Tyrosine kinase inhibitor CEP-701 blocks the NTRK1/NGF receptor and limits the invasive capability of prostate cancer cells in vitro

In the prostate, cellular growth and differentiation are finely regulated by a complex interaction between stromal and epithelial cells under the control of both autocrine and paracrine regulatory factors such as the nerve growth factor (NGF). However, the role of NGF and its receptors including the high-affinity p-140 TrkA and the low-affinity p75 NTR receptors remains controversial. Moreover prostate tissues stored other neutrophins such as NT3, NT4 and brain derived neutrophic factor (BDNF) as well as the corresponding receptors (NTRs). Different members of NTRs are expressed during prostate cancer (PCa) progression, suggesting their involvement in cell proliferation, anoikis protection and malignancy. Therefore, we analyzed the expression of NTRs including NTRK1 (TrkA), NTRK2 (TrkB), NTRK3 (TrkC) and p75 NGFR in a panel of 7 well-characterized PCa cell lines and 12 cell derivatives from PC3 (4), DU145 (2), CWR22R (4) and LnCap (2) cell lines possessing different proliferative/invasive capabilities. We evaluated also the role of NGF, BDNF and NT3 in the modulation of cell migration and invasion and, finally, the effects of a pan Trk inhibitor, CEP-701 which has been included in some clinical trials for the treatment of PCa. We observed the following: i) TrkA and TrkB expression was significantly higher in AR-negative compared to AR-positive cells; ii) TrkA and TrkB expression was related to the invasive capacity/malignancy of PCa cells; iii) p75 NGFR could be considered a tumor suppressor gene which is present at high levels only in AR-positive cells; and iv) that NGF and BDNF (targeting TrkA/p75 NTR and TrKB, respectively) induced cell migration and this was inhibited by the CEP-701 treatment. In conclusion, the malignancy of PCa seems to be accompanied by increased TrkA and TrkB signaling (with a reduction of p75 NGFR expression) and CEP-701 could be used to reduce the metastasis formation in advanced PCa. CEP-701 is a trademark of Cephalon Inc., West Chester, PA, USA.     

Expression of Trk tyrosine kinase receptor is a biologic marker for cell proliferation and perineural invasion of human pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a widely known severe malignancy with a poor prognosis. Perineural invasion extending to the extra-pancreatic nerve plexus, a significant concern in the treatment is frequently present in this cancer. We analyzed immunohistochemical expression of neurotrophins (NGF, BDNF, NT-3) and the cognate receptors, Trk tyrosine kinase receptor family (TrkA, B, C) and p75NGFR in 28 surgically resected PDAC specimens. A comparative study between several clinicopathologic factors and Trk receptors revealed a significant correlation between increased expression of TrkA and cancer proliferation, as well as TrkC and cancer invasion, including venous and perineural invasion. The present findings revealed a novel mechanism in PDAC progression that is mediated via a NTs-Trk interaction.     

Expression of the neurotrophin receptor TrkA down-regulates expression and function of angiogenic stimulators in SH-SY5Y neuroblastoma cells

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors. Mechanisms regulating the expression of angiogenic factors in tumor cells are largely unknown. High expression of the neurotrophin receptor TrkA in neuroblastomas (NBs) is associated with a favorable prognosis, whereas TrkB is mainly expressed on aggressive, MYCN-amplified NBs. To investigate the biological effects of TrkA and TrkB expression on angiogenesis in NB, we examined the expression of angiogenic factors in the human NB cell line SY5Y and its TrkA and TrkB transfectants. In comparison with parental SY5Y cells, mRNA and protein levels of the examined angiogenic factors were significantly reduced in SY5Y-TrkA cells, whereas SY5Y-TrkB cells did not demonstrate a significant change. Conditioned medium of TrkB transfectants and parental SY5Y cells induced endothelial cell proliferation and migration, but this effect was completely absent in SY5Y-TrkA cells. TrkA expression also resulted in severely impaired tumorigenicity in a mouse xenograft model and was associated with reduced angiogenic factor expression and vascularization of tumors, as determined by immunohistochemistry and an in vivo Matrigel assay. TrkA expression inhibits angiogenesis and tumor growth in SY5Y NB cells by down-regulation of angiogenic factors, whereas expression of TrkB does not down-regulate the production of these angiogenic factors. The biologically different behavior of TrkA- and TrkB-expressing NBs may be explained in part by their effects on angiogenesis.     

