Receptor Tyrosine Kinase TrkA Research Articles

Repotrectinib: First Approval.

Repotrectinib (AUGTYRO™) is a next-generation, oral, small-molecule kinase inhibitor of proto-oncogene tyrosine-protein kinase ROS1 (ROS1) and tropomyosin receptor tyrosine kinases (TRKs) TRKA, TRKB, and TRKC. It is being developed by Turning Point Therapeutics, a wholly owned subsidiary of Bristol-Myers Squibb (BMS), for the treatment of locally advanced or metastatic solid tumours, including non-small cell lung cancer (NSCLC). Repotrectinib is a next-generation tyrosine kinase inhibitor rationally designed to inhibit ROS1 and TRK fusion, including in the presence of resistance mutations such as solvent-front mutations. In November 2023, repotrectinib received its first approval in the USA for the treatment of adults with locally advanced or metastatic ROS1-positive NSCLC. Repotrectinib is under regulatory review in China and the EU for NSCLC. Clinical studies of repotrectinib are ongoing in several countries in patients with NSCLC and other solid tumours (including primary central nervous system cancer) across both adult and paediatric patient populations. In addition, preclinical investigation of repotrectinib in multiple myeloma is underway in the USA. This article summarizes the milestones in the development of repotrectinib leading to this first approval for the treatment of locally advanced or metastatic ROS1-positive NSCLC.

Influenza A virus activates cellular Tropomyosin receptor kinase A (TrkA) signaling to promote viral replication and lung inflammation.

Influenza A virus (IAV) infection causes acute respiratory disease with potential severe and deadly complications. Viral pathogenesis is not only due to the direct cytopathic effect of viral infections but also to the exacerbated host inflammatory responses. Influenza viral infection can activate various host signaling pathways that function to activate or inhibit viral replication. Our previous studies have shown that a receptor tyrosine kinase TrkA plays an important role in the replication of influenza viruses in vitro, but its biological roles and functional mechanisms in influenza viral infection have not been characterized. Here we show that IAV infection strongly activates TrkA in vitro and in vivo. Using a chemical-genetic approach to specifically control TrkA kinase activity through a small molecule compound 1NMPP1 in a TrkA knock-in (TrkA KI) mouse model, we show that 1NMPP1-mediated TrkA inhibition completely protected mice from a lethal IAV infection by significantly reducing viral loads and lung inflammation. Using primary lung cells isolated from the TrkA KI mice, we show that specific TrkA inhibition reduced IAV viral RNA synthesis in airway epithelial cells (AECs) but not in alveolar macrophages (AMs). Transcriptomic analysis confirmed the cell-type-specific role of TrkA in viral RNA synthesis, and identified distinct gene expression patterns under the TrkA regulation in IAV-infected AECs and AMs. Among the TrkA-activated targets are various proinflammatory cytokines and chemokines such as IL6, IL-1β, IFNs, CCL-5, and CXCL9, supporting the role of TrkA in mediating lung inflammation. Indeed, while TrkA inhibitor 1NMPP1 administered after the peak of IAV replication had no effect on viral load, it was able to decrease lung inflammation and provided partial protection in mice. Taken together, our results have demonstrated for the first time an important biological role of TrkA signaling in IAV infection, identified its cell-type-specific contribution to viral replication, and revealed its functional mechanism in virus-induced lung inflammation. This study suggests TrkA as a novel host target for therapeutic development against influenza viral disease.

Exploring the Pain Attenuating Potential of Imatinib in Chronic Constriction Injury Model of Neuropathic Pain

Background: Preclinical studies have identified three members of tyrosine kinase receptor family (Trk), TrkA, TrkB, and TrkC in dorsal root of ganglia (DRG), which carry sensory neural signals to the central nervous system from the peripheral nervous system in neuropathic pain. Imatinib is a selective tyrosine kinase inhibitor and widely employed as anti-cancer drug in myeloid leukemia and gastrointestinal stromal tumor. The present study was designed to investigate the potential of imatinib in neuropathic pain. Method: Neuropathic pain was induced by chronic constriction injury in rats by putting four loose ligatures around sciatic nerve. The extent of neuropathic pain was assessed by noting paw withdrawal threshold (mechanical hyperalgesia) in pin prick test, paw withdrawal latency in hot plate test (heat hyperalgesia) and paw withdrawal duration in acetone drop test (cold allodynia) before surgery and on 14th day post surgery. Result: There was a significant development of cold allodynia, mechanical and heat hyperalgesia on 14th day in CCI-subjected rats. Administration of imatinib (25 mg/kg and 50 mg/kg) significantly abolished the behavioural deficits (hyperalgesia and allodynia) induced by CCI in a dose-dependent manner. Conclusion: The observed beneficial effects of imatinib in reduction of behavioral deficits in CCI-subjected rats may be possibly attributed to tyrosine kinase inhibition in dorsal root ganglia neurons associated with the neuropathic pain.

