High TrkA Research Articles

First-time in-human study of VMD-928, an oral allosteric TrkA selective inhibitor targeting TrkA protein overexpression, in patients with solid tumors or lymphoma.

3081 Background: Tropomyosin receptor kinase A (TrkA) is a protein encoded by the NTRK1 gene. Upregulation of TrkA signal transduction pathways, which can be caused by either NTRK1 gene fusions or intact TrkA protein overexpression, are oncogenic for multiple tumor types. This is clinically validated by demonstrated efficacy of ATP-competitive pan-TrkA/B/C inhibitors, larotrectinib and entrectinib for treatment of advanced solid tumors harboring NTRK1/2/3 gene fusions. VMD-928 is the first oral small-molecule TrkA (NTRK1) selective inhibitor with differentiated allosteric (ATP non-competitive) and irreversible mechanisms of action, acting as a molecular glue which sticks two TrkA proteins together and dose-dependently inhibits TrkA functions and downstream effectors, e.g. activated ERK, a hallmark of cancer. We conducted a first time in human phase 1 trial and completed the dose escalation phase. Methods: This is an open label, Phase 1 study investigating oral VMD-928 in adults with advanced solid tumors or lymphoma. The primary objective is to assess the safety and tolerability of VMD-928 and determine the recommended phase 2 dose. Secondary objectives include characterizing the pharmacokinetics (PK) and pharmacodynamics as well as assessing antitumor activity. Results: Non-biomarker-selected patients (n = 20) were accrued to 4 dose escalation cohorts ranging from 300 mg/day to 2400 mg/day. Three patients were accrued to the 2400 mg dose level with one DLT of elevated bilirubin, AST and ALT. The trough concentrations (Ctrough, ng/mL) ranged from 5.3 to 727. The dose was de-escalated to 1200 mg per day in divided doses and fifteen heavily pretreated patients were accrued with the following tumor types: adenoid cystic carcinoma, cholangiocarcinoma, lung cancer, pancreatic cancer, parotid, and squamous cell carcinoma of head and neck. There were no DLT’s at this dose and one patient with adenoid cystic carcinoma had prolonged stable disease. Common adverse events related to therapy were dark stool (35%), elevated liver enzymes (25%, primarily at 2400 mg/day), fatigue, nausea or vomiting, and decreased appetite (20% each). Conclusions: VMD-928 was well tolerated with mainly gastrointestinal side effects. The recommended phase 2 dose (RP2D) is 600 mg twice (1200 mg) per day. The study is currently accruing in expansion cohorts to evaluate efficacy in biomarker-selected patients with tumors of TrkA protein overexpression. Tumor types with reported high TrkA protein expression including thymic carcinoma (98% with TrkA protein expression without NTRK1/2/3 gene fusions), mesothelioma (81%), squamous cell carcinoma of head and neck (80%), ovarian (80%), hepatocellular (72%), and squamous cell carcinoma of the lung (71%) are being accrued. Clinical trial information: NCT03556228.

Investigating Trk Protein Expression between Oropharyngeal and Non-oropharyngeal Squamous Cell Carcinoma: Clinical Implications and Possible Roles of Human Papillomavirus Infection.

PurposeThe relationship between head and neck squamous cell carcinoma (HNSCC) and subtypes of tropomyosin-related kinase (Trk) has not been studied in-depth. In this study, we evaluated the expression patterns of TrkA, TrkB, and panTrk and their clinicopathological significance as well as association with p16 expression and human papilloma virus (HPV) status.Materials and MethodsTotal of 396 radically resected oropharyngeal (n=121) and non-oropharyngeal (n=275) HNSCCs were included. Immunohistochemistry for TrkA, TrkB, and panTrk was performed. In addition, p16 immunohistochemistry was performed to assess the HPV status. Using HPV-negative HNSCC cell lines, FaDu and CAL27, HPV type 16 E6/E7 gene was transfected, and then changes of TrkA and TrkB expression were analyzed.ResultsIn the clinical samples of HNSCC, high expression of TrkA and panTrk were more associated with oropharyngeal and p16 positive squamous cell carcinoma (SCC). In patients with completely resected (R0-resected) oropharyngeal SCC, high TrkA expression was related to superior overall survival and recurrence-free survival (RFS). In patients with R0-resected oral cavity SCC, high panTrk was related to poor RFS. In HPV type E6/E7 gene-transfected FaDu and CAL27 cell lines, increase of TrkA expression was observed.ConclusionIt seems that expression pattern of panTrk and TrkA differed according to anatomical sites of HNSCC and was closely related to p16 expression and patient prognosis. Trk expression should be considered in the context of anatomical site, p16 expression or HPV status and Trk subtypes.

