NTRK fusions and Trk proteins: what are they and how to test for them.

The NTRK genes include a family of three genes, NTRK1, NTRK2, and NTRK3, which are associated with fusions with a variety of partner genes, leading to upregulation of three proteins, TrkA, TrkB, and TrkC. NTRK fusions occur in a variety of solid tumors: at high incidence in secretory carcinoma of the breast and salivary glands, congenital mesoblastic nephroma, and infantile fibrosarcoma; at intermediate incidence in thyroid carcinoma, particularly postradiation carcinomas and a subset of aggressive papillary carcinomas, Spitzoid melanocytic neoplasms, pediatric midline gliomas (particularly pontine glioma), and KIT/PDGFRA/RAS negative gastrointestinal stromal sarcomas; and at a low incidence in many other solid tumors. With new FDA-approved treatments available and effective in treating patients whose tumors harbor NTRK fusions, testing for these fusions has become important. A variety of technologies can be used for testing, including FISH, PCR, DNA, and RNA-based next-generation sequencing, and immunohistochemistry. RNA-based next-generation sequencing represents the gold standard for the identification of NTRK fusions, but FISH using break-apart probes and DNA-based next-generation sequencing also represent adequate approaches. Immunohistochemistry to detect increased levels of Trk protein may be very useful as a screening technology to reduce costs, although it alone does not represent a definitive diagnostic methodology.

Effect of mating on mRNA and protein expression of beta nerve growth factor and its receptor, TrKA, in the oviduct of llama (Lama glama).

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of ß-nerve growth factor (ß-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. ß-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both ß-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative ß-NGF abundance was higher in the UTJ of copulated females (p < .05). ß-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the ß-NGF/TrKA system and that an increase in ß-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas.

The Receptor Tyrosine Kinase TrkA Is Increased and Targetable in HER2-Positive Breast Cancer.

The tyrosine kinase receptor A (NTRK1/TrkA) is increasingly regarded as a therapeutic target in oncology. In breast cancer, TrkA contributes to metastasis but the clinicopathological significance remains unclear. In this study, TrkA expression was assessed via immunohistochemistry of 158 invasive ductal carcinomas (IDC), 158 invasive lobular carcinomas (ILC) and 50 ductal carcinomas in situ (DCIS). TrkA was expressed in cancer epithelial and myoepithelial cells, with higher levels of TrkA positively associated with IDC (39% of cases) (p < 0.0001). Interestingly, TrkA was significantly increased in tumours expressing the human epidermal growth factor receptor-2 (HER2), with expression in 49% of HER2-positive compared to 25% of HER2-negative tumours (p = 0.0027). A panel of breast cancer cells were used to confirm TrkA protein expression, demonstrating higher levels of TrkA (total and phosphorylated) in HER2-positive cell lines. Functional investigations using four different HER2-positive breast cancer cell lines indicated that the Trk tyrosine kinase inhibitor GNF-5837 reduced cell viability, through decreased phospho-TrkA (Tyr490) and downstream AKT (Ser473) activation, but did not display synergy with Herceptin. Overall, these data highlight a relationship between the tyrosine kinase receptors TrkA and HER2 and suggest the potential of TrkA as a novel or adjunct target for HER2-positive breast tumours.

Nerve growth factor from Indian Russell's viper venom (RVV-NGFa) shows high affinity binding to TrkA receptor expressed in breast cancer cells: Application of fluorescence labeled RVV-NGFa in the clinical diagnosis of breast cancer.