Differential cross-regulation of TrkA and TrkC tyrosine kinase receptors with p75

The neurotrophins neurotrophin-3 (NT-3), brain-derived growth factor (BDNF) and nerve growth factor (NGF) bind to the p75 receptor, but each neurotrophin also binds a more selective Trk receptor (e.g. TrkA-NGF and TrkC-NT-3). The biochemical signals following engagement of either Trk or p75 with ligands are well understood, but long-term biological outcomes (trophic, proapoptotic or differentiative) remain unclear because they are cell/tissue specific. For example, Trk receptors are usually trophic but when overexpressed they can be proapoptotic in neuroblastomas and medulloblastomas. We hypothesized that coexpression of Trk and p75 receptors may lead to cross-regulation of signals and different biological outcomes; and used receptor-selective ligands to study cross-regulation by these receptors. We show that in the absence of Trk activation, expression of TrkC is permissive of p75 trophic and differentiation signals induced by p75 ligands, whereas expression of TrkA abolishes trophic and differentiation signals induced by p75 ligands. In contrast, in the presence of Trk activation, p75 ligands can regulate TrkA-mediated survival and TrkC-mediated differentiation. Therefore, a complex homeostasis of p75-selective and Trk-selective signals may determine the fate of cells expressing both receptors.     

Resistance to chemotherapy mediated by TrkB in neuroblastomas

Neuroblastoma is a common childhood tumor derived from the peripheral nervous system. Favorable neuroblastomas usually express TrkA, the receptor for nerve growth factor (NGF), whereas unfavorable, MYCN-amplified neuroblastomas usually express TrkB and its ligand, brain-derived neurotrophic factor (BDNF). Here, we provide evidence that the TrkB-BDNF pathway is associated with enhanced survival and resistance to chemotherapy in neuroblastoma. We transfected the neuroblastoma line SH-SY5Y, which has endogenous expression of BDNF, with a full-length TrkB expression vector, and obtained clones with moderate or high levels of expression. Cells were exposed in vitro to chemotherapy agents used to treat neuroblastomas: doxorubicin, etoposide (VP16), and cisplatin. Chemoresistance was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell survival and by ELISA for cell death. In all cases, the TrkB-expressing subclones were more resistant to treatment than the parent line. Furthermore, when the TrkB tyrosine kinase was blocked with the Trk-specific inhibitor CEP-2563, or by neutralizing antibody to BDNF, sensitivity to chemotherapy was significantly increased. We also found constitutive phosphorylation of AKT at the Ser-473 site in TrkB transfectants, whereas there was only a minimal level of constitutive phosphorylation of AKT in SY5Y cells. These results show that the TrkB-BDNF pathway provides a survival advantage when exposed to DNA-damaging reagents, and, therefore, this autocrine pathway may play an important role in mediating the drug-resistant phenotype associated with TrkB-expressing neuroblastomas. Activation of PI3K/AKT survival pathway may contribute to the increased drug resistance in TrkB-expressing neuroblastomas.     

TrkB inhibition by GNF-4256 slows growth and enhances chemotherapeutic efficacy in neuroblastoma xenografts

Purpose

Neuroblastoma (NB) is one of the most common and deadly pediatric solid tumors. NB is characterized by clinical heterogeneity, from spontaneous regression to relentless progression despite intensive multimodality therapy. There is compelling evidence that members of the tropomyosin receptor kinase (Trk) family play important roles in these disparate clinical behaviors. Indeed, TrkB and its ligand, brain-derived neurotrophic factor (BDNF), are expressed in 50–60 % of high-risk NBs. The BDNF/TrkB autocrine pathway enhances survival, invasion, metastasis, angiogenesis and drug resistance.

Methods

We tested a novel pan-Trk inhibitor, GNF-4256 (Genomics Institute of the Novartis Research Foundation), in vitro and in vivo in a nu/nu athymic xenograft mouse model to determine its efficacy in inhibiting the growth of TrkB-expressing human NB cells (SY5Y-TrkB). Additionally, we assessed the ability of GNF-4256 to enhance NB cell growth inhibition in vitro and in vivo, when combined with conventional chemotherapeutic agents, irinotecan and temozolomide (Irino–TMZ).

Results

GNF-4256 inhibits TrkB phosphorylation and the in vitro growth of TrkB-expressing NBs in a dose-dependent manner, with an IC₅₀ around 7 and 50 nM, respectively. Furthermore, GNF-4256 inhibits the growth of NB xenografts as a single agent (p < 0.0001 for mice treated at 40 or 100 mg/kg BID, compared to controls), and it significantly enhances the antitumor efficacy of irinotecan plus temozolomide (Irino–TMZ, p < 0.0071 compared to Irino–TMZ alone).