The implications of TrkA and MET aberrations in de novo salivary duct carcinoma

Salivary duct carcinoma (SDC) is an aggressive carcinoma with poor prognosis. Although anti-HER2 therapy is a potential treatment option for HER2-positive SDC, other potential therapeutic targets are not known, in particular for HER2-negative cases. In this study, the recently identified receptors tyrosine kinases MET and tropomyosin-receptor kinase (Trk) were investigated as potential therapeutic targets. A total of 28 consecutive, surgically resected, de novo SDC cases were selected after evaluating histology and immunohistochemical expression of androgen receptor. Immunohistochemical expression of c-erb2, TrkA, TrkB, TrkC, and c-MET was analyzed, and the genetic status of the HER2 and MET genes was investigated through dual-color silver in situ hybridization. High expression of c-MET or Trk was defined as that above the median value. Among the 28 SDC cases, 64.3% (18/28) were HER2-positive. c-MET expression varied, with a median H-score of 65 (range, 0 to 200). Copy number gain and amplification of MET were noted in 57.1% (16/28) and 10.7% (3/28) of cases, respectively. TrkA was variably expressed, with a median H-score of 100 (range, 0 to250). High TrkA expression was significantly related to an inferior overall survival rate in HER2-negative SDC. High expression of TrkA and c-MET and MET copy number gain/amplification were frequent events in SDC, and high expression of TrkA revealed the tendency to be related to poor prognosis in HER2-negative SDC. TrkA and MET may be possible therapeutic targets in SDC, especially in HER2-negative SDC.

Invariant natural killer T infiltration in neuroblastoma with favorable outcome.

Tumor immunity has been suggested to play a key role in clinical and biological behavior of neuroblastomas. Given that CD1-restricted invariant natural killer T (iNKT) cells enhance both innate and acquired tumor immunity, we investigated the expression of the iNKT-cell-specific T-cell receptor Vα24-Jα18 in neuroblastoma tissues and its correlation with clinical and biological characteristics. Using real- time quantitative PCR, we quantified the expression of Vα24-Jα18 in untreated tumor samples from 107 neuroblastoma cases followed in our institution and analyzed the correlation between the presence of infiltrated iNKT cells and clinical characteristics or patients' outcome. Vα24-Jα18 receptor was detected in 62 untreated cases (57.9%). The expression was significantly higher in stages 1, 2, 3, or 4S (P = 0.0099), in tumors with low or intermediate risk (P = 0.0050), with high TrkA expression (P = 0.0229), with favorable histology (P = 0.0026), with aneuploidy (P = 0.0348), and in younger patients (P = 0.0036). The overall survival rate was significantly higher in patients with iNKT-cell infiltration (log-rank; P = 0.0089). Since tumor-infiltrating iNKT cells were predominantly observed in neuroblastomas undergoing spontaneous differentiation and/or regression, we suggest that iNKT cells might play a key role in these processes.

