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Keywords:

  • neuroblastoma;
  • Akt;
  • brain-derived neurotrophic factor;
  • TrkB;
  • perifosine;
  • etoposide

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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 2011;. © 2011 American Cancer Society.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood that is derived from neural crest precursor cells.1 It accounts for nearly 8% of pediatric malignancies, yet is responsible for approximately 15% of all pediatric cancer deaths.2, 3 Spontaneous regression, differentiation, and a good response to current therapeutic regimens such as surgery and chemotherapy are common in infants or those with low-risk tumors. However, children, typically those aged > 18 months, fail to achieve sustained responses to intensive multimodality chemotherapy and stem cell transplantation.4 Although patients may initially have a response to chemotherapy, they eventually develop disease recurrence with multifocal metastatic disease and resistance to chemotherapy. Despite the aggressive treatment, the prognosis of these patients is poor and their long-term survival is < 40%.4 More effective treatment strategies are urgently needed for this high-risk group of patients with NB.

A better understanding of the genetics and biologic behavior of poor-prognosis NB tumors should lead to new molecular targets that can be used to develop more specific, more effective, and less toxic cancer therapies. After reports that brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB are often detected in NB tumors in patients with an unfavorable prognosis,5 we identified that BDNF activation of TrkB attenuated the sensitivity of NB cells to chemotherapy.6 We later identified phosphatidylinositol-3 (PI3) kinase and its downstream target, Akt serine/threonine kinase, as critical mediators of activated TrkB-induced chemoresistance in NB cells.7, 8 In addition to BDNF/TrkB, specific ligand-induced activation of other tyrosine kinase receptors, such as insulin-like growth factor receptor (IGFR),9 platelet-derived growth factor receptor (PDGFR),10 and vascular endothelial growth factor receptor (VEGFR),11 are known to activate Akt and affect NB cell survival. Studies have shown that IGF protects NB cells from chemotherapy-induced or osmolarity-induced cell death in NB cells via activation of Akt.12 PDGF, a potent mesenchymal cell-derived mitogen, also activates Akt in PDGFR-expressing NB cells and stimulates cell proliferation, chemotaxis, and neurite extension. PDGFR inhibitors block PDGFR-induced phosphorylation of Akt and induce apoptosis in NB cells.10 Gastrin-releasing peptide receptor is a G protein-coupled receptor whose upregulation in patients with advanced stage NB is accompanied by increased levels of activated Akt.13 Anaplastic lymphoma kinase (ALK) is a tyrosine kinase and its mutation or amplification is associated with Akt activation in patients with poor-prognosis NB.14 In drug resistance models, cathespin D-induced resistance to doxorubicin is mediated by activated Akt.15 Recently, a study of tumors from 116 patients with NB demonstrated that activated Akt is correlated with poor prognosis and advanced disease stage in patients with primary NB, as well as those with unfavorable biological markers in NB such as MYCN amplification and 1p36 aberrations.16 It is obvious that Akt is a critical survival signaling node in many signal transduction pathways and the association between activated Akt and poor prognosis and advanced stage disease in patients with NB makes it an important target for treatment.

Although Akt inhibitors have been developed and tested in many adult tumor model systems,17 to the best of our knowledge our recent study was the first to systematically evaluate an Akt inhibitor as a single agent in pediatric NB.18 Because therapeutic studies implicating a role for Akt in chemoresistance in NB have been performed for the most part using in vitro models, in the current study we used a tetracycline(TET)-regulated TrkB expression system to demonstrate that increased expression of TrkB is associated with elevated levels of activated Akt in vivo and reduced sensitivity to etoposide. Moreover, the current study demonstrated that using the Akt inhibitor perifosine increased the sensitivity of NB tumors to etoposide and caused regression of NB tumors.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Cell Culture

Human NB cells (TB3) that have a transfected TET-regulated rat TrkB7 and NGP, LAN5, and SY5Y cells were used in the current study. Puromycin (0.5 μg/mL) and TET (1 μg/mL) were added to the TB3 cells for maintenance of selection pressure and repression of the TrkB gene. TB3 and NGP cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), 2 mM of glutamine, and antibiotics as described previously.7