Conclusions

Our data suggest that GNF-4256 is a potent and specific Trk inhibitor capable of significantly slowing SY5Y-TrkB growth, both in vitro and in vivo. More importantly, the addition of GNF-4256 significantly enhanced the antitumor efficacy of Irino–TMZ, as measured by in vitro and in vivo growth inhibition and increased event-free survival in a mouse xenograft model, without additional toxicity. These data strongly suggest that inhibition of TrkB with GNF-4256 can enhance the efficacy of current chemotherapeutic treatment for recurrent/refractory high-risk NBs with minimal or no additional toxicity.

     

Biological effects of TrkA and TrkB receptor signaling in neuroblastoma

The Trk family consists of three receptor tyrosine kinases, each of which can be activated by one or more of four neurotrophins-NGF, BDNF, NT3 and NT4. Neurotrophins mediate their multiple effects through a number of distinct intracellular signaling cascades regulating such diverse biological responses as cell survival, proliferation and differentiation in normal and neoplastic neuronal cells. Expression of Trk receptors also plays an important role in the biology and clinical behavior of neuroblastomas. High expression of TrkA is present in neuroblastomas with favorable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavorable, aggressive neuroblastomas. This short review discusses recent data on the biological roles of TrkA and TrkB signaling in neuroblastoma.     

Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells

Activation of the neurotrophin receptor TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events leading to neuronal differentiation in vitro and might play an important role in the differentiation of favorable neuroblastomas (NB) in vivo. To study TrkA signal transduction pathways and their effects on differentiation in NB, we stably expressed wild-type TrkA and five different TrkA mutants in the NGF unresponsive human NB cell line SH-SY5Y. Resulting clones were characterized by TrkA mRNA and protein expression, and by autophosphorylation of the receptor. Introduction of wild-type TrkA restored NGF responsiveness of SH-SY5Y cells, as demonstrated by morphological differentiation, activation of mitogen-activated protein kinases (MAPK) and induction of immediate-early genes. Expression of TrkA in the absence of NGF resulted in growth inhibition of transfectants compared to parental cells, whereas NGF-treatment increased their proliferation rate. Analysis of downstream signal transduction pathways indicated that NGF-induced differentiation was dependent on TrkA kinase activity. Our data suggest that several redundant pathways are present further downstream, but activation of the RAS/MAPK signaling pathway seems to be of major importance for NGF mediated differentiation of NB cells. Our results also show that the signaling effector SH2-B is a substrate of NGF-mediated Trk signaling in NB, whereas it is not activated by NGF in rat pheochromocytoma PC12 cells. This might explain the differences we observed in TrkA signaling between neuroblastoma and PC12 cells. Further insight into TrkA signaling may suggest new options for the treatment of NB.     

Neurotrophin Effects on Neuroblastoma Cells: Correlation with Trk and p75NTR Expression and Influence of Trk Receptor Bodies

Neurotrophins are key signalling molecules in the development of the nervous system. They elicit diverse cellular responses such as proliferation, differentiation, survival and apoptosis. Neurotrophins (NTs) bind to two different classes of cell surface receptors, Trk receptor tyrosine kinases and p75NTR, both of which are expressed by neuroblastoma cells. Neurotrophin signalling via Trks was shown to promote both survival and differentiation of neuroblastoma cells in vitro. The expression of certain Trk receptors is considered to be a prognostic indicator. The p75NTR receptor is the founding member of the Fas/TNF-R family, which is best known for its function in the induction of apoptosis. Its function in neuroblastomas is thus far poorly understood. We analysed neurotrophin receptor (NTR) expression of neuroblastoma cells by surface biotinylation assays and applied recombinant nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 to these cell lines assessing their survival and proliferation in long-term assays lasting 6 days. NGF increased proliferation of Neuro 2a cells, which express p75NTR but no TrkA receptors on their surface. On the other hand, SK-N-BE cell proliferation was decreased after NGF treatment, even though these cells also express p75NTR but no TrkA receptors on their surface. Interestingly, neurotrophin-scavenger proteins (TrkB-Fc and TrkC-Fc) as well as chemical blockers of Trk receptor signalling (K252a, Wortmannin, PD98059) slowed down the proliferation of both cell lines in medium containing serum. Taken together, our results indicate that p75NTR activation has diverse effects on neuroblastomas, depending on the specific neuroblastoma clone. In addition, our studies point towards TrkB-Fc or TrkC-Fc receptor bodies as useful tools to influence the survival of neuroblastoma cells.     