Abstract LB-328: SHP-1, p53 and Y674/Y675-phosphorylated-trkA: a molecular pathway and prognostic marker for neuroblastoma

Abstract Neuroblastomas, which have sympathoadrenergic origin, are the most frequent childhood extracranial tumors. Good prognosis tumors (favourable cytogenetics, differentiated) express nerve growth factor (NGF) receptor tyrosine kinase trkA, whereas poor prognosis neuroblastomas (adverse cytogenetics, Myc amplification, poor differentiation) express neurotrophin receptor tyrosine kinase trkB. On NGF stimulation, trkA is activated by tyrosine phosphorylation and outcome is cell/tissue-dependent, and can signal for differentiation. We have shown that in rat sympathoadrenergic PC12 cells, wild-type (wt)p53 expression induces trkA tyrosine phosphorylation and trkA dependent signalling, promoting their NGF independent differentiation. In breast cancer tumors and neuroblastomas, high trkA levels are associated with good prognosis. In both tumour types the p53 mutation incidence is 20-25%. The tyrosine phosphatase SHP-1 dephosphorylates trkA tyrosines 674/675(Y674/Y675). We have shown that in breast cancer wt-p53 represses SHP-1 expression and induces trkA-Y674/Y675 phosphorylation, leading to NGF-independent trkA activation. This signals to suppress breast cancer proliferation by promoting cell-cycle arrest. Furthermore, in breast cancer, coexpression of phosphorylated trkA-Y674/Y675, wt-p53 and low SHP-1 levels, correlates with prolonged 15 years disease free survival (DFS). Our experiments provide a mechanism for trkA expression correlation with good prognosis in breast cancer and emphasise the importance of this mechanism in cancer. As neuroblastomas contain different cell populations, it has been difficult to identify prognostic biomarkers that would predict patient outcome. We have shown that wt-p53 repression of SHP-1 leads to trkA-Y674/Y675 phosphorylation and neuroblastoma differentiation. Neuroblastoma differentiation is associated with improved DFS. Thus we asked if, the presence of wt-p53 and SHP-1 downregulation, together with phosphorylated trkA, in neuroblastoma samples, correlates with favourable outcome. Confirmation of this mechanism would validate new molecular targets for neuroblastoma. Neuroblastoma arrays containing 169 samples were stained for p53, SHP1 and phosphorylated trkA-Y674/Y675 expression. Multivariate analysis showed low p53 levels associated with 65% 5 years DFS (hazard-ratio (HR)=0.35, p=0.09), whereas, high SHP1 expression was associated with a worse DFS (HR=1.37, p=0.028). Phosphorylated trkA-Y674/Y675 expression correlated with 88% 5 years DFS (HR=0.12, (p=0.001)). As differentiation is associated with improved survival, the differentiation markers MAP2 and Vimentin were included. Expression of MAP2 and Vimentin correlated with good DFS (71% (HR=0.29, p=0.027) and 35% (HR=0.65, p=0.014)). Myc amplification correlated with reduced DFS (HR=4.84, p=0.001) as expected. Importantly, multivariate-analysis of patients with tumors harbouring p53 together with high SHP-1 levels and phosphorylated trkA-Y674/Y675 showed 69% 5 years DFS (HR=0.29, p=0.001). Favourable DFS was seen when MAP2 and Vimentin (HR=0.53, p=0.032 and HR=0.51, p=0035) were included. Further statistical analysis which considers tumor cytogenetics will be presented. Our results will allow refinement of the prognostic tools available for neuroblastoma with direct impact on child healthcare. Citation Format: Gehad Youssef, Cheryl Gillet, Dyanne Rampling, Mirza Chagtay, Alex Virasami, Neil Sebire, John Anderson, Ximena Montano. SHP-1, p53 and Y674/Y675-phosphorylated-trkA: a molecular pathway and prognostic marker for neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-328. doi:10.1158/1538-7445.AM2014-LB-328

The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria, within the context of a tumour stem cell-like phenotype.

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.

Modulation of Nerve Growth Factor receptors in human monocytes and their influence in pulmonary inflammatory diseases