Nerve growth factor (NGF) is a minor and neglected component of snake venom. Present study describes the purification and characterization of a NGF isoform (RVV-NGFa) from Indian Russell's viper venom (RVV). RVV-NGFa showed a protonated molecular ion [MH+] at m/z 17388.725 Da. The RVV-NGFa induced neuritogenesis in PC-12 cells but did not show cytotoxicity in mammalian cells, hemolytic activity, platelet modulation, and interference in blood coagulation system which are the characteristic pharmacological properties of RVV. By ELISA and immunofluorescence microplate reader assay, the RVV-NGFa showed appreciable binding to TrkA receptor expressed in breast cancer MDA-MB-231 and MCF-7 cells; nevertheless, pre-incubation of cells with anti-TrkA (and not TrkB or TrkC) or anti-p75NTR antibody significantly decreased (p < 0.05) this binding. The RVV-NGFa demonstrated insignificant binding with non-cancerous cells (HEK-293, L6) lacking TrkA receptor. The binding of RVV-NGFa to TrkA receptor of breast cancer cells resulted in internalization of ligand (RVV-NGFa)-receptor (TrkA) complex to cell cytoplasm in a time-dependent manner. The spectrofluorometric study demonstrated an interaction between RVV-NGFa and cytosolic domain of the purified TrkA receptor. The fluorescence (FITC)-tagged RVV-NGFa depicted a strong fluorescence signal that was observed under a fluorescence microscope and determined by fluorescence microplate reader assay post binding to breast cancer cells; but no fluorescence signal was detected after incubating FITC-RVV-NGFa with non-cancerous L6 and HEK-293 cells. The clinical application of FITC/fluorescence nanoparticle tagged RVV-NGFa for the ex vivo and in vivo diagnosis of breast cancer is highly promising.

Spatio-temporal expression profile of NGF and the two-receptor system, TrkA and p75NTR, in experimental autoimmune encephalomyelitis.

BACKGROUND: Nerve growth factor (NGF) and its receptors, tropomyosin receptor kinase A (TrkA) and pan-neurotrophin receptor p75 (p75NTR), are known to play bidirectional roles between the immune and nervous system. There are only few studies with inconclusive results concerning the expression pattern and role of NGF, TrkA, and p75NTR (NGF system) under the neuroinflammatory conditions in multiple sclerosis (MS) and its mouse model, the experimental autoimmune encephalomyelitis (EAE). The aim of this study is to investigate the temporal expression in different cell types of NGF system in the central nervous system (CNS) during the EAE course. METHODS: EAE was induced in C57BL/6 mice 6-8 weeks old. CNS tissue samples were collected on specific time points: day 10 (D10), days 20-22 (acute phase), and day 50 (chronic phase), compared to controls. Real-time PCR, Western Blot, histochemistry, and immunofluorescence were performed throughout the disease course for the detection of the spatio-temporal expression of the NGF system. RESULTS: Our findings suggest that both NGF and its receptors, TrkA and p75NTR, are upregulated during acute and chronic phase of the EAE model in the inflammatory lesions in the spinal cord. NGF and its receptors were co-localized with NeuN+ cells, GAP-43+ axons, GFAP+ cells, Arginase1+ cells, and Mac3+ cells. Furthermore, TrkA and p75NTR were sparsely detected on CNPase+ cells within the inflammatory lesion. Of high importance is our observation that despite EAE being a T-mediated disease, only NGF and p75NTR were shown to be expressed by B lymphocytes (B220+ cells) and no expression on T lymphocytes was noticed. CONCLUSION: Our results indicate that the components of the NGF system are subjected to differential regulation during the EAE disease course. The expression pattern of NGF, TrkA, and p75NTR is described in detail, suggesting possible functional roles in neuroprotection, neuroregeneration, and remyelination by direct and indirect effects on the components of the immune system.

Primary sensory neurons expressing tropomyosin receptor kinase A in the rat trigeminal ganglion.

Tropomyosin receptor kinase A (trkA), a high affinity receptor for nerve growth factor (NGF), has been implicated in neuronal survival, neurite outgrowth and inflammatory pain. So far, the characterization of the primary sensory neurons that express trkA, and are thus potentially affected by NGF, has remained incomplete. The goal of this study was to investigate the trkA-expressing neurons and fibers in the rat trigeminal ganglion and its sensory root using light- and electron-microscopic immunohistochemistry and quantitative analysis. TrkA-immunopositive (+) trigeminal neurons varied from small to large. Double immunofluorescent staining showed that about 28%, 33% and 3% of the trkA(+) neurons coexpressed SP, CGRP and IB4, respectively. About 11% of the trkA(+) neurons also coexpressed parvalbumin. Electron microscopy revealed that trkA was expressed in all types of fibers: While the large majority of the trkA(+) fibers were unmyelinated (35.3%) and small myelinated (<20 µm2 in cross-sectional area; 45.5%), a still considerable fraction (19.2%) was large myelinated. These findings indicate that all types of trigeminal neurons (ones with unmyelinated, small myelinated or large myelinated fibers) may be regulated by NGF/trkA signaling.