Expression of TrkA,TrkB and TrkC in human neuroblastomas

There is considerable interest in the role of the TRK family of neurotrophin receptors in regulating the survival, growth and differentiation of normal and neoplastic nerve cells. Indeed, there is increasing evidence that TRK genes play an important role in the biology and clinical behavior of neuroblastomas, tumors of the peripheral nervous system. Evidence from several independent studies suggests that high expression of TrkA is an indicator of favorable outcome, and there is an inverse correlation between TrkA expression and N-myc amplification. In addition, some primary neuroblastomas differentiate in vitro in the presence of NGF but die in its absence. We have evidence that coexpression of full-length TrkB and BDNF is associated with N-myc amplification and may represent an autocrine survival pathway. Conversely, truncated TrkB is expressed predominantly in differentiated tumors. Finally, TrkC is expressed in favorable neuroblastomas, essentially all of which also express TrkA. In summary, the study of neurotrophin receptor expression and function in neuroblastomas may provide important insights into the role that these pathways play in the pathogenesis and clinical behavior of this tumor. Ultimately, these pathways may provide attractive targets for the development of therapy aimed at inducing differentiation or programmed cell death in these tumors.     

Trk inhibitor attenuates the BDNF/TrkB-induced protection of neuroblastoma cells from etoposide in vitro and in vivo

TrkB activation by brain-derived neurotrophic factor (BDNF) contributes to chemo-resistance in neuroblastoma (NB). AZD6918 is a novel potent and selective inhibitor of the Trk tyrosine kinases. In this study we evaluated the effect of AZD6918 on the sensitivity of TrkB-expressing NB cells or tumors to etoposide, a topoisomerase II inhibitor. TrkB-expressing NB cells were treated with AZD6918 and etoposide in the presence or absence of BDNF in vitro and cell survival was determined. NB xenograft tumors were treated with AZD6918 and etoposide, either alone or in combination in vivo, and the anti-tumor growth effect or mice survival advantage was evaluated. Our study showed that AZD6918 induced cell death as a single agent and attenuated BDNF/TrkB-induced protection from etoposide in vitro. Although AZD6918 alone didn''t show anti-tumor growth effect or survival advantage in vivo, a combination of AZD6918 and etoposide had a statistically significant stronger anti-tumor growth effect and survival advantage compared to treatment with either agent alone. Our data indicate that as a Trk inhibitor AZD6918 increased the sensitivity of NB to etoposide. These results extend the spectrum of cytotoxic drugs whose efficacy is increased in combination with Trk inhibitors and support the combination of Trk inhibitors and cytotoxic drugs for NB treatment.     

The selective Trk inhibitor AZ623 inhibits brain-derived neurotrophic factor-mediated neuroblastoma cell proliferation and signaling and is synergistic with topotecan

BACKGROUND:

TrkB expression is associated with poor prognosis for patients with neuroblastoma. AZ623 is a novel potent and selective inhibitor of the Trk family of tyrosine kinases. The authors hypothesized that AZ623 would inhibit TrkB‐mediated signaling in neuroblastoma tumor cells and would be synergistic when combined with chemotherapy.

METHODS:

Neuroblastoma cell lines were screened for TrkB receptor mRNA expression and for their proliferation rates in response to brain‐derived neurotrophic factor (BDNF). The effects of AZ623 on Trk receptor phosphorylation, signaling, and cell growth were evaluated in BDNF‐treated neuroblastoma cells. Mice with human neuroblastoma xenograft tumors were treated with AZ623 alone and in combination with topotecan, and tumor growth rates were determined during and after treatment.

RESULTS:

Neuroblastoma cell lines expressed various levels of the TrkB receptor and demonstrated increased proliferation in response to BDNF. BDNF treatment stimulated TrkB phosphorylation and downstream signaling that could be inhibited by AZ623. Neuroblastoma cells demonstrated in vitro sensitivity to AZ623, with concentration that inhibits 50% (IC50) values between 0.8 to 7 μM. AZ623 treatment was found to inhibit BDNF‐mediated neuroblastoma cell proliferation. Mice with human neuroblastoma xenograft tumors demonstrated tumor growth inhibition when treated with AZ623 and with AZ623 combined with topotecan. Limited tumor regrowth was noted in mice with tumors treated with AZ623 combined with topotecan after treatment discontinuation.