Neurotrophins (NTs) are a family of growth/survival factors with well-established functions in the nervous system. In the last decade, novel biological actions, from oncogenicity to inflammation, have been attributed to these factors. In particular, Nerve Growth Factor (NGF) acts through two different classes of receptors: the high affinity transducing TrKA receptor, associated to proliferation/survival, and the low affinity p75 receptor that, depending on its cross talk with TrKA, induces either apoptosis or survival. Recently, NGF and its receptors have been detected in healthy pulmonary tissues and seem to be involved in pulmonary inflammatory diseases. Since the expression of these NGF receptors in circulating monocytes is controversial, our initial aim was to investigate the role of these receptors both in pulmonary tissues and in peripheral blood monocytes of patients with Chronic Obstructive Pulmonary Disease (COPD). We therefore analyzed 38 healthy control subjects divided in two groups based on their smoking status. Serendipitously, the data obtained in these two control groups may have a value of their own, with potential implications in preventive medicine. We show here that in healthy subjects, smoking induces an early increase in p75 expression in monocytes, while TrKA seems unaffected. Furthermore, our control subjects could be divided in three subsets according to the constitutive TrKA expression in monocytes: TrKA-negatives, -intermediates (up to 50%) or -high (> 50%), independently on their smoking status. Since TrKA activation promotes inflammation, we hypothesize that subjects with high-TrKA monocytes could be more prone to pulmonary inflammatory diseases, such as COPD. In fact, all 28 COPD patients in our series belonged to the high-TrKA subset, while all long term heavy smokers with no evidence of pneumologic diseases belonged to TrKA-negative subset. In conclusion, our data support the hypothesis that, since TrKA expression promotes survival, elevated levels of TrKA-positive monocytes may render subjects more prone to long term inflammatory diseases (e.g., COPD). Moreover, in patients constitutively expressing high levels of TrKA in monocytes, the smoking-dependent increase in p75 may in turn further extend monocytes survival, contributing to a chronic inflammation. Furthermore, the early increase in p75 expression in monocytes following smoking may support the hypothesis that p75 determination might represent a novel marker for passive smoking. Acknowledgments. This study was totally supported by Fondazione Cassa di Risparmio di Terni

Repression of SHP-1 expression by p53 leads to trkA tyrosine phosphorylation and suppression of breast cancer cell proliferation

The nerve growth factor (NGF) receptor, trkA, the tumour suppressor p53 and the phosphatase SHP-1 are critical in cell proliferation and differentiation. SHP-1 is a trkA phosphatase that dephosphorylates trkA at tyrosines (Y) 674 and 675. p53 can induce trkA activation and tyrosine phosphorylation in the absence of NGF stimulation. In breast cancer tumours trkA expression is associated with increased patient survival. TrkA protein expression is higher in breast-cancer cell lines than in normal breast epithelia. In cell lines (but not in normal breast epithelia) trkA is functional and can be NGF-stimulated to promote cell proliferation. This study investigates the functional relationship between trkA, p53 and SHP-1 in breast-cancer, and reveals that in wild-type (wt) trkA expressing breast-cancer cells both endogenous wtp53, activated by therapeutic agents, and transfected wtp53 repress expression of SHP-1 through the proximal CCAAT sequence of the SHP-1-P1-promoter and the transcription factor NF-Y. In these cells trkA-Y674/Y675 phosphorylation is detected when SHP-1 protein levels decrease in a wtp53-dependent manner. Proliferation and cell-cycle assays, with cells expressing endogenous or transfected wt-trkA and a temperature-sensitive p53 grown at 32 degrees C (when p53 is in the wt configuration), show suppressed cell proliferation. Suppression is not detected when grown at 37 degrees C (when p53 is in the mutant configuration). A release from suppression is observed when these cells are transiently transfected with wt-SHP-1 and grown at 32 degrees C. Suppression is also detected when, as control, wt-trkA-expressing cells are transiently transfected with SHP-1-siRNA, but not when a dominant-negative (DN) mutant trkA is used to abolish wt-trkA activity. Importantly, suppression is not seen with control trkA-negative breast-cancer cells (expressing wtp53, wt-SHP-1 and undetectable trkA), transfected with Y674F/Y675F mutant-trkA. BrdU-incorporation experiments reveal lack of incorporation in cells expressing wt-trkA and wtp53, or wt-trkA and SHP-1-siRNA. However, BrdU is incorporated in the presence of Y674F/Y675F mutant trkA or DN mutant trkA. These results indicate that p53 repression of SHP-1 expression leads to trkA-Y674/Y675 phosphorylation and trkA-dependent suppression of breast-cancer cell proliferation. These data provide an explanation as to why high trkA levels are associated with favourable prognosis.