Expression of ß-NGF and high-affinity NGF receptor (TrKA) in llama (Lama glama) male reproductive tract and spermatozoa.

ß-Nerve growth factor (ß-NGF) is a seminal plasma element, responsible for inducing ovulation in camelids. The main organ of ß-NGF production remains nondescript. The aims of this study were to (a) characterize gene expression and protein localization of ß-NGF and its main receptor tyrosine kinase receptor A (TrKA) in the llama male reproductive tract, and (b) determine whether the seminal ß-NGF interacts with ejaculated sperm by localizing ß-NGF and TrKA in epididymal, ejaculated, and acrosome-reacted (AR) sperms and, additionally, by identifying ß-NGF presence in sperm-adsorbed proteins (SAP). Both ß-NGF and TrkA transcripts are widely expressed along the male reproductive tract, with a higher expression level of ß-NGF at prostate (p < 0.05). ß-NGF immunolabeling was only positive for prostate, whereas TrKA label was present in epithelial and muscular cells of testis, prostate, bulbourethral glands, and epididymis. Using an immunofluorescent technique, ß-NGF was colocalized with TrKA in the middle piece of ejaculated and AR sperm. However, only TrKA was observed in epididymal sperm indicating that ß-NGF could have a seminal origin. This was also confirmed by the identification of four ß-NGF isoforms in SAP. This study extends the knowledge about the participation of ß-NGF/TrkA in llama reproduction, providing evidence that may have roles in the regulation of sperm physiology.

Evaluation of pan-TRK immunohistochemistry in infantile fibrosarcoma, lipofibromatosis-like neural tumour and histological mimics.

AIMS: Infantile fibrosarcoma is characterised by intersecting fascicles of spindle cells and ETV6-NTRK3 gene fusion in most cases. Given histological overlap with other spindle-cell tumours, the diagnosis can be challenging and often requires molecular confirmation. A recently developed pan-TRK antibody shows promise for identifying tumours with NTRK fusions. The purpose of this study was to evaluate the potential diagnostic utility of pan-TRK immunohistochemistry for infantile fibrosarcoma. METHODS AND RESULTS: We evaluated whole-tissue sections from 210 cases, including 15 infantile fibrosarcomas; five each lipofibromatosis-like neural tumour and lipofibromatosis; 10 each primitive myxoid mesenchymal tumour of infancy (PMMTI) and low-grade myofibroblastic sarcoma; 15 each fibrous hamartoma of infancy (FHI), myofibroma/myofibromatosis and desmoid-type fibromatosis; and 20 each low-grade fibromyxoid sarcoma, synovial sarcoma, spindle-cell rhabdomyosarcoma, malignant peripheral nerve sheath tumour, fibrosarcomatous dermatofibrosarcoma protuberans (F-DFSP) and nodular fasciitis. Immunohistochemistry was performed using a rabbit monoclonal pan-TRK antibody. Immunoreactivity for pan-TRK was observed in all 15 (100%) infantile fibrosarcomas, including diffuse immunoreactivity (>50% of cells) in 14 (93%) cases. Pan-TRK was positive in all five (100%) lipofibromatosis-like neural tumours. Of the 190 histological mimics, diffuse pan-TRK immunoreactivity was noted in 16 (8%) cases, including five PMMTI, five FHI (highlighting predominantly the primitive myxoid spindle-cell components), three F-DFSP, one low-grade myofibroblastic sarcoma, one myofibroma and one spindle-cell rhabdomyosarcoma. CONCLUSIONS: Diffuse pan-TRK immunoreactivity is a highly sensitive but not entirely specific diagnostic marker for infantile fibrosarcoma, and may be helpful in selecting patients for TRK-targeted therapy. As expected, lipofibromatosis-like neural tumours, which harbour NTRK1 fusions, also show diffuse pan-TRK immunoreactivity.

TRKA expression and NTRK1 gene copy number across solid tumours.

AIMS: Neurotrophic Tropomyosin Kinase Receptor 1 (NTRK1) gene encodes for the protein Tropomyosin-related kinase A (TRKA). Deregulated activity of TRKA has been shown to have oncogenic potential. We present here the results of an immunohistochemical (IHC) observational cohort study of TRKA expression together with gene copy number (GCN) assessment in various solid tumours. METHODS: Formalin-fixed, paraffin-embedded consecutive samples of different tumour types were tested for TRKA expression. Samples showing TRKA IHC staining in at least 10% of cells were analysed by fluorescence in situ hybridisation to assess NTRK1 gene rearrangements and/or individual GCN gain. All patients underwent this molecular assessment within the phase I ALKA-001 clinical trial. RESULTS: 1043 samples were tested and annotation for histology was available in 1023. Most of the samples were colorectal adenocarcinoma (CRC) (n=550, 52.7%) and lung adenocarcinoma (n=312, 29.9%). 24 samples (2.3%) were biliary tract carcinoma (BTC). Overall, 17 (1.6%) samples were characterised by TRKA IHC expression (four weak, eight moderate, five strong): 9/17 lung adenocarcinoma, 3/17 CRC, 3/17 BTC, 1/17 thyroid cancer and 1/17 cancer of unknown primary. Of these, 1/17 with strong TRKA IHC staining displayed NTRK1 gene rearrangement and 15/17 NTRK1 GCN gain by FISH. No correlation was found between intensity of TRKA IHC staining and number of copies of NTRK1. CONCLUSIONS: TRKA expression can be found in 1.6% of solid tumours and can be paralleled by NTRK1 gene rearrangements or mostly GCN gain. The prognostic and translational therapeutic impact of the latter remains to be established.

Human vs. Mouse Nociceptors - Similarities and Differences.

The somatosensory system allows us to detect a diverse range of physical and chemical stimuli including noxious ones, which can initiate protective reflexes to prevent tissue damage. However, the sensation of pain can - under pathological circumstances - outlive its usefulness and perpetrate ongoing suffering. Rodent model systems have been tremendously useful to help understand basic mechanisms of pain perception. Unfortunately, the translation of this knowledge into novel therapies has been challenging. We have investigated similarities and differences of human and mouse peptidergic (TRKA expressing) nociceptors using dual-color fluorescence in situ hybridization of dorsal root ganglia. By comparing the transcripts of a selected group of well-established nociceptive markers, we observed significant differences for some of them. We found co-expression of Trpv1, a key player for sensitization and inflammatory pain, with TrkA in a larger population in humans compared to mice. Similar results could be obtained for Nav1.8 and Nav1.9, two voltage gated sodium channels implicated in pathological forms of pain. Additionally, co-expression of Ret and TrkA was also found to be more abundant in human neurons. Moreover, the neurofilament heavy polypeptide was detected in all human sensory DRG neurons compared to a more selective expression pattern observed in rodents. To our knowledge, this is the first time that such detailed comparative analysis has been performed and we believe that our findings will direct future experimentation geared to understand the difficulties we face in translating findings from rodent models to humans.

BDNF: An Oncogene or Tumor Suppressor?

Neurotrophins are a family of growth factors that are vital to the proper development of the central nervous system. Their effects on cells are governed by the expression and activation of the tyrosine kinase receptors TrkA, TrkB and TrkC. TrkB has been immensely implicated in mediating neuronal migration, development and differentiation. It has also been shown to protect several neuronal cell types from an array of cytotoxic stressors after activation by its conjugate ligand brain-derived neurotrophic factor (BDNF). Over the past two decades, it has been shown that TrkB and BDNF are up-regulated in many types of cancers, conferring aggressive phenotypes underpinned by their resistance to several standard chemotherapeutic agents. This resistance to chemotherapy is modulated by the downstream targets of the TrkB receptor which include the well-characterized PI3K /Akt growth pathway, a hallmark of uncontrolled cancer cell growth and proliferation. Pre-clinical efforts to develop inhibitors of this receptor are promising, and such inhibitors also seem to sensitize cancer cells to standard chemotherapies. However, new evidence suggests that BDNF overexpression in the hypothalamus has immunoaugmenting properties, eliciting an increased anti-tumor immune response and reducing the activity of several proteins that would normally confer resistance to chemotherapeutic agents. In the current work, we provide a global analysis of the physiological consequences of TrkB receptor activation in vitro and discuss the dynamic consequences of TrkB activation in vivo. Finally, we propose a clinically-feasible option for increasing BDNF expression in the hypothalamus to more readily utilize the oncolytic effects of BDNF.

The dynamics of trkA expression in the bovine ovary are associated with a luteotrophic effect of ovulation-inducing factor/nerve growth factor (OIF/NGF).

BACKGROUND: Ovulation-inducing factor in semen (OIF/NGF) influences ovulation and CL form and function in camelids and, remarkably, in cows. To test the hypothesis that the luteotrophic effect of OIF/NGF is mediated by an increase in trkA receptors in the ovulatory follicle and early CL, a study was designed to characterize the spatial and temporal distribution of trkA in ovarian follicles and CL at known stages of the bovine estrous cycle. METHODS: Sexually mature cattle (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6 or in the pre-ovulatory period just before or after exogenous LH treatment. After a complete interovulatory interval, the cows were re-assigned to a different day-group on which the remaining ovary was removed. Sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were processed for immunofluorescent detection and quantification of trkA receptor. RESULTS: TrkA immuno-fluorescence in ovarian tissues was restricted to follicles and the CL (no reaction in stroma or vessels), and was restricted to the cytoplasm (no nuclear staining). The trkA staining intensity, area of staining, and proportion of cells stained was greater in both theca and granulosa layers of dominant follicles than in that of subordinate follicles (P ≤ 0.05) in all day-groups except the Pre-LH group. Among dominant follicles, a progressive reduction in the immuno-positive reaction was detected from Day 2 to Day 6. Among subordinate follicles, immuno-reactivity remained low and unchanged except a rise in the Pre-LH group. The number of immuno-positive cells was greater in early developing CL (Days 2 and 4 combined) than in mature or regressing stage CL (Day 6, Pre- and Post-LH combined; P = 0.01). The intracellular distribution of trkA was more diffuse and widespread in dominant than subordinate follicles, particularly on Day 2 and Post-LH (P < 0.05). CONCLUSIONS: Distinct differences in trkA expression between dominant and subordinate follicles, particularly when circulating progesterone is minimal (early luteal development and after luteolysis) is consistent with a local role of OIF/NGF in follicle selection and early luteogenesis.

Trk inhibition reduces cell proliferation and potentiates the effects of chemotherapeutic agents in Ewing sarcoma.

Ewing sarcoma (ES) is a highly aggressive pediatric cancer that may arise from neuronal precursors. Neurotrophins stimulate neuronal devlopment and plasticity. Here, we found that neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as their receptors (TrkA and TrkB, respectively) are expressed in ES tumors. Treatment with TrkA (GW-441756) or TrkB (Ana-12) selective inhibitors decreased ES cell proliferation, and the effect was increased when the two inhibitors were combined. ES cells treated with a pan-Trk inhibitor, K252a, showed changes in morphology, reduced levels of ß-III tubulin, and decreased mRNA expression of NGF, BDNF, TrkA and TrkB. Furthermore, combining K252a with subeffective doses of cytotoxic chemotherapeutic drugs resulted in a decrease in ES cell proliferation and colony formation, even in chemoresistant cells. These results indicate that Trk inhibition may be an emerging approach for the treatment of ES.

Surgical intestinal manipulation increases gene expression of TrkA, CGRP, and PAR-2 IN dorsal root ganglia in the rat.