CONCLUSIONS:

AZ623 is a novel selective Trk inhibitor that inhibits BDNF‐mediated signaling and neuroblastoma cell proliferation. AZ623 treatment inhibits the growth of human neuroblastoma xenograft tumors, and treatment with AZ623 combined with topotecan results in the prolonged inhibition of tumor regrowth. On the basis of these results, further preclinical development is warranted. Cancer 2011. © 2010 American Cancer Society.     

Brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from chemotherapy-induced apoptosis via phosphatidylinositol 3'-kinase pathway

Neuroblastoma (NB) tumors expressing high levels of brain-derived neurotrophic factor (BDNF) and TrkB are associated with poor 5-year survival outcomes. Our previous studies indicated that BDNF blocked the cytotoxic effects of vinblastine on NB cells. Here we evaluated the ability of BDNF to decrease the chemosensitivity of NB cells to a number of common chemotherapeutic agents. Two SH-SY5Y NB cell lines (TB3 and TB8) expressing TrkB under the control of a tetracycline (Tet)-repressible promoter element were generated, and used to assess apoptosis resulting from treatment with cisplatin, doxorubicin, etoposide, and vinblastine. BDNF treatment of high TrkB-expressing TB8 (Tet-) and TB3 (Tet-) cells blocked drug-induced cell death in a dose-dependent manner. Only high-dose BDNF (100 ng/ml) could block the effects of chemotherapy in low TrkB-expressing cells. The ability of BDNF to rescue the cells from chemotherapeutic agent-induced cell death was inhibited by treatment with the Trk tyrosine kinase inhibitor K252a or the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002, but not by the mitogen-activated protein kinase kinase inhibitor PD98059 or the peritoneal lymphocyte gamma inhibitor U73122, indicating that both TrkB and PI3K activities are required for the survival-promoting effects of BDNF. BDNF also protected TrkB-expressing NGP and KCNR NB cells from chemotherapeutic agent-induced cell death, and LY294002 inhibited this protection. These results suggest that TrkB and BDNF can contribute to the chemoresistance of poor prognosis tumors, and that suppression of PI3K activity might improve the ability of these agents to induce the death of NB tumors.     

A possible role of BDNF in prostate cancer detection

Many studies have demonstrated that both normal and malignant prostate cells respond to a variety of growth factors, while several significant differences were found between normal and tumoural cells. The aim of this study was to focus on the localization and distribution of the immuno-reactivity for neurotrophins (NTs) and neurotrophin receptors (NTRs) in normal, hyperplastic and prostate cancer cells, obtained from 40 subjects. We studied samples obtained from 16 prostate cancer (PC, retropubic radical prostatectomy), 20 benign prostatic hyperplasia (BPH, supra-pubic prostatectomy) and normal peripheral prostate tissue from four fresh male cadavers. Samples were examined via immunohistochemical techniques in order to detect the expression of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and their own receptors TrkA, p75, TrkB and TrkC. We observed a high expression of BDNF and TrkB in PC and BPH, though no immuno-reactivity was found for p75. Low expression was reported by other NTs and NTRs in the normal peripheral prostate zone, BPH and PC. These data suggest a possible predictive role for NTs and NTRs, especially for BDNF and TrkB, in the diagnosis and/or management of prostate cancer. The absence of p75 expression confirms its supposed role in apoptotic phenomenon.     

Perifosine-induced inhibition of Akt attenuates brain-derived neurotrophic factor/TrkB-induced chemoresistance in neuroblastoma in vivo

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.     

K252a对表达TrkB的NB细胞耐药性的影响

目的:使用全反式维甲酸(ATRA)诱导SH-SY5Y NB(SY5Y)细胞的TrkB表达,加入BDNF激活TrkB/BDNF信号传导通路使TrkB磷酸化,然后用特异性酪氨酸激酶抑制剂K252a阻断该通路,观察NB细胞对化疗药物-顺铂(CDDP)敏感性的变化.方法:用ATRA诱导SY5Y细胞的TrkB高表达后,用BDNF、CDDP和TrkB信号传导通路的特异性阻断剂-K252a处理,MTT法检测细胞的存活率变化;流式细胞技术(FCM)检测细胞凋亡率;应用免疫组化法检测细胞磷酸化TrkB(p-TrkB)的表达水平.结果:ATRA能特异性诱导TrkB的表达,加入BDNF后可激活TrkB/BDNF信号传导通路并能引起TrkB的自动磷酸化;K252a能够阻断TrkB的磷酸化和TrkB/BDNF信号传导通路;经CDDP处理的SY5Y细胞的存活率下降、凋亡率上升.结论:阻断TrkB/BDNF信号传导通路可提高共表达TrkB/BDNF的NB细胞的化疗敏感性.因此,应用TrkB的抑制剂治疗NB有望提高该病的临床治愈率.     