Expression of the TrkA or TrkB receptor tyrosine kinase alters the double-strand break (DSB) repair capacity of SY5Y neuroblastoma cells

In the childhood tumor neuroblastoma, high expression of the TrkA neurotrophin receptor is associated with a favorable prognosis and a lack of structural chromosomal changes, whereas TrkB is expressed in aggressive neuroblastomas demonstrating high genomic instability. The ability to repair DNA double-strand breaks (DSBs) is considered a central determinant of chromosomal stability with nonhomologous end joining (NHEJ) being the major pathway of DSB repair in vertebrates. Here, we used the SH-SY5Y human neuroblastoma cell line ectopically expressing either TrkA or TrkB as a model system to analyze the impact of Trk receptor expression on NHEJ-mediated DSB repair. In a cell-free NHEJ assay, SY5Y-TrkA cells displayed a significantly higher efficiency for NHEJ compared to SY5Y-TrkB cells. To detect possible underlying mechanisms, gene expression data (Affymetrix U95A microarray chips) obtained from the same SY5Y-TrkA/TrkB model system were reanalyzed focussing on genes involved in DNA repair. Expression of XRCC4, a central component of NHEJ, was significantly upregulated in SY5Y-TrkA compared to SY5Y-TrkB cells. Expression data were confirmed using real-time PCR and western blotting. Additionally, XRCC4 expression was enhanced in most primary neuroblastomas with high TrkA expression. The TrkA-induced increase in NHEJ activity could be reverted by XRCC4 knock-down, confirming the induction of XRCC4 by TrkA to be essential for the observed phenotype. Our data provide the first evidence for a functional relationship between tyrosine kinase receptor signaling and NHEJ-mediated DSB repair in cancer cells, potentially contributing to their genomic stability.

Intense Isolectin-B4 Binding in Rat Dorsal Root Ganglion Neurons Distinguishes C-Fiber Nociceptors with Broad Action Potentials and High Nav1.9 Expression

Binding to isolectin-B4 (IB4) and expression of tyrosine kinase A (trkA) (the high-affinity NGF receptor) have been used to define two different subgroups of nociceptive small dorsal root ganglion (DRG) neurons. We previously showed that only nociceptors have high trkA levels. However, information about sensory and electrophysiological properties in vivo of single identified IB4-binding neurons, and about their trkA expression levels, is lacking. IB4-positive (IB4+) and small dark neurons had similar size distributions. We examined IB4-binding levels in >120 dye-injected DRG neurons with sensory and electrophysiological properties recorded in vivo. Relative immunointensities for trkA and two TTX-resistant sodium channels (Nav1.8 and Nav1.9) were also measured in these neurons. IB4+ neurons were classified as strongly or weakly IB4+. All strongly IB4+ neurons were C-nociceptor type (C-fiber nociceptive or unresponsive). Of 32 C-nociceptor-type neurons examined, approximately 50% were strongly IB4+, approximately 20% were weakly IB4+ and approximately 30% were IB4-. Adelta low-threshold mechanoreceptive (LTM) neurons were weakly IB4+ or IB4-. All 33 A-fiber nociceptors and all 44 Aalpha/beta-LTM neurons examined were IB4-. IB4+ compared with IB4- C-nociceptor-type neurons had longer somatic action potential durations and rise times, slower conduction velocities, more negative membrane potentials, and greater immunointensities for Nav1.9 but not Nav1.8. Immunointensities of IB4 binding in C-neurons were positively correlated with those of Nav1.9 but not Nav1.8. Of 23 C-neurons tested for both trkA and IB4, approximately 35% were trkA+/IB4+ but with negatively correlated immunointensities; 26% were IB4+/trkA-, and 35% were IB4-/trkA+. We conclude that strongly IB4+ DRG neurons are exclusively C-nociceptor type and that high Nav1.9 expression may contribute to their distinct membrane properties.

Expression of nerve growth factor receptors is correlated with progression and prognosis of human pancreatic cancer

The aim of the present study was to investigate the prognostic value of the two types of nerve growth factor receptors (NGFR), namely high-affinity receptor TrkA and low-affinity receptor p75NGFR, in pancreatic cancer. The mRNA expression of NGFR for TrkA and p75NGFR was examined in 56 human primary pancreatic cancers using real-time quantitative reverse transcription-polymerase chain reaction. Nerve growth factor (NGF) receptors were found in all tumor specimens. It appears that the growth of pancreatic cancer cells stimulated by NGF depended on the expression levels and the ratio of TrkA to p75NGFR. TrkA and p75NGFR were negatively correlated and both were associated with abdominal or back pain and perineural invasion. Regarding this, patients with high TrkA expression levels exhibited more frequent perineural invasion and a higher degree of pain, whereas the results of p75NGFR were opposite. For Cox univariate analyses in the overall survival study, high expression of p75NGFR was associated with longer overall survival, but TrkA exhibited opposite effects and included an effect on perineural invasion and pain. Histoprognostic grading, tumor size and node involvement were not prognostic factors. In Cox multivariate analyses, TrkA and p75NGFR were both prognostic parameters. The present study found that the expression of TrkA in pancreatic cancer is a marker of tumor aggressiveness. Conversely, we also found that elevated p75NGFR expression is associated with a favorable prognosis. We demonstrated that NGF exerts both stimulatory and inhibitory effects on pancreatic cancers, with the overall effect determined by the expression levels and the ratio of TrkA to p75NGFR.

Low expression of human tubulin tyrosine ligase and suppressed tubulin tyrosination/detyrosination cycle are associated with impaired neuronal differentiation in neuroblastomas with poor prognosis.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups. However, the precise mechanism underlying this phenomenon remains largely unknown. To understand the molecular and genetic bases of neuroblastoma, we have generated its cDNA libraries and identified a human ortholog of tubulin tyrosine ligase gene (hTTL/Nbla0660) as a differentially expressed gene at high levels in a favorable subset of the tumor. Tubulin is subjected to several types of evolutionarily conserved posttranslational modification, including tyrosination and detyrosination. Tubulin tyrosine ligase catalyzes ligation of the tyrosine residue to the COOH terminus of the detyrosinated form of alpha-tubulin. The measurement of hTTL mRNA expression in 74 primary neuroblastomas by quantitative real-time reverse transcription-PCR revealed that its high expression was significantly associated with favorable stages (1, 2 and 4s; p = 0.0069), high TrkA expression (p = 0.002), a single copy of MYCN (p < 0.00005), tumors found by mass screening (p = 0.0042), nonadrenal origin (p = 0.0042) and good prognosis (p = 0.023). The log-rank test showed that high expression of hTTL was an indicator of favorable prognosis (p = 0.026). Immunohistochemical analysis using specific antibodies generated by us demonstrated that tyrosinated tubulin (Tyr-tubulin), detyrosinated tubulin (Glu-tubulin) and hTTL as well as Delta2-tubulin were positive in favorable tumors, whereas only Delta2-tubulin was positive in the tumors with MYCN amplification. In an RTBM1 neuroblastoma cell line, hTTL was increased after treating the cells with bone morphogenetic protein 2 (BMP2) or all-trans retinoic acid (RA), which induced neuronal differentiation. These results suggest that the deregulated tubulin tyrosination/detyrosination cycle caused by decreased expression of hTTL is associated with inhibition of neuronal differentiation and enhancement of cell growth in the primary neuroblastomas with poor outcome.

Differential effects of TrkA or TrkB expression on DNA repair capacity might contribute to the genomic stability of SY5Y neuroblastoma cells

The capacity to repair DNA double strand breaks (DSB) is crucial for genomic stability. The non-homologous end-joining pathway (NHEJ) for DNA repair is an important caretaker of the mammalian genome. High expression of TrkA is associated with favorable prognosis of neuroblastoma and a lack of structural chromosomal changes, whereas TrkB is expressed on aggressive neuroblastomas with high genomic instability. We here examined the contribution of TrkA or TrkB expression to the regulation of the NHEJ-pathway in neuroblastoma using SY5Y cells transfected with TrkA or TrkB. NHEJ activity was determined in cell-free extracts by a plasmid rejoining assay. Consistent with higher resistance to irradiation, SY5Y-TrkA extracts demonstrated a high rejoining activity in comparison to parental cells. In contrast, NHEJ activity was reduced in SY5Y-TrkB extracts. Microarray data obtained in the same model revealed up-regulation of XRCC4, a central effector gene of NHEJ, in SY5Y-TrkA cells. Expression data were confirmed by quantitative PCR and western blot. XRCC4 expression was also up-regulated in primary neuroblastomas with high TrkA expression. These data suggest a contribution of Trk receptors to the genomic stability in SY5Y neuroblastoma cells by mediating DSB-repair capacity. Further functional analyses of the NHEJ pathway may shed light on regulation of genomic stability in neuroblastoma and might partially explain current hypothetic models of neuroblastoma evolution.

Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma.

Neuroblastoma (NB) is a common pediatric tumor that exhibits a wide range of biological and clinical heterogeneity. EPH (erythropoietin-producing hepatoma amplified sequence) family receptor tyrosine kinases and ligand ephrins play pivotal roles in neural and cardiovascular development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6) and its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is associated with low-stage NB (stages 1, 2, and 4S) and high TrkA expression. In this study, we showed that EFNB2 and TrkA expressions were associated with both tumor stage and age, whereas EPHB6 and EFNB3 expressions were solely associated with tumor stage, suggesting that these genes were expressed in distinct subsets of NB. Kaplan-Meier and Cox regression analyses revealed that high-level expression of EPHB6, EFNB2, and EFNB3 predicted favorable NB outcome (P<0.005), and their expression combined with TrkA expression predicted the disease outcome more accurately than each variable alone (P<0.00005). Interestingly, if any one of the four genes (EPHB6, EFNB2, EFNB3, or TrkA) was expressed at high levels in NB, the patient survival was excellent (>90%). To address whether a good disease outcome of NB was a consequence of high-level expression of a "favorable NB gene," we examined the effect of EPHB6 on NB cell lines. Transfection of EPHB6 cDNA into IMR5 and SY5Y expressing little endogenous EPHB6 resulted in inhibition of their clonogenicity in culture. Furthermore, transfection of EPHB6 suppressed the tumorigenicity of SY5Y in a mouse xenograft model, demonstrating that high-level expressions of favorable NB genes, such as EPHB6, can in fact suppress malignant phenotype of unfavorable NB.

Prognostic impact of telomerase activity in patients with neuroblastoma.

Neuroblastoma is one of the most common malignant neoplasms occurring among children. The prognosis for this disease is strongly associated with age, disease stage, histology, and some biologic features. It has been reported that telomerase, a ribonucleoprotein enzyme, which maintains the telomere length in immortal cells, is related to disease stage and other biologic features. The purpose of this study was to evaluate the prognostic value of telomerase activity compared to TrkA expression in 65 patients with neuroblastoma. Telomerase activity and TrkA expression were examined in tissue samples collected between 1980 and 1994 from 65 patients by polymerase chain reaction-based telomerase activity. TrkA expression was examined by immunoblotting using a rabbit anti-gp140 proto-trk polyclonal antibody. Low telomerase activity was found in 22 of 30 (73.3%) patients with Stages 1, 2, or 4S neuroblastomas; 7 of 13 (53.8%) with Stage 3; and 8 of 22 (36.3%) with stage 4; no telomerase activity was detected in 7 of 22 (31.8%) patients with Stage 4 neuroblastoma. The 5-year event-free survival (EFS) rate was 86.5% for patients with low telomerase activity, while it was 53.8% for patients with high telomerase activity. By the combination of telomerase activity and TrkA expression, the 5-year EFS rate was highest among patients with a high TrkA expression and a low or non-existent telomerase activity (91.7%), and it was lowest among patients with a low TrkA expression and a high telomerase activity (29.6%). Thus, it appears that telomerase activity would be a useful prognostic factor for neuroblastoma, especially when used in combination with the TrkA expression.

Different effects of TrkA expression in neuroblastoma cell lines with or without MYCN amplification.

Expression of the neurotrophin receptor TrkA is associated with a favorable prognosis in neuroblastoma (NB) and promotes growth inhibition and neuronal differentiation. Aggressive, MYCN-amplified NB tumors express little or no TrkA mRNA, suggesting that MYCN overexpression may inhibit TrkA expression. To study the interactions of TrkA expression and MYCN amplification in NB, we stably expressed the TrkA receptor in the MYCN single copy cell lines SH-SY5Y and NB69 as well as in the MYCN amplified cell lines CHP134 and IMR5. All four transfected cell lines demonstrated high TrkA expression and similar activation of the TrkA receptor and of mitogen-activated protein kinases as well as induction of immediate-early genes in response to nerve growth factor (NGF). Introduction of TrkA restored NGF responsiveness of SH-SY5Y and NB69 cells, as demonstrated by morphologic differentiation, growth inhibition, and enhanced survival in serum-free medium. However, no morphologic, growth, or survival responses to NGF were detected in MYCN-amplified CHP134 and IMR5 TrkA transfectants. Thus, transfection of TrkA into MYCN amplified NB cell lines only partly restored the TrkA/NGF signaling pathway, suggesting additional inhibitory effects of MYCN overexpression on TrkA signaling.

Biology and genetics of human neuroblastomas.

Neuroblastomas have a variety of clinical behaviors, from spontaneous regression or differentiation to early metastasis and death. We have examined a variety of genetic variables that might explain or predict the clinical behavior. We have studied DNA or RNA from a number of children enrolled in clinical trials with the major pediatric oncology cooperative groups. We propose that neuroblastomas may be classified into three subsets with distinct biological features and clinical behavior. The first subset consists of those tumors with hyperdiploid modal karyotypes and high TRK-A expression. Patients with these tumors are usually infants with low stages of disease and a very favorable outcome. The second group consists of tumors that have a near-diploid DNA content, usually with 1p allelic loss or other structural changes, but they lack MYCN amplification, and TRK-A expression is low. The patients are generally older, with advanced stages of disease and an intermediate outcome. The third group is characterized by tumors with MYCN amplification, 1p allelic loss, and low or absent TRK-A expression. The patients are 1-5 years of age and have advanced stages of disease, rapid tumor progression, and a very poor prognosis. Current evidence suggests the tumor types are genetically distinct, and one type seldom if ever evolves into another. Identification of these genetic and clinical subsets permits a more accurate prediction of outcome. This, in turn, allows more appropriate selection of therapeutic intensity to minimize side effects in those with a favorable outcome but optimize the chance of cure in those requiring aggressive treatment.

Gene expression of neuropeptide Y and neuropeptide-Y1 receptor in relation to proto-oncogene tyrosine kinase A, nerve-growth-factor low-affinity-receptor and the transcription factor N-myc in human neuroblastomas

The expression of neuropeptide Y (NPY) and NPY-Y1 receptor (NPY-Y1R) in relation to that of tyrosine kinase A (trkA), nerve-growth-factor low-affinity-receptor (LNGFR) and the transcription factor N-myc was studied in 26 neuroblastomas and one ganglioneuroma by quantitative Northern-blot analysis. A correlation of NPY-Y1R with LNGFR (r = 0.85, p < 0.01) and trkA (r = 0.38, p < 0.05), respectively, could be shown, while no correlation between NPY and its receptor NPY-Y1R was observed. Comparison of a high and a low level NPY expressing group revealed that the high NPY expressing group also had high LNGFR and high trkA levels while the low NPY expressing group had low LNGFR and low trkA levels which were significantly different (NPY: p = 0.035, trkA: p = 0.008, LNGFR p = 0.004). Dividing the tumors in a high and a low N-myc expressing group showed that the low N-myc expressing group contained both high and low trkA expressing tumors while the high N-myc expressing group exclusively were low level trkA expressing tumors. The frequency distribution in both groups concerning trkA expression showed a significant difference (p < 0.01). In conclusion the coexpression of NPY-Y1R and LNGFR or trkA may indicate a similar gene regulation during ontogenesis of the peripheral nervous system.