BACKGROUND: Surgical handling of the bowel evokes degranulation of peritoneal mast cells (PMC). Nonetheless, role of PMCs in postoperative ileus (POI) is somewhat controversial. We aimed to investigate if intestinal manipulation elicits changes in afferent mediators related to MC activation and alteration of gastrointestinal (GI) motility. METHODS: Postoperative ileus was induced by intestinal manipulation in Sprague-Dawley rats. Additionally, compound 48/80 (C48/80) and ketotifen were used to modulate MC activity. Rat mast cell protease 6 (RMCP-6, ELISA) release was determined in peritoneal lavage 20 min after intestinal manipulation. At 24 h, GI transit was determined. Gene expression of calcitonin gene-related peptide (CGRP), protease-activated receptor-2 (PAR-2), nerve growth factor (NGF), and TrkA receptor was determined (PCR) in dorsal root ganglia (DRG). Ileal wall inflammation was assessed by myeloperoxidase (MPO) activity, interleukin-6 expression (IL-6). KEY RESULTS: Intestinal manipulation and exposure to C48/80-induced degranulation of PMCs delayed GI transit and up-regulated IL-6 and MPO activity. Intestinal manipulation, but not C48/80, up-regulated CGRP, PAR-2, and NGF/TrkA in DRGs. Ketotifen only improved gastric emptying and fecal output. Up-regulation of CGRP and TrkA expression in DRG was not prevented by ketotifen. CONCLUSIONS & INFERENCES: Postoperative ileus is accompanied by activation of CGRP, NGF-TrkA, and PAR-2 in DRGs. Our results suggest that these mediators could be a target in further POI studies in order to find new therapeutic targets for this medical condition.

Spitz Tumors: Comparison of Histological Features in Relationship to Immunohistochemical Staining for ALK and NTRK1.

Spitz tumors are a group of melanocytic neoplasms with distinct morphological features that tend to affect young individuals. Distinguishing benign from malignant Spitz tumors can be challenging, but cytogenetic and molecular tests have contributed to improvements in diagnostic accuracy. Spitz tumors harbor diverse genetic alterations, including mutations in HRAS, loss of BAP1, or kinase fusions in ROS1, NTRK1, ALK, BRAF, and RET genes. Limited data exist on the correlation between histopathological features and kinase fusions. Here, we describe the histopathological features of 105 Spitz tumors (Spitz nevi and atypical Spitz tumors), comparing lesions according to their immunoreactivity for ALK or NTRK1. Intersecting fascicular growth of fusiform melanocytes was seen in all but one ALK-positive tumor (27 of 28 or 96.4%), whereas it was infrequent in NTRK1-positive tumors (5 of 20 or 25.0%) and tumors negative for both ALK and NTRK1 (96.4% vs 25.0% vs 8.7%, P < .0027). There was a trend toward ALK-positive tumors being amelanotic compared with NTRK1-positive tumors and combined ALK-/NTRK1-negative tumors (89.3% vs 45% vs 47.4%, respectively, P = .1023) and lacking epithelioid cell morphology (0% vs 45.0% vs 41%, respectively, P = .6985). In conclusion, this study confirms that although not specific, the growth pattern of intersecting fascicles of amelanotic fusiform melanocytes is strongly associated with ALK expression.

Prognostic value of tropomyosin-related kinases A, B, and C in gastric cancer.

PURPOSE: Tropomyosin-related kinase (Trk) receptors play critical roles in tumor development and are considered attractive targets for cancer therapy. We investigated correlations of the expression of TrkA, TrkB, and TrkC with clinicopathological features and outcomes in gastric cancer. METHODS: Tumor samples were obtained from 221 patients with gastric cancer who underwent gastrectomy between 2003 and 2007. The expression of TrkA, TrkB, and TrkC was analyzed using immunohistochemical staining. The relationship of their expression to clinicopathological factors and outcomes was assessed. RESULTS: High expression of TrkA, TrkB, or TrkC was significantly associated with histopathology (p = 0.022, p < 0.001, and p < 0.001). High expression of TrkA was significantly correlated with variables related to tumor progression, including lymph node metastasis (p = 0.024) and distant metastasis or recurrence (p < 0.001). Distant metastasis or recurrence was found in a significantly higher proportion of patients with high expression of TrkC than in those with low expression (p = 0.036). High expression of TrkA was significantly associated with poorer relapse-free survival (RFS) in univariate analysis (p = 0.001). High expression of TrkA or TrkC was significantly associated with poorer disease-specific survival (DSS) in univariate analysis (p < 0.001 and p = 0.008). In multivariate analysis, TrkA was an independent predictor of RFS [hazard ratio (HR), 2.294; 95 % confidence interval (CI), 1.309-4.032; p = 0.004] and DSS (HR, 2.146; 95 % CI, 1.195-3.861; p = 0.011). Expression of TrkB was not associated with RFS or DSS in univariate analysis. CONCLUSIONS: Our results demonstrated that TrkA expression was associated with tumor progression and poor survival, and was an independent predictor of poor outcomes in gastric cancer patients.

LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells.

Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without ß-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

Overexpression of NTRK1 Promotes Differentiation of Neural Stem Cells into Cholinergic Neurons.

Neurotrophic tyrosine kinase type 1 (NTRK1) plays critical roles in proliferation, differentiation, and survival of cholinergic neurons; however, it remains unknown whether enhanced expression of NTRK1 in neural stem cells (NSCs) can promote their differentiation into mature neurons. In this study, a plasmid encoding the rat NTRK1 gene was constructed and transfected into C17.2 mouse neural stem cells (NSCs). NTRK1 overexpression in C17.2 cells was confirmed by western blot. The NSCs overexpressing NTRK1 and the C17.2 NSCs transfected by an empty plasmid vector were treated with or without 100 ng/mL nerve growth factor (NGF) for 7 days. Expression of the cholinergic cell marker, choline acetyltransferase (ChAT), was detected by florescent immunocytochemistry (ICC). In the presence of NGF induction, the NSCs overexpressing NTRK1 differentiated into ChAT-immunopositive cells at 3-fold higher than the NSCs transfected by the plasmid vector (26% versus 9%, P < 0.05). The data suggest that elevated NTRK1 expression increases differentiation of NSCs into cholinergic neurons under stimulation of NGF. The approach also represents an efficient strategy for generation of cholinergic neurons.

Testicular expression of NGF, TrkA and p75 during seasonal spermatogenesis of the wild ground squirrel (Citellus dauricus Brandt).

The nerve growth factor (NGF) not only has an essential effect on the nervous system, but also plays an important role in a variety of non-neuronal systems, such as the reproductive system. The aim of this study was to investigate the seasonal changes in expression of NGF and its receptors (TrkA and p75) in testes of the wild ground squirrel during the breeding and nonbreeding seasons. Immunolocalization for NGF was detected mainly in Leydig cells and Sertoli cells in testes of the breeding and nonbreeding seasons. The immunoreactivity of TrkA was highest in the elongated spermatids, whereas p75 in spermatogonia and spermatocytes in testes of the breeding season. In the nonbreeding season testes, TrkA showed positive immunostainings in Leydig cells, spermatogonia and primary spermatocytes, while p75 showed positive signals in spermatogonia and primary spermatocytes. Consistent with the immunohistochemical results, the mean mRNA and protein level of NGF and TrkA were higher in the testes of the breeding season, and then decreased to a relatively low level in the nonbreeding season. In addition, the concentration of plasma gonadotropins and testosterone were assayed by radioimmunoassay (RIA), and the results showed a significant seasonal change between the breeding and nonbreeding seasons. To conclude, these results of this study provide the first evidence on the potential involvement of NGF and its receptor, TrkA and p75 in the seasonal spermatogenesis and testicular function change of the wild ground squirrel.

Expression of NGF, BDNF and their high-affinity receptors in ovine mammary glands during development and lactation.

The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their high-affinity tyrosine kinase receptors TrkA and TrkB was investigated by immunohistochemical method in the mammary gland of ewes from prepubertal stage to involution. NGF and BDNF protein expressions were strong during development of glands at prepubertal stage and during pregnancy and decreased during lactation and involution. The expressions localized in both stromal and parenchymal cells of developing gland were mainly arranged in the apical side of secretory cells during lactation. These observations were also confirmed at transcriptional level by RT-PCR analyses. The highest expression of all genes significantly occurred at prepubertal stage. NGF was then down-regulated from pregnancy to involution, and no statistical differences were observed among these stages. The receptor TrkA was also under-expressed from pregnancy to involution, and its expression significantly differed between pregnancy and 30 days of lactation and also between 30 and 60 days of lactation. BDNF was significantly down-regulated at 60 days of lactation in comparison with prepubertal stage and again between pregnancy and 30 days of lactation. The relative abundance of its receptor, TrkB, showed also a significant down-regulation at 60 days of lactation in comparison with pregnancy and involution. Among the myriad of other molecular signals involved in the mammary gland cycle, the local production of neuropeptides and their receptors could be of interest in understanding their potential role in mammary biology.