Anti-apoptotic role and clinical relevance of neurotrophins in diffuse large B-cell lymphomas

Background:

Diffuse large B-cell lymphoma (DLBCL) is a fatal malignancy that needs to identify new targets for additional therapeutic options. This study aimed to clarify the clinical and biological significance of endogenous neurotrophin (nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF)) in DLBCL biopsy samples and cell lines.

Methods:

We analysed expression of NGF, BDNF, and their receptors (Trk, p75^NTR) in 51 biopsies and cell lines by immunohistochemistry, immunofluorescence, and western blotting. To investigate the biological role of BDNF/TrkB/p75^NTR axis, effects of neurotrophin signalling inhibition were determined on tumour cell survival and vascular endothelial growth factor (VEGF) secretion. The pharmacological pan-Trk inhibitor K252a was used for in vitro and in vivo studies.

Results:

A BDNF/TrkB axis was expressed in all biopsies, which was independent of the germinal centre B-cell (GCB)/non-GCB profile. p75^NTR, TrkB, and BDNF tumour scores were significantly correlated and high NGF expression was significantly associated with MUM1/IRF4, and the non-GCB subtype. Diffuse large B-cell lymphoma cell lines co-expressed neurotrophins and their receptors. The full-length TrkB receptor was found in all cell lines, which was also phosphorylated at Tyr-817. p75^NTR was associated to Trk and not to its cell death co-receptor sortilin. In vitro, inhibition of neurotrophin signalling induced cell apoptosis. K252a caused cell apoptosis, decreased VEGF secretion, and potentiated rituximab effect, notably in less rituximab-sensitive cells. In vivo, K252a significantly reduced tumour growth and potentiated the effects of rituximab in a GCB-DLBCL xenograft model.

Conclusions:

This work argues for a pro-survival role of endogenous neurotrophins in DLBCLs and inhibition of Trk signalling might be a potential treatment strategy for rituximab resistant subgroups.     

Signal transduction pathways through TRK-A and TRK-B receptors in human neuroblastoma cells.

Little is known about the signal transduction pathways of TRK family receptors in neuroblastoma (NB) cells. In this study, an NB cell line, designated MP-N-TS, was established from an adrenal tumor taken from a 2-year-old boy. This cell line expressed both TRK-A and TRK-B receptors, which is rare in a single NB cell line. Therefore, the MP-N-TS cell line was used to determine whether the signal transduction through these constitutive receptors is functional. Three neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-4 / 5 (NT-4 / 5), induced tyrosine phosphorylation of panTRK, and BDNF and NT-4 / 5 induced tyrosine phosphorylation of TRK-B. Tyrosine phosphorylation of panTRK and / or TRK-B by the neurotrophins was inhibited in the presence of a tyrosine kinase inhibitor K252a. Tyrosine phosphorylation of Src homologous and collagen (Shc), extracellular signal-regulated kinase (ERK)-1 and ERK-2, and phospholipase C-gamma1 (PLC-gamma1) was increased by the three neurotrophins and the increase was inhibited in the presence of K252a. Activation of Ras, detected as the GTP-bound form of Ras, was induced by the three neurotrophins. The neurotrophins did not modulate the expressions of TRK-A or TRK-B mRNA, but they did induce the expression of c-fos mRNA. Exogenous NGF induced weak neurite outgrowth, whereas exogenous BDNF and NT-4 / 5 induced distinct neurite outgrowth. Exogenous BDNF and NT-4 / 5 increased the number of viable cells, while NGF did not. Our results demonstrate that the signal transduction pathways through TRK-A and TRK-B in MP-N-TS cells are functional and similar, and the main downstream signaling pathways from the three neurotrophins are mitogen-activated protein kinase (MAPK) cascades through Shc, activated Ras, ERK-1 and ERK-2, and the transduction pathway through PLC-gamma1. Further, BDNF and NT-4 / 5 increased cell viability. The MP-N-TS cell line should be useful for clarifying the TRK-A and TRK-B signaling pathways responsible for the different prognoses in patients with NB.     

Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

Background  

Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC) and p75^NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75^NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75^NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75^NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue.