blocks endogenous as well as IL-6 induced STAT3 activation. AZD1480 decreases cell viability in 7/7NB, 7/7RMS and 2/2 ESFT cell lines (median EC50 is 1.5 μM, ranging from 0.36-5.37μM). AZD1480 induces cell growth inhibition and caspase-dependent apoptosis in vitro and decreases expression of STAT3 target genes, including cell cycle regulators CyclinD1, 3 and CDC25A, anti-apoptotic genes Bcl-2 and survivin, the metastasis-related factor TIMP-1 and c-Myc. In vivo studies showed AZD1480 significantly decreased tumor growth and prolonged overall survival in tumor-bearing mice. Tumors from AZD1480-treated mice showed inhibition of activated STAT3 as well as decreased expression of STAT3 downstream targets. Our study provides strong evidence of the anti-tumor growth potency of JAK inhibitor AZD1480 in pediatric solid tumors, providing proof-of principle that inhibition of the JAK/STAT3 signal transduction could be a promising therapeutic target for high-risk pediatric solid tumors.
Oncotarget 03/2013; · 4.78 Impact Factor
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ABSTRACT: Activation of Akt is a marker of decreased event-free or overall survival in neuroblastoma patients. MK-2206, a novel allosteric Akt inhibitor, is now tested in clinical trials in adult cancers. In this study, effect of MK-2206 on tumor growth and murine survival, alone or in combination, with etoposide or rapamycin was evaluated.
The anticell proliferation effect of MK-2206 was tested in eight neuroblastoma cell lines by MTS assay. Caspase-3/7 activity, cell-cycle analysis, and reactive oxygen species (ROS) production were determined. Effect of MK-2206 combined with etoposide or rapamycin was evaluated in vitro and in vivo. Akt phosphorylation was measured by Western blotting in neuroblastoma cells and tumors.
In vitro, MK-2206 treatment inhibited neuroblastoma cell proliferation, which was accompanied by a cell line selective G(1) arrest of cell cycle or production of ROS. A synergistic effect between MK-2206 and etoposide was detected in four tested neuroblastoma cell lines via caspase-dependent apoptosis, whereas increased inhibition of cell growth induced by combination of MK-2206 and rapamycin was mediated by ROS production. In vivo, MK-2206 alone decreased tumor growth and increased murine survival at dose that inhibited Akt phosphorylation in tumors. MK-2206, in combination with etoposide or rapamycin, caused a significant decrease in tumor growth and increase of murine survival compared with MK-2206 alone.
Akt inhibition by MK-2206 increased the efficacy of etoposide or rapamycin. Our study supports future clinical evaluation of MK-2206 in combination with conventional cytotoxic therapy or with rapamycin in high-risk neuroblastoma patients.
Clinical Cancer Research 05/2012; 18(13):3603-15. · 7.74 Impact Factor
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Chunxi Wang,
Zhihui Liu,
Chan-Wook Woo, Zhijie Li,
Lifeng Wang,
Jun S Wei,
Victor E Marquez,
Susan E Bates,
Qihuang Jin,
Javed Khan,
Kai Ge,
Carol J Thiele
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ABSTRACT: Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb protein histone methyltransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibiton with 3-deazaneplanocin A increased CASZ1 expression, inhibited NB cell growth, and induced neurite extension. Similarly, EZH2(-/-) mouse embryonic fibroblasts (MEF) displayed 3-fold higher levels of CASZ1 mRNA compared with EZH2(+/+) MEFs. In cells with increased expression of CASZ1, treatment with histone deacetylase (HDAC) inhibitor decreased expression of EZH2 and the Polycomb Repressor complex component SUZ12. Under steady-state conditions, H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR, and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors.
Cancer Research 11/2011; 72(1):315-24. · 7.86 Impact Factor
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ABSTRACT: Neuroblastoma (NB) tumors expressing high levels of brain-derived neurotrophic factor (BDNF) and its receptor TrkB or activated Akt are associated with decreased event-free or overall survival in patients with NB. In the current study, the effect of perifosine, an Akt inhibitor, on the chemosensitivity of TrkB-expressing NB cells or tumors was evaluated.
A tetracycline-regulated TrkB-expressing isogenic NB cell model system was tested. In this system, NB cells were treated with etoposide and/or perifosine both in vitro and in vivo. Inhibition of the target by perifosine was evaluated by Western blot analysis or kinase activity assay. Cell survival and tumor growth were investigated.
In vitro BDNF treatment induced Akt phosphorylation and rescued cells from etoposide-induced cell death in cells with high TrkB expression, but not in cells with low TrkB expression. Pretreatment of high TrkB-expressing TB3 cells with perifosine blocked BDNF/TrkB-induced Akt phosphorylation and inhibited BDNF's protection of TB3 cells from etoposide treatment. In vivo, tumors with high TrkB expression were found to have elevated levels of phosphorylated Akt and were less sensitive to etoposide treatment compared with tumors with low TrkB expression. Mice treated with a combination of perifosine and etoposide were found to have a statistically significant decrease in tumor growth compared with mice treated with either etoposide or perifosine alone. Activation of Akt through the BDNF/TrkB signaling pathway induced chemoresistance in NB in vivo.
Perifosine-induced inhibition of Akt increased the sensitivity of NB to chemotherapy. The results of the current study support the future clinical evaluation of an Akt inhibitor combined with cytotoxic drugs for the improvement of treatment efficacy.
Cancer 05/2011; 117(23):5412-22. · 4.77 Impact Factor
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ABSTRACT: Communication between the vasculature and nervous system is important during embryogenesis but the molecular mechanisms mediating this are ill-defined. We evaluated the molecular mechanisms by which Nerve Growth Factor (NGF) and Brain-derived neurotrophic factor (BDNF) regulate VEGF production. NGF activation of TrkA causes a marked increase in VEGF secretion by neuronal cells. The NGF induced increase in VEGF is accompanied by an increase in HIF-1α. Pharmacologic inhibitors of the Trk tyrosine kinase, PI-3 kinase and mTOR paths prevent NGF stimulated increases in HIF-1α and VEGF. NGF induced increase in VEGF transcription is dependent on a hypoxia response element (HRE) in the VEGF promoter. Mutation of the HRE or siRNA mediated silencing of HIF-1α expression blocks NGF induced increases in VEGF transcription. In primary cultures of TrkA expressing neurons from dorsal root ganglion, NGF induces VEGF expression that is accompanied by increases in HIF-1α but not HIF-2α expression. In CGN neurons, BDNF induces VEGF that is dependent on induction of HIF-1α. Our study indicates that neurotrophin activation of Trk stimulates an increase in VEGF transcription that is mediated by induction of HIF-1α.
Molecular and Cellular Neuroscience 02/2011; 46(2):498-506. · 3.66 Impact Factor
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ABSTRACT: Activated AKT is a marker of decreased event-free or overall survival in neuroblastoma (NB) patients. The aim of this study was to investigate the effect of perifosine, a nontoxic AKT inhibitor, as a single agent on NB cell growth in vitro and in vivo.
Four human NB cell lines (AS, NGP, BE2, and KCNR) were treated with increasing concentrations of perifosine, and a quantitative analysis of cell death (apoptosis) was performed by using MTS and caspase-3/7 activity assays. Survival of mice carrying xenograft NB tumors that were treated with perifosine (n = 6-7 mice per group) was compared with that of untreated mice (n = 7 mice per group) using Kaplan-Meier analysis. Tumor volumes were calculated to determine the effect of perifosine on NB tumor growth. Phosphorylation of AKT and expression of cleaved caspase-3 were measured in proteins from the tumors. All statistical tests were two-sided.
Perifosine, at 30 muM concentration, decreased AKT phosphorylation and increased apoptosis in all four NB cell lines in vitro. Perifosine-treated mice bearing xenograft NB tumors had longer survival than untreated mice (untreated vs treated, median survival: AS, 13 days, 95% confidence interval [CI] = 11 to 16 days vs not reached, P = .003; NGP, 22 days, 95% CI = 20 to 26 days vs not reached, P = .013; BE2, 24 days, 95% CI = 21 to 27 days vs not reached, P < .001; and KCNR, 18 days, 95% CI = 18 to 21 days vs not reached, P < .001). Perifosine treatment induced regression in AS tumors, growth inhibition in BE2 tumors, and slower growth in NGP and KCNR tumors. Inhibition of AKT phosphorylation and induction of caspase-dependent apoptosis were noted in tumors of perifosine-treated mice in all four in vivo NB tumor models.
Perifosine inhibited the activation of AKT and was an effective cytotoxic agent in NB cells in vitro and in vivo. Our study supports the future clinical evaluation of perifosine for the treatment of NB tumors.
CancerSpectrum Knowledge Environment 06/2010; 102(11):758-70. · 14.07 Impact Factor
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ABSTRACT: As histone deacetylase inhibitors such as romidepsin (depsipeptide, FK228) complete successful Phase I clinical trials in pediatric solid tumors, it is important that their mechanisms of action are delineated in order to inform the development of subsequent clinical trials as single agents or in combination therapies. In this study, we evaluate the effect of romidepsin as a single agent on a number of different neuroblastoma (NB) cell lines. We find that the growth of 6/6 human NB tumor cell lines but not an immortalized fibroblast cell line (NIH3T3) is inhibited by romidepsin (IC(50) = 1-6.5 ng/ml) after 72 h of treatment. Romidepsin shows selective dose-dependent cytotoxicity in both single copy and N-myc amplified NB cell lines, in cell lines with wild type or mutant p53 and those containing Alk mutations. The decrease in cell proliferation is accompanied by caspase-dependent apoptosis as shown by PARP cleavage, an accumulation of cells in the sub-G(1) phase of the cell cycle and the ability of a pan-caspase inhibitor to reduce cell death. Romidepsin inhibits the growth of subcutaneous NB xenografts in a dose dependent manner in immunocompromised mice. Furthermore, romidepsin induces expression of genes such as p21 and expression of p75 and NTRK (TrkA) which are more highly expressed in the tumors from NB patients that have a good prognosis. These studies support continued investigations into the therapeutic activity of romidepsin in NB.
Cell cycle (Georgetown, Tex.) 05/2010; 9(9):1830-8. · 5.36 Impact Factor
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ABSTRACT: In the beginning, Trk was an oncogene. Yet Neurotrophin-Trk signaling came to preeminence in the field of neurobiology. Now it is appreciated that Trks regulate important processes in nonneuronal cells and, in addition to their impact on tumors of neural origin, may contribute to the pathogenesis of carcinomas, myelomas, and prostate and lymphoid tumors. Although mutations and rearrangements of Trk are seen only sporadically in human cancers, such as medullary thyroid carcinoma, a number of recent studies indicate that expression of TrkB contributes to tumor pathology. In neuroblastoma, TrkA expression marks good prognosis which TrkB and Brain-derived neurotrophic factor (BDNF) expression marks poor prognosis. Activation of the BDNF/TrkB signal transduction pathway also stimulates tumor cell survival and angiogenesis and contributes to resistance to cytotoxic drugs and anoikis, enabling cells to acquire many of the characteristic features required for tumorigenesis. Small molecule inhibitors, such as Cephalon's CEP-701, are in phase 1 and 2 clinical trials, and a series of AstraZeneca Trk inhibitors are poised to enter the clinic. As monotherapy, inhibitors may be effective only in tumors with activating Trk mutations. Important clinical follow-up will be the assessment of Trk inhibitors in combination with standard chemo- or radiotherapy or other signal transduction pathway inhibitors.
Clinical Cancer Research 09/2009; 15(19):5962-7. · 7.74 Impact Factor
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ABSTRACT: Neuroblastoma (NB) is a neural crest precursor cell-derived extracranial solid tumor in children. Patients with a poor prognosis are often resistant to chemotherapy and have tumors that express the neuronal growth/survival factor brain-derived neurotrophic factor and its tyrosine kinase receptor, TrkB. In this article, the authors discuss a growth/survival factor-stimulated mechanism leading to chemoresistance in NB that is mediated by the PI3K/Akt signaling pathway. Besides brain-derived neurotrophic factor/TrkB, other growth/survival factors and their receptors also activate the PI3K/Akt pathway and have the potential to mediate chemoresistance in NB. These findings raise the possibility of a new therapeutic approach in NB that would target Akt, the common downstream mediator of multiple growth/survival factor signaling pathways, to enhance the efficacy of chemotherapeutics. Several classes of Akt inhibitors, including phosphatidylinositol ether lipid analogs, alkylphospholipid analogs, allosteric Akt kinase inhibitors, HSP90 inhibitor and HIV protease inhibitors are discussed.
Expert opinion on therapeutic targets 01/2008; 11(12):1611-21. · 3.72 Impact Factor
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ABSTRACT: Elucidating signaling pathways that mediate cell survival or apoptosis will facilitate the development of targeted therapies in cancer. In neuroblastoma tumors, brain-derived neurotrophic factor (BDNF) and its receptor TrkB are associated with poor prognosis. Our previous studies have shown that BDNF activation of TrkB induces resistance to chemotherapy via activation of phosphoinositide-3-kinase (PI3K)/Akt pathway. To study targets of PI3K/Akt that mediate protection from chemotherapy, we focused on glycogen synthase kinase-3beta (GSK-3beta), which is a known modulator of apoptosis. We used pharmacologic and genetic methods to study the role of GSK-3beta in the BDNF/TrkB/PI3K/Akt protection of neuroblastoma from chemotherapy. BDNF activation of TrkB induced the Akt-dependent phosphorylation of GSK-3beta, resulting in its inactivation. Treatment of neuroblastoma cells with inhibitors of GSK-3beta, LiCl, GSK-3beta inhibitor VII, kenpaullone, or a GSK-3beta-targeted small interfering RNA (siRNA) resulted in a 15% to 40% increase in neuroblastoma cell survival after cytotoxic treatment. Transfection of neuroblastoma cells with a constitutively active GSK-3beta S9A9 caused a 10% to 15% decrease in cell survival. Using real-time, dynamic measurements of cell survival, we found that 6 to 8 h after etoposide treatment was the period during which critical events regulating the induction of cell death or BDNF/TrkB-induced protection occurred. During this period, etoposide treatment was associated with the dephosphorylation and activation of GSK-3beta in the mitochondria that was blocked by BDNF activation of TrkB. These data indicate that the inactivation of GSK-3beta contributes to the BDNF/TrkB/PI3K/Akt protection of neuroblastoma cells from chemotherapy.
Molecular Cancer Therapeutics 01/2008; 6(12 Pt 1):3113-21. · 5.23 Impact Factor
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ABSTRACT: Patients whose neuroblastoma tumors express high levels of brain-derived neurotrophic factor (BDNF) and TrkB have an unfavorable prognosis. Our previous studies indicated that BDNF activation of the TrkB signal transduction pathway blocked the cytotoxic effects of chemotherapeutic drugs via the phosphatidylinositol 3-kinase pathway. Akt is an important downstream target of phosphatidylinositol 3-kinase and functions to regulate cell survival, proliferation, and protein synthesis. In this study, we examined whether Akt is required and sufficient to mediate BDNF/TrkB protection of neuroblastoma cells from chemotherapy. Transient transfection of a constitutively active Akt (Akt-Myr) into TrkB-expressing SY5Y cells (TB8 cells) increases Akt activation and attenuates the cell death induced by chemotherapeutic reagents in the absence of BDNF. Furthermore, expression of a dominant-negative Akt (Akt-K179A) blocks the ability of BDNF to rescue TB8 cells from chemotherapy-induced cell death. Pharmacologic inhibition of Akt, with PIA6, a phosphatidylinositol ether lipid analogue (PIA), blocks BDNF-induced phosphorylation of Akt and the downstream target of Akt. PIA6 sensitizes neuroblastoma cells to chemotherapy and attenuates BDNF protection of neuroblastoma cells from chemotherapy-induced cell death. These results indicate that Akt is a key signaling component by which BDNF activation of the TrkB signal transduction pathway protects neuroblastoma cells from chemotherapy-induced cell death. This study raises the possibility that novel pharmacologic inhibitors of Akt may enhance the effectiveness of chemotherapeutic agents in the treatment of neuroblastoma tumors.
Cancer Research 04/2005; 65(6):2070-5. · 7.86 Impact Factor