Reagents and Antibodies

Recombinant human BDNF was obtained from PeproTech, Inc (Rocky Hill, NJ). Puromycin and TET were purchased from Sigma Chemical Company, Inc (St Louis, Mo). Etoposide was obtained from Bedford Laboratories (Bedford, Ohio). The Akt inhibitor perifosine was a gift from Keryx Biopharmaceuticals (New York, NY). The pan Trk antibody, TrkB antibody, and phosphorylated tyrosine (P-Tyr) antibody were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, Calif). The total Akt antibody (T-Akt), phosphorylated Akt (P-Akt, Ser473, Thr308) antibody, total S6 antibody (T-S6), P-S6 (Ser235/236) antibody, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody, and an Akt kinase assay kit were obtained from Cell Signaling Technology (Beverly, Mass).

Treatment

TB3 cells were cultured in the presence of TET (1 μg/mL) for 3 days to repress TrkB expression. To study the activation of TrkB, TB3 cells were treated with BDNF (100 ng/mL) for 15 minutes. To study the response to etoposide or perifosine, TB3 cells were treated with different concentrations of etoposide or perifosine for 24 hours and cell survival was assessed using an MTS assay. To study perifosine's blockage of BDNF-induced phosphorylation of Akt, TB3 cells were pretreated with perifosine (2.5 μm, 5 μm) for 6 hours or 24 hours, and then treated with BDNF (100 ng//mL) for 15 minutes. To study perifosine's effect on BDNF-induced cell survival, TB3 cells were first treated with perifosine (5 μM) for 6 hours, followed by treatment with BDNF and etoposide for 24 hours.

Real-Time Quantitative Polymerase Chain Reaction

Total RNA was isolated using a Qiagen Extraction Kit procedure (Valencia, Calif). Equal amounts of total RNA (1 μg) were reverse-transcribed using the Superscript First-Strand Synthesis System for reverse transcriptase-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, Calif), and the resulting first-strand cDNA was diluted and used as a template in real-time, quantitative PCR analysis. All measurements were performed in duplicate. Actin served as an internal control and was used to normalize variances in input cDNA. The following gene-specific primer pair was designed: TrkB-sense: 5′-CTCAGCAAATCGCAGCAGG-3′; TrkB-antisense: 5′-AGTAGTCGGTGCTGTATA-3′. The specificity of each primer was determined using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) module. Detection of TrkB expression was performed with SYBR Green (Applied Biosystems, Carlsbad, Calif) and an ABI PRISM 7700 Sequence Detection System (Applied Biosystems).

Immunoprecipitation/Western Blot Analysis

After treatment, TB3 cells were harvested and protein was extracted with protein lysis buffer as described previously.7 Protein concentrations were determined using a Bradford assay kit (Bio-Rad Laboratories, Hercules, Calif). For immunoprecipitation of TrkB and P-Tyr, 500 μg of protein from each lysate were immunoprecipitated with polyclonal anti–pan-Trk rabbit antibody and protein A agarose (Life Technologies, Inc, Carlsbad, Calif). Immunoprecipitates were electrophoresed in 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) gels, transferred to nitrocellulose, and subsequently probed with an anti–P-Tyr antibody or TrkB antibody. For Western blot analysis, 30 μg of protein from each lysate were loaded onto 10% SDS-PAGE gels, transferred to nitrocellulose, and probed with the anti–Akt antibody, anti–P-Akt (Ser473) antibody, anti–S6 antibody, or anti–P-S6 (Ser235/236) antibody. Signals were detected using enhanced chemiluminescence reagents (Amersham Life Science, Arlington Heights, Ill).

Akt Kinase Assay

TB3 cells were lysed in lysis buffer provided by the Akt kinase assay kit. Protein concentrations were determined using the Bradford assay kit (Bio-Rad Laboratories). Akt kinase activity was assessed using an Akt kinase assay kit (Cell Signaling Technology Inc) according to the manufacturer's instructions. Briefly, protein (200 μg) from lysate samples was immunoprecipitated with P-Akt (Ser473) antibody conjugated with beads overnight at 4°C. The immunoprecipitates were washed twice with lysis buffer and twice with kinase buffer. The samples were resuspended in 50 μL of kinase buffer containing 1 μL of 10 mM ATP and kinase substrate GSK-3 fusion protein and allowed to proceed at 30°C for 30 minutes. Reaction products were resolved by 10% SDS-PAGE gels followed by Western blot analysis with a phosphorylated GSK-3α/β antibody (P–GSK-3α/β antibody).

Immunohistochemical Staining

Tumor tissues were fixed in 10% formalin, embedded with paraffin, and processed to slides with a thickness of 5 μm. The sections were stained with hematoxylin and eosin for histology observation, or slides were stained with P-Akt (Ser473; 1:50 dilution) antibody or P-S6 (Ser235/236) antibody following the manufacturer's instructions. The immune complex was visualized using3,3′-diaminobenzidine (DAB). The sections were counterstained with hematoxylin and mounted and observed under a microscope at a magnification of × 20 or × 63.

Cell Survival Analysis

The MTS assay (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt assay) was performed according to the manufacturer's instructions. The percentage of cell survival (survival rate) was calculated by dividing the absorbance value of the treated samples by the absorbance value of the untreated control within each group. All experiments were repeated 2 to 3 times.

In Vivo Animal Model

TB3 and NGP cells were cultured in RPMI-1640 and 10% FBS media, harvested, washed with Hank balanced salt solution (HBSS), and resuspended in HBSS and Matrigel (Trevigen Inc, Gaithersburg, Md). A total of 100 μL of cell suspension containing 4 × 106 TB3 cells or 2 × 106 NGP cells were implanted into the subcutaneous tissue of the right flank of female nude mice ages 4-5 weeks (Taconic, Germantown, NY). For TB3 xenografts, mice were given water supplemented with placebo (sucrose) or TET (with sucrose) 1 week before tumor implantation and this was continued throughout the experiment. Treatment was initiated when tumors reached approximately 200 mm3. Etoposide was given 3 times a week for approximately 3 weeks at doses of 10 mg/kg and 20 mg/kg by intraperitoneal injection. Perifosine was given by oral gavage for 30 days. For the combination of etoposide and perifosine, the mice were given etoposide (10 mg/kg) 3 times a week, whereas perifosine was administered at a dose of 15 mg/kg (TB3 tumors) or 17 mg/kg (NGP tumors) by oral gavage daily for 30 days. Protein and RNA were extracted from tumors to detect TrkB levels in TB3 tumors. The dimensions of the resulting tumors were determined at least 3 times a week using a digital caliper and the tumor volume (mm3) was calculated as (L × W2)/4, in which L indicates length in mm and W indicates width in mm. All mice were euthanized by asphyxiation with regulated carbon dioxide, and their tumors were excised and immediately frozen at −80°C or fixed in 10% formalin.

Statistical Analysis

Comparisons between the 2 groups were performed using the Student t test; comparisons among ≥ 3 groups were performed using 1-way analysis of variance with Bonferroni correction. The results were shown as the means ± the standard error.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

TET-Regulated TrkB-Expressing Cell Line

To study the role of BDNF/TrkB-Akt in the response of NB to chemotherapy, we used a TET (TET off)-suppressible TrkB expression cell line, TB3.7 The presence of TET inhibited expression of TrkB, whereas the absence of TET induced expression of TrkB. In the absence of TET, there was a 3.6-fold increase in TrkB mRNA levels (Fig. 1A) and a 2.5-fold increase in TrkB protein levels (Fig. 1B) (densitometric analysis of the Western blot analysis result). Consistent with previous results,7 BDNF treatment induced an increase in the phosphorylation of TrkB only in TrkB-expressing cells (TET-) (Fig. 1B), and this was accompanied by an increase in levels of P-Akt (Ser473 and Thr308) (3.8-fold and 2-fold, respectively) (Fig. 1C). An Akt activity assay detected increased Akt activity indicated by P–GSK-3α/β (Ser21/9) in TrkB-expressing cells treated with BDNF (Fig. 1C). There was no difference in the levels of phosphorylated TrkB (P-TrkB) or P-Akt between TET+ and TET- conditions in the absence of BDNF. These data indicate that in our model system, TET regulated TrkB mRNA and protein levels and BDNF stimulation induced an increase in TrkB phosphorylation and induction of its downstream target Akt in NB cells with high TrkB expression.

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Figure 1. TrkB expression is regulated by tetracycline (TET) in vitro and affects the neuroblastoma (NB) cell response to chemotherapy. TB3 cells were cultured for 3 days in the presence or absence of TET (1 μg/mL) and (A) total RNA was extracted and treated with brain-derived neurotrophic factor (BDNF) (100 ng/mL) for 15 minutes. (B and C) Total protein was then extracted, (D) treated with etoposide (Etop), or (E and F) a combination of BDNF and Etop for 24 hours. (A) Total RNA (1 μg) was reverse-transcribed (RT) into cDNA followed by quantitative polymerase chain reaction (PCR) analysis for TrkB mRNA. (B) Protein lysates (500 μg/mL) were immunoprecipitated with anti–pan-Trk antibody and subjected to Western blot analysis for evaluation of TrkB and phosphorylated tyrosine (P-Tyr). (C) Total protein (30 μg) was immunoblotted for phosphorylated Akt (P-Akt) (Ser473), P-Akt (Thr308), and total Akt (T-Akt). In vitro Akt kinase activity was assessed by immunoprecipitating with P-Akt (Ser473) antibody, and GSK-3 was then used as a substrate to measure Akt activity indicated by phosphorylated GSK-3α/β (Ser21/9). (D, E, and F) The MTS assay was used to determine cell survival at the end of treatment. IP indicates immunoprecipitation; IB, immunoblotting; TET+, media with TET; TET-, media without TET. *P < .05 for the combination of BDNF plus etoposide versus etoposide alone.

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To compare the response to etoposide between cells with high TrkB expression and those with low TrkB expression, TB3 cells were treated with etoposide in the presence or absence of TET. Etoposide induced a decrease in cell survival in both high and low TrkB-expressing cells, but there was no difference noted with regard to their sensitivity to etoposide (Fig. 1D). To evaluate the response to etoposide after activation of the TrkB signaling pathway, we pretreated the cells with BDNF before the initiation of etoposide. The addition of BDNF to the cells with low TrkB expression did not appear to change the response to etoposide (Fig. 1E), but the addition of BDNF to the cells with high TrkB expression caused a nearly 40% increase in cell survival after treatment with etoposide (Fig. 1F).

The Akt Inhibitor Perifosine Blocked BDNF-Induced Protection of TB3 Cells From Etoposide

In a previous study, we demonstrated that constitutively active Akt attenuated the cytotoxic effects of etoposide on NB cells and the use of a small-molecule inhibitor targeting the Pleckstrin homology (PH) domain of Akt restored sensitivity to etoposide.8 Using an Akt inhibitor, perifosine, which is currently in phase 2 clinical trials in adults and has been approved by the US Food and Drug Administration for the treatment of NB, we treated the high TrkB-expressing TB3 cells with perifosine and evaluated cell growth. We found that perifosine treatment induced cell death in a dose-dependent manner at 24 hours (Fig. 2A), which was accompanied by an increase in caspase 3/7 activity (Fig. 2A). We selected concentrations of perifosine (2.5 μM and 5 μM) that did not alter cell growth at 24 hours to assess whether perifosine inhibited BDNF/TrkB-induced phosphorylation of Akt. Pretreatment of high TrkB-expressing TB3 cells with perifosine (2.5 μM or 5 μM) followed by BDNF treatment led to a 22% or 36% block, respectively, of the increase in P-Akt (Ser473) at 6 hours. At 24 hours, perifosine at a dose of 2.5 μM induced a 60% and a dose of 5 μM induced a 100% block in a BDNF-induced increase in P-Akt (Ser473) (Fig. 2B). Similar results were found in another phosphorylated site of Akt (Thr308) (Fig. 2B). Pretreatment with perifosine blocked BDNF activation of TrkB-induced phosphorylation of S6, a downstream target of activated Akt (Fig. 2B). A 24-hour pretreatment with perifosine completely blocked Akt activity, as indicated by a decrease in phosphorylation of the Akt substrate GSK-3α/β (Ser21/9) as assessed using an Akt activity assay (Fig. 2B). To determine whether perifosine altered BDNF/TrkB protection of TB3 cells from etoposide-induced cell death, we pretreated high TrkB-expressing TB3 cells with a concentration of perifosine (5 μM), which inhibits activation of both Akt and its downstream target S6, followed by etoposide (1 μg/mL) treatment in the absence or presence of BDNF (100 ng/mL) for an additional 24 hours. The results indicated that etoposide-induced cell death was blocked by pretreatment with BDNF. However, prior treatment with perifosine blocked the BDNF/TrkB-mediated rescue of TB3 cells from etoposide-induced cell death (Fig. 2C). These data indicate that perifosine blocks Akt phosphorylation and attenuates BDNF/TrkB-induced resistance to etoposide.

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Figure 2. Perifosine blocked brain-derived neurotrophic factor (BDNF)/TrkB-induced protection of TB3 cells from etoposide. (A) TB3 cells cultured in the absence of tetracycline (TET) were treated with different concentrations of perifosine for 24 hours. The MTS assay was used to detect cell survival (Left) or if treated with perifosine for 16 hours then caspase 3/7 activity was detected (Right). (B) TB3 cells cultured in the absence of TET were treated with perifosine (2.5 μm, 5 μm) for 6 hours or 24 hours followed by a 15-minute treatment with BDNF (100 ng/mL); total proteins were extracted. The expression of phosphorylated Akt (P-Akt) (Ser473), P-Akt (Thr308), total Akt (T-Akt), P-S6 (Ser235/236), and total S6 (T-S6) were analyzed by Western blot analysis. In vitro Akt kinase activity was assessed as described in Figure 1C. (C) TB3 cells cultured in the absence of TET were first pretreated with perifosine (5 μM) for 6 hours and then treated with BDNF (100 ng/mL) for 1 hour followed by etoposide (1 μg/mL) treatment for 24 hours. The MTS assay was used to assess cell survival. IP indicates immunoprecipitation; IB, immunoblotting; TET+, media with TET; TET-, media without TET.

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TET-Regulated TrkB-Expressing Tumors In Vivo

To determine whether increased TrkB expression and resistance to chemotherapy determined in in vitro models were relevant in an in vivo model system, we injected TB3 cells into nude mice that had been given access to water with TET plus sucrose (TET+) or water with sucrose alone (TET-). The results indicated that the levels of TrkB mRNA in tumors from TET- mice were nearly 3-fold higher than TrkB levels in tumors from TET+ mice. The levels of TrkB protein expression and the levels of P-Tyr in tumors from TET- mice were higher than the levels detected in tumors from TET+ mice (Fig. 3B). Hematoxylin and eosin staining of tumor tissues demonstrated that tumors from both groups of mice had the characteristic histology of small, round, blue cell tumors of childhood typical of NB tumors (Fig. 3C). Immunohistochemical staining analyses demonstrated that the relative levels of P-Akt were higher in tumors expressing high TrkB from TET- mice compared with those in low TrkB-expressing tumors from TET+ mice (Fig. 3D). These data indicate that in the absence of TET, TrkB expression is induced in vivo and this is associated with increased expression of P-Akt.

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Figure 3. Tetracycline (TET)-regulated TrkB-expressing system in vivo is shown. Subcutaneous xenografts were established as described. (A) mRNA was extracted from the tumor tissues. Total RNA (1 μg) was reverse-transcribed into cDNA followed by quantitative polymerase chain reaction analysis for TrkB mRNA levels. (B) Total protein was extracted from tumor tissues, and protein lysates (500 μg/mL) were immunoprecipitated with anti–pan-Trk antibody and subjected to Western blot analysis for the evaluation of TrkB and phosphorylated tyrosine (P-Tyr). (C and D) Tumor tissues were stained with (C) hematoxylin and eosin or (D) phosphorylated Akt (P-Akt) (Ser473) by immunohistochemical staining. IP indicates immunoprecipitation; IB, immunoblotting; TET+, water with TET plus sucrose; TET-, water with sucrose alone (original magnifications, × 63 [upper row] and ×20 [lower row]).

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Tumors With High TrkB Expression Are More Resistant to Etoposide Than Tumors With Low TrkB Expression

To study the sensitivity of tumors with low and high TrkB expression to etoposide in vivo, we initiated etoposide treatment in mice (at doses of 10 mg/kg and 20 mg/kg) when tumor volumes reached approximately 200 mm3. In the mice with low TrkB-expressing tumors (TET+), treatment with etoposide at either a dose of 10 mg/kg or a dose of 20 mg/kg caused an up to 47% decrease in tumor growth compared with placebo treatment (Fig. 4A). However, in the mice with tumors demonstrating high expression of TrkB (TET-), only etoposide at a dose of 20 mg/kg caused an up to 39% decrease in tumor growth, whereas the growth of tumors treated with etoposide at a dose of 10 mg/kg was not found to be significantly different from tumor growth in the mice treated with placebo (Fig. 4B). These data are consistent with a model in which the tumors with high TrkB expression are more resistant to etoposide treatment than the tumors with low TrkB expression.

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Figure 4. Tumors with high TrkB expression are less sensitive to etoposide than tumors with low TrkB expression. Mice with tumors were treated with vehicle or etoposide at a dose of 10 mg/kg or 20 mg/kg for 21 days. The size of the tumors from the group treated with etoposide was compared with that of the control group. TET+ indicates water with TET plus sucrose; TET-, water with sucrose alone. #P < .05 for the group treated with 10 mg/kg of etoposide versus the control group. *P < .05 for the group treated with 20 mg/kg of etoposide versus the control group.

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Perifosine Sensitized High TrkB-Expressing TB3 Tumors to Etoposide Treatment

Because high TrkB-expressing TB3 tumors have elevated levels of P-Akt (Fig. 3D) and are more resistant to etoposide (Fig. 4), we next examined whether the Akt inhibitor perifosine would affect the sensitivity of high TrkB-expressing tumors to etoposide. We first tested the response of tumors to different doses of perifosine (10 mg/kg, 15 mg/kg, and 20 mg/kg). There was an antitumor growth effect of perifosine at a dose of 20 mg/kg, but treatment with 10 mg/kg or 15 mg/kg of perifosine did not appear to cause a statistically significant difference in tumor growth compared with the placebo treatment (Fig. 5A). When we examined the tumors using immunohistochemical staining to assess expression of P-Akt (Ser 473) and P-S6 (Ser235/236) in mice treated with 15 mg/kg of perifosine, we found decreased expression of P-Akt and P-S6 in these tumors compared with tumors from placebo-treated mice (Fig. 5B). Therefore, we evaluated whether perifosine at a dose (15 mg/kg/day) sufficient to inhibit Akt activation would alter the sensitivity of TB3 tumors to etoposide (10 mg/kg). Although neither drug at these doses, as a single agent, caused a statistically significant alteration in tumor growth, the combination of perifosine (15 mg/kg) and etoposide (10 mg/kg) resulted in an 80% decrease in tumor growth compared with the placebo (Fig. 5C). These data indicate that perifosine treatment inhibits Akt phosphorylation in tumor tissues and increases the sensitivity of TB3 NB tumors to etoposide treatment. To determine whether perifosine could synergize with etoposide in another TrkB-expressing NB cell line, we tested 3 cell lines (NGP, LAN5, and SY5Y) and found that NGP cells expressed TrkB and activated Akt (Fig. 6 A). The in vivo sensitivities of NGP xenografts to etoposide (Fig. 6B) and perifosine (Fig. 6C) were established. Mice were then treated with a combination of etoposide (10 mg/kg) and perifosine (17 mg/kg) at doses that individually did not have a statistically significant effect on tumor growth. We found that this combination of etoposide and perifosine induced an 85% inhibition of tumor growth compared with placebo (Fig. 6D).

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Figure 5. Perifosine (Peri) increased the sensitivity of high TrkB-expressing TB3 tumors to treatment with etoposide. (A) Mice were treated with vehicle or Peri at doses of 10 mg/kg, 15 mg/kg, or 20 mg/kg for 30 days. The tumor size in the treated groups was compared with that of the control group. #P < .05 for the group treated with Peri (20 mg/kg) versus the control group. (B) Tumor tissues were stained with phosphorylated Akt (P-Akt) (Ser473) and P-S6 (Ser235/236) by immunohistochemical staining (original magnification, × 63). (C) Mice were treated with vehicle, Peri (15 mg/kg), etoposide (Etop) (10 mg/kg), or a combination of Peri (15 mg/kg) and Etop (10 mg/kg). The tumor size in treated groups was compared with that of the control group. * P < .05 for group treated with Peri plus Etop versus the control group. TET- indicates water with sucrose alone.

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Figure 6. Perifosine (Peri) increased the sensitivity of NGP tumors to treatment with etoposide (Etop). (A) Proteins from NGP, LAN5, and SY5Y cells were extracted and expression of TrkB, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphorylated Akt (P-Akt) (Ser473), and total Akt (T-Akt) was analyzed by Western blot analysis. (B, C, and D) NGP tumors were treated with (B) Etop (10 mg/kg or 20 mg/kg) or (C) Peri (10 mg/kg, 17 mg/kg, or 24 mg/kg) individually or (D) a combination of Etop (10 mg/kg) and Peri (17 mg/kg). The tumor size in treated groups was compared with that of the control group. Panel B: *P < .05 for the group treated with Etop (20 mg/kg) versus the control group. Panel C: #P < .05 for the group treated with Peri (24 mg/kg) versus the control group. Panel D: *P < .05 for the group treated with Peri (17 mg/kg) and Etop (10 mg/kg) versus the control group.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

In the current study, we established an in vivo tumor model system using a TET-repressible TrkB-expressing isogenic cell model to study the influence of the TrkB/Akt signaling pathway on the response of NB tumors to chemotherapy. We found tumor xenografts with elevated TrkB had increased levels of activated TrkB (P-Tyr-TrkB) and Akt (P-Akt). Consistent with our in vitro studies, tumors expressing elevated activated TrkB and Akt were found to be more resistant to etoposide. Finally, the sensitivity of NB tumors expressing elevated TrkB levels to etoposide was restored if mice were treated with a concentration of perifosine sufficient to inhibit activated Akt in tumor xenografts. These findings indicate that targeting Akt in NB tumors may increase their sensitivity to cytotoxic therapy.

Clinical observation has indicated that TrkB and BDNF are expressed in a subset of aggressive or unfavorable NB.19 To understand how activation of the BDNF/TrkB pathway affected the biology of NB tumors, we and other investigators determined that BDNF activation of TrkB did not significantly alter NB cell growth but enhanced cell survival under stress conditions such as serum starvation.20, 21 Later, we reported that BDNF activation of TrkB induced NB cell resistance to the cytotoxic drug vinblastine in vitro6 and subsequent studies demonstrated similar results using cytotoxic drugs such as etoposide, cisplatin, and paclitaxel.7, 8 Given the role of TrkB in the biological and clinical behavior of NB, it was reasonable to pursue inhibition of the TrkB receptor as an important adjunct to therapy. Lestaurtinib (CEP-701) is a Trk-selective tyrosine kinase inhibitor that blocks activated Trk and has shown efficacy alone or in combination with conventional chemotherapy in an NB cell model system using constitutive TrkB expression.22, 23 Recently, another Trk inhibitor, AZ623, was found to inhibit activation of TrkB in vitro and NB cell growth in vivo.24 However, neither of these studies demonstrated the inhibition of target in vivo. In addition to Trk, other receptors tyrosine kinases (RTK) such as VEGFR, epidermal growth factor receptor, and IGFR1 have also been implicated in NB pathogenesis or malignant behavior.9-11 Use of a Trk inhibitor would only target those cells dependent on activated Trks, whereas cells not dependent on Trks could survive and even those that may initially be dependent on Trks may develop resistance or co-opt other tyrosine receptor kinases for survival. Therefore, the advantage of targeting the Akt pathway over tyrosine receptor kinase-specific targeting strategies is that it would have a broader specificity against multiple tyrosine receptor kinases that activate the PI3 kinase/Akt pathway and stimulate tumor cell survival. Therefore, it would not be necessary to know if a given tumor relied on a specific tyrosine kinase receptor, as long as its survival and/or other malignant characteristics were dependent on Akt.25

To our knowledge to date, several types of Akt inhibitors have been investigated, including phosphatidylinositol analog inhibitors, allosteric Akt kinase inhibitors, ATP-competitive inhibitors, and alkylphospholipids.26 However, the use of these inhibitors is limited either by high toxicity or low bioavailability and stability in vivo.26 Perifosine, an alkylphospholipid, acts at the cell membrane, which makes it distinct from conventional chemotherapeutic drugs that mainly target DNA. In adult clinical trials, perifosine has been found to have no hematological toxicity and the only dose-limiting toxicity was gastrointestinal, which was readily ameliorated with prophylactic medicine.27 More recently, we found that perifosine had an antitumor growth effect in all NB xenograft tumor models tested and this effect was noted using NB cell lines that carried several genetic alterations that characterize NB tumors, such as MYCN amplification, TP53 mutation, and ALK mutation. The phase 1 clinical trial of perifosine in pediatric solid tumors used 3 doses of perifosine (25 mg/m2/day, 50 mg/m2/day, and 75 mg/m2/day), with serum levels reaching 17 to 32 μM and no dose-limiting toxicities observed.28 Our preclinical perifosine dosing of 24 mg/kg/day would be equivalent to a phase 1 dosing of 72 mg/m2/day,29 and in our in vitro studies, doses of perifosine from 20 to 30 μM induced at least a 70% induction of cell death in all NB cell lines evaluated. From these data, it can be noted that the doses used in the preclinical in vitro and in vivo models are achievable in patients. Although clinical response is not an aim of phase 1 trials, approximately 67% of patients with stage 4 (defined by International Neuroblastoma Staging System) multiply recurrent NB achieved stable disease for up to 55 weeks.28

As effective as a targeted monotherapy may be, it is usually enhanced when combined with traditional cytotoxic reagents. We found a synergistic response and an 80% decrease in tumor growth when mice bearing xenografts were administered the combination of perifosine and etoposide using doses at which either agent alone had no significant effect on tumor growth. Using a dose of perifosine (15 mg/kg/day, equal to 45 mg/m2/day) that inhibited Akt activity but had little effect on tumor growth in TB3 tumors, we found the sensitivity of NB tumors to treatment with etoposide was greatly increased. We did not observe any side effects using these low doses of perifosine and etoposide (10 mg/kg) in combination. An important goal of therapy is to not only increase treatment efficacy but also decrease toxicity. The results of the current study are similar to those reported using the Trk inhibitor.22, 23 To our knowledge, no study to date has reported the combined use of an Akt inhibitor and chemotherapeutic drugs in pediatric solid tumors in vivo, although perifosine has been shown to increase the sensitivity of medulloblastoma to radiation in vitro.30 In adult cancer studies, perifosine is reported to sensitize endometrial cancer cells to cisplatin therapy.31 In clinical trials, perifosine is reported to have a synergistic antitumor growth effect when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)32 in the treatment of patients with multiple myeloma. Based on the results of our preclinical studies and those in adults, we have reason to believe that treatment with perifosine in combination with conventional therapy will be a good strategy with which to improve the treatment efficacy in patients with NB.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Supported by the Intramural Research Program, Center for Cancer Research at the National Cancer Institute, National Institutes of Health.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

We thank Keryx Biopharmaceuticals and Dr. Enrique Poradosu (Keryx Biopharmaceuticals) and Dr. Sherry S. Ansher (Cancer Therapy Evaluation Program) for coordinating the supply of perifosine. We thank Lauren Marks for programmatic assistance and other members of the Cellular and Molecular Biology Section at the National Cancer Institute for their thoughtful review of this study.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. Acknowledgements
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES
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