Targeting TRK: A fast-tracked application of precision oncology and future directions.

The NTRK genes encode the tropomyosin-related receptor tyrosine kinases TrkA, TrkB and TrkC. TRK receptors regulate the proliferation, differentiation, and survival of many neuronal and non-neuronal glial cells during embryogenesis, thus playing a critical role in synaptic plasticity and the development of nociceptive pathways. Recurrent genomic alterations in NTRK genes, typically fusions involving the 3' region encoding the kinase domain juxtaposed to 5' sequences from numerous partner genes, occur at a low frequency in a wide diversity of adult and pediatric cancers. The contributions of the resulting constitutively activated kinase to oncogenesis and cancer progression are being elucidated. Larotrectinib and entrectinib are potent first-generation TRK inhibitors with IC50 values in the nanomolar range across cancer cell lines harboring NTRK fusions. Larotrectinib is highly selective for TRK receptors, whereas entrectinib also potently inhibits ROS1 and ALK. Clinical trials of both drugs demonstrated significant and durable responses in patients with tumors harboring NTRK alterations, leading to first of its kind cancer agnostic FDA approvals in the United States for drugs targeting a genomic alteration. Unfortunately, acquired resistance inevitably develops. The second-generation TRK inhibitors selitrectinib and repotrectinib are designed to overcome known mechanisms of resistance.

Abstract 1979: JAK2/STAT3 and TrkA pathways are frequently co-activated in triple-negative and HER2-enriched breast cancers and the co-activation correlates with an increased potential of metastasis

Abstract Breast cancer is the leading cause of cancer-related deaths in American women. Despite the current standard-of-care, which implements tumor resection, radiotherapy, and chemotherapy, triple-negative and HER2-positive breast cancer patients often relapse and present with recurrent disease, for which there is no cure. Thus, there is an urgent need to identify new molecular targets to improve patient response to anti-cancer therapies. The JAK2-STAT3 and TrkA receptor tyrosine kinase signaling pathways have been separately implicated in metastatic breast cancers, but information about their crosstalk remains limited. Activated Janus kinase 2 (JAK2) receptor tyrosine kinase phosphorylates signal transducer and activator of transcription 3 (STAT3) transcription factor, inducing STAT3 nuclear translocation and transcriptional activity. Abnormal activation of STAT3-mediated transcription has been identified as a pro-tumorigenic event in breast cancers and promotes progression through induction of breast cancer stem cells, invasion, and angiogenesis. Tropomyosin receptor kinase A (TrkA) frequently forms oncogenic fusion proteins and its overexpression has been shown to drive malignant transformation of breast cancer cells. JAK2 inhibitors, while approved for treatment of myeloproliferative neoplasms, are currently in clinical trials for triple-negative breast cancers. Currently, there are two FDA-approved TrkA inhibitors for treatment of NTRK1 fusion-positive solid tumors, one of which is considered a tumor-agnostic inhibitor. However, whether these two important pathways are co-activated in breast cancers and whether the co-activation is associated with overall progression of breast cancer have not been investigated. Herein, we report that STAT3 and TrkA are more co-activated in triple-negative and HER2-enriched breast cancers, compared to luminal subtypes, as determined by immunohistochemical staining of 33 invasive breast carcinomas and datamining data of over 1,500 breast cancer patients. We also observed that high co-activation of both JAK2-STAT3 and TrkA pathways significantly shortens overall metastasis-free survival of both triple-negative and HER2-enriched breast cancer patients when compared to patients with low co-activation. Similarly, we also find that JAK2-STAT3 and TrkA co-activation also significantly shortens brain, lung, and bone metastasis-free survival in both of these breast cancer subtypes, suggesting a critical role for JAK2-STAT3 and TrkA in distant metastasis of aggressive breast cancers. Taken together, our findings indicate a novel signaling crosstalk between JAK2-STAT3 and TrkA pathways in triple-negative and HER2-enriched breast cancers, and the use of their co-activation as a prognostic indicator for metastatic breast cancers of both subtypes. Citation Format: Angelina T. Regua, Noah R. Aguayo, Sara Abu Jalboush, Daniel L. Doheny, Sara G. Manore, Dongqin Zhu, Grace L. Wong, Austin Arrigo, Calvin J. Wagner, Yang Yu, Karen Baylon, Alexandra Thomas, Michael D. Chan, Jimmy Ruiz, Guangxu Jin, Roy E. Strowd, Peiqing Sun, Linda J. Metheny-Barlow, Jiayuh Lin, Hui-Wen Lo. JAK2/STAT3 and TrkA pathways are frequently co-activated in triple-negative and HER2-enriched breast cancers and the co-activation correlates with an increased potential of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1979.

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 and its receptor tyrosine kinase TrkA are overexpressed in cervical squamous cell carcinoma.

Nerve growth factor (NGF) and its receptors are increasingly implicated in cancer progression, but their expression in cervical cancer is unclear. The objective of this study was to define the protein expression of NGF, its precursor (proNGF), as well as their receptors, the tyrosine kinase receptor TrkA, the common neurotrophin receptor p75NTR and the pro‐neurotrophin receptor sortilin in cervical cancer. Immunohistochemistry was performed in a cohort of cervical cancers (n = 287), including the two major subtypes of the disease: squamous cell carcinomas (SCC) and adenocarcinomas (AC). Normal cervical tissues (n = 28) were also analyzed. Protein expression was determined by computer‐based digital quantification of staining intensity and comparative statistical analyses were made with clinicopathological parameters including histological subtype, age, grade, tumor size, lymph node invasion, and stage. The expression of NGF, proNGF, TrkA, p75NTR, and sortilin was higher in cervical cancer compared to normal cervical tissues. NGF and TrkA were found overexpressed in SCC compared to AC (P = .0006 and P < .0001, respectively). The expression of NGF (P = .0053), proNGF (P = .0022), and p75NTR (P = .0002), but not that of TrkA or sortilin, was associated with increasing grade in SCC. In addition, nerve infiltration into the tumor microenvironment was assessed using the pan‐neuronal marker PGP9.5. Infiltrating nerves were detected in 27% of cervical tumors and expressed TrkA. Functional investigations using the HELA cervical cancer cell line indicated that the Trk tyrosine kinase inhibitor GNF‐5837 reduced cell viability through decreased ERK1/2 activation. Together, these data reveal the overexpression of NGF and TrkA in cervical SCC, suggesting a potential therapeutic value of targeting the NGF‐TrkA signaling pathway in this subtype of cervical cancer.

Molecular insight on the altered membrane trafficking of TrkA kinase dead mutants

We address the contribution of kinase domain structure and catalytic activity to membrane trafficking of TrkA receptor tyrosine kinase. We conduct a systematic comparison between TrkA-wt, an ATP-binding defective mutant (TrkA-K544N) and other mutants displaying separate functional impairments of phosphorylation, ubiquitination, or recruitment of intracellular partners. We find that only K544N mutation endows TrkA with restricted membrane mobility and a substantial increase of cell surface pool already in the absence of ligand stimulation. This mutation is predicted to drive a structural destabilization of the αC helix in the N-lobe by molecular dynamics simulations, and enhances interactions with elements of the actin cytoskeleton. On the other hand, a different TrkA membrane immobilization is selectively observed after NGF stimulation, requires both phosphorylation and ubiquitination to occur, and is most probably related to the signaling abilities displayed by the wt but not mutated receptors. In conclusion, our results allow to distinguish two different TrkA membrane immobilization modes and demonstrate that not all kinase-inactive mutants display identical membrane trafficking.

Mapping of the tyrosine kinase receptors trkA (NTRK1), trkB (NTRK2) and trkC(NTRK3) to human chromosomes 1q22, 9q22 and 15q25 by fluorescence in situ hybridization.

trk (NTRK) genes encode tyrosine kinase transmembrane receptors that are stimulated by neurotrophins, and are responsible for the transduction of signals controlling neuropoiesis and neuron survival in the central and peripheral nervous system, trkA gene has earlier been assigned to three different loci on chromosome 1. To resolve these conflicting results, and confirm the localization of trkB and trkC, probes specific to each of these related genes were constructed and used in fluorescent in situ hybridization on human metaphase cells. Our results indicate that trkA, trkB and trkC are located in chromosome bands 1q22, 9q22 and 15q25, respectively.

Tumor necrosis factor-related apoptosis-inducing ligand reduces the expression of the neuroprotective Na+ /Ca2+ exchanger isoform NCX3 in human neuroblastoma SH-SY5Y cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cytokine belonging to the TNF superfamily, is regarded as a mediator of neurotoxicity. The constitutively expressed ion exchanger Na+ /Ca2+ exchanger isoform-3 (NCX3) has been shown to protect neurons from injury. Its expression is induced by nerve growth factor (NGF) through activation of its tyrosine kinase receptor trkA. The latter, in turn, activates downstream kinases, such as extracellular signal-regulated kinase (ERK) and the survival-related kinase protein kinase B (AKT). Here, we verified whether TRAIL could influence the expression of NCX3 via modulation of the NGF/trkA system. Differentiated human neuroblastoma SH-SY5Y cells were incubated with TRAIL and, subsequently, the expression of the NCX3 protein was studied at different times by means of western blot analysis. Then, the expression of the phosphorylated forms of either trkA, ERK or AKT was analyzed at identical intervals. Western blot analysis revealed that the expression of NCX3 protein decreased in a time-dependent fashion in SH-SY5Y cells treated with TRAIL, to reach its minimum at 48 h. On the other hand, p-trkA, p-ERK, and p-AKT expression was increased in cells treated with TRAIL after 6 and 16 h; then it declined to nearly undetectable levels after 48 h. Results indicate that the increase in TRAIL expression occurring during neuronal damage may be responsible of NCX3 down-regulation and weakens its neuroprotective effects. The TRAIL system could thus represent a potential target for treatment of neuronal damage characterized by NCX3 function impairment.

TRK Fusion Cancers in Children: A Clinical Review and Recommendations for Screening.

Chromosomal translocations involving the NTRK1, NTRK2, and NTRK3 genes (TRK fusions), which encode the neurotrophin tyrosine kinase receptors TRKA, TRKB, and TRKC, can result in constitutive activation and aberrant expression of TRK kinase. Certain cancers almost universally harbor TRK fusions, including infantile fibrosarcoma, cellular congenital mesoblastic nephroma, secretory breast cancer, and mammary analog secretory carcinoma of the salivary gland. TRK fusions have also been identified at lower frequencies across a broad range of other pediatric cancers, including undifferentiated sarcomas, gliomas, papillary thyroid cancers, spitzoid neoplasms, inflammatory myofibroblastic tumors, and acute leukemias. Here we review the prevalence and diseases associated with TRK fusions and methods of detection of these fusions in light of the recent development of selective TRK inhibitors, such as larotrectinib, which demonstrated a 75% response rate across children and adults with TRK fusion cancers. We provide recommendations for screening pediatric tumors for the presence of TRK fusions, including the use of immunohistochemistry or fluorescence in situ hybridization for patients with tumors likely to harbor TRK fusions. Further, we recommend next-generation sequencing for tumors that have a relatively low prevalence of TRK fusions, both to identify patients who may benefit from TRK inhibition and to identify other targetable oncogenic drivers that exist in the same tumor types.

MicroRNA-141 binds to the nerve growth factor receptor associated protein 1 gene and restores the erectile function of diabetic rats through down-regulating the nerve growth factor/neurotrophin receptor p75 (NGF/p75NTR) signaling.

Erectile dysfunction (ED) is one of the major complications in diabetes mellitus (DM). We have previously reported that the nerve growth factor (NGF)/tyrosine kinase receptor (TrkA) signaling is actively involved in DM-induced ED (DMED). Here, we investigate the effect of micro-RNA-141 (miR-141) on the NGF/p75 neurotrophin receptor (p75NTR) signaling and erectile function of diabetic rats. Sprague-Dawlay (SD) rats were used to establish a DMED model. The dual-luciferase reporter gene assay was first performed to identify the nerve growth factor receptor-associated protein 1 (NGFRAP1) gene as the target gene of miR-141. The regulatory mechanisms underlying miR-141 governing NGFRAP1 in vivo were then validated by modulating the expressions of miR-141 and knocking down NGFRAP1. The expressions of miR-141 were decreased while the expressions of NGFRAP1, NGF, and p75NTR were increased in DMED. miR-141 and downregulation of NGFRAP1, respectively, increased the density of corpus cavernosum smooth muscle and the ratio of intracavernosal pressure (ICP)/mean arterial blood pressure (MAP) and promoted the expression of α-actin and desmin as well. miR-141 also upregulated the expressions of NGFRAP1 in DMED, and knockdown of NGFRAP1 inhibited the productions of NGF and p75NTR. Furthermore, miR-141 suppressed the NGF/p75NTR signaling via binding to NGFRAP1. NGF/p75NTR signaling actively participates in the pathogenesis of DMED. miR-141 binds to NGFRAP1 and restores the erectile function of diabetic rats via downregulation of NGF/p75NTR signaling.

Biological and Genetic Features of Neuroblastoma and Their Clinical Importance.

Neuroblastoma derived from primitive cells of the sympathetic nervous system typically develops in the adrenal medulla or paraspinal ganglia. Neuroblastoma usually occurs sporadically, but familial cases are also observed. ALK and PHOX2B germline mutations can cause hereditary neuroblastoma, while a common genetic variation in chromosome 6p22 is associated to sporadic neuroblastoma. However, the aetiology of sporadic neuroblastoma is not exactly known. This embryonic malignancy generally represents the second most common solid tumour after central nervous system tumours throughout the world in childhood. Neuroblastoma is a complex disease that has different clinical courses, from metastatic spread to spontaneous regression. Spontaneous regression can occur without therapy in primary or metastatic site. Potential regression mechanisms primarily involve apoptosis, hypermethylation of subtelomeric DNA, immune response and Nerve Growth Factor (NGF) deprivation. Neuroblastoma is a heterogeneous tumour that can show many different chromosomal abnormalities; e.g. MYCN amplification, 1p deletion, unbalanced translocations involving chromosome 17, aneuploidies and Loss of Heterozygosity (LOH) events. Tyrosine kinase receptors TrkA, TrkB and TrkC, their ligands NGF, Brain-derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT-3), and Aurora Kinase A (AURKA) play a regulatory role in differentiation, apoptosis, cell proliferation, tumourigenesis, angiogenesis or metastasis of neuroblastoma. TrkA expression is associated with differentiation or regression, depending on presence or absence of NGF, whereas TrkB and BDNF are mostly expressed in aggressive neuroblastomas with MYCN amplification. MYCN is amplified in 18-38% of neuroblastoma cases. MYCN amplification mechanism remains to be completely clarified. This paper reviews the biological/genetic features and their clinical importance in neuroblastoma.

Activation of β- and α2-adrenergic receptors stimulate tubulin polymerization and promote the association of Gβγ with microtubules in cultured NIH3T3 cells

Microtubules (MTs) constitute a crucial part of the cytoskeleton and are essential for cell division and differentiation, cell motility, intracellular transport, and cell morphology. Precise regulation of MT assembly and dynamics is essential for the performance of these functions. Although much progress has been made in identifying and characterizing the cellular factors that regulate MT assembly and dynamics, signaling events in this process is not well understood. Gβγ, an important component of the G protein-coupled receptor (GPCR) signaling pathway, has been shown to promote MT assembly in vitro and in cultured NIH3T3 and PC12 cells. Using the MT depolymerizing agent nocodazole, it has been demonstrated that the association of Gβγ with polymerized tubulin is critical for MT assembly. More recently, Gβγ has been shown to play a key role in NGF-induced neuronal differentiation of PC12 cells through its interaction with tubulin/MTs and modulation of MT assembly. Although NGF is known to exert its effect through tyrosine kinase receptor TrkA, the result suggests a possible involvement of GPCRs in this process. The present study was undertaken to determine whether agonist activation of GPCR utilizes Gβγ to promote MT assembly. We used isoproterenol and UK 14,304, agonists for two different GPCRs (β- and α2-adrenergic receptors, respectively) known to activate Gs and Gi respectively, with an opposing effect on production of cAMP. The results demonstrate that the agonist activation of β- and α2-adrenergic receptors promotes the association of Gβγ with MTs and stimulates MT assembly in NIH3T3 cells. Interestingly, the effects of these two agonists were more prominent when the cellular level of MT assembly was low (30% or less). In contrast to MT assembly, actin polymerization was not affected by isoproterenol or UK 14, 304 indicating that the effects of these agonists are limited to MTs. Thus, it appears that, upon cellular demand, GPCRs may utilize Gβγ to promote MT assembly. Stimulation of MT assembly appears to be a novel function of G protein-mediated signaling.

The neurotrophic tyrosine kinase receptor TrkA and its ligand NGF are increased in squamous cell carcinomas of the lung

The neurotrophic tyrosine kinase receptor TrkA (NTRK1) and its ligand nerve growth factor (NGF) are emerging promoters of tumor progression. In lung cancer, drugs targeting TrkA are in clinical trials, but the clinicopathological significance of TrkA and NGF, as well as that of the precursor proNGF, the neurotrophin co-receptor p75NTR and the proneurotrophin co-receptor sortilin, remains unclear. In the present study, analysis of these proteins was conducted by immunohistochemistry and digital quantification in a series of 204 lung cancers of different histological subtypes versus 121 normal lung tissues. TrkA immunoreactivity was increased in squamous cell carcinoma compared with benign and other malignant lung cancer histological subtypes (p < 0.0001). NGF and proNGF were also increased in squamous cell carcinoma, as well as in adenocarcinoma (p < 0.0001). In contrast, p75NTR was increased across all lung cancer histological subtypes compared to normal lung (p < 0.0001). Sortilin was higher in adenocarcinoma and small cell carcinoma (p < 0.0001). Nerves in the tumor microenvironment were negative for TrkA, NGF, proNGF, p75NTR and sortilin. In conclusion, these data suggest a preferential therapeutic value of targeting the NGF-TrkA axis in squamous cell carcinomas of the lung.

Stellate Transcriptomics Reveal Consistent Patterns of Gene Expression Across Species

IntroductionSympathetic hyperactivity is a key feature of human hypertension as well as animal models of hypertension and cardiovascular disease. Sympathetic hyperactivity can result from cell‐autonomous changes in neurons that innervate cardiovascular target tissues. Stellate ganglia are of particular interest because the majority of sympathetic neurons innervating the heart reside there. Transcriptomics provides a powerful tool for identifying genomic changes that contribute to sympathetic dysfunction in disease.AimTo compare the transcriptomes of healthy stellate ganglia from adult rat and mouse.MethodRNA‐sequencing was carried out on individual stellate ganglia from 16‐week male Wistar rats or 18‐week male C57Bl6J mice. Mouse samples were depleted of ribosomal RNA prior to library preparation, while the rat library was prepared used OligoDT to enrich for mRNAs. Sequencing was carried out in an Illumina HiSeq 4000 at the High‐Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (Oxford) or the Massively Parallel Sequencing Shared Resource (OHSU). The rat transcriptome was aligned to the reference genome and transcripts were quantified using the Salmon‐DESeq2 method, which relies on exon matching. The mouse transcriptome was aligned to the reference genome by the OHSU Bioinformatics &amp; Computational Biology Core using STAR, which includes non‐coding RNAs.ResultsA comparison of these independent analyses of gene expression reveals a significant degree of consistency regarding the most highly expressed genes, despite differences in the data processing pipeline. Genes closely associated with sympathetic neuron function were among the most highly expressed genes in both rat and mouse. We identified high levels of genes encoding proteins required for norepinephrine synthesis tyrosine hydroxylase (Th), L‐DOPA decarboxylase (Ddc), and dopamine beta hydroxylase (dbh), as well as genes encoding transporters for vesicular packaging (slc18a2, vesicular monoamine transporter 2) and removal of extracellular catecholamines (slc6a2, norepinephrine transporter, NET). High levels of mRNA encoding the sympathetic co‐transmitter Neuropeptide Y (Npy) were also observed.Genes encoding the Nerve Growth Factor receptors Ngfr (p75 neurotrophin receptor) and Trk1 (TrkA tyrosine kinase receptor) were abundantly expressed in rat and mouse stellate ganglia. Genes encoding structural proteins such as actins and tubulins were abundant in both species, as were genes encoding proteins involved in synaptic transmission including Snap25 (synaptosomal‐associated protein 25), Syt1 (synaptotagmin 1), and several nicotinic acetylcholine receptor subunits. The details of transcript counts differed between the two methods but the overall pattern of gene expression between species was quite consistent.ConclusionThese data will provide a normal baseline for identification of changes that occur in diseases associated with sympathetic over‐activity such as hypertension and cardiovascular disease.Support or Funding InformationNIH OT2TR001984, OHSU School of Medicine Dean's Fund, The Wellcome TrustThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dimeric Dipeptide Mimetics of NGF and BDNF Are Promising Agents for Post-Stroke Therapy

The dimeric dipeptide mimetics of the brain derived neurotrophic factor (BDNF) loops 1 and 4 and nerve growth factor (NGF) loop 4 were designed and synthesized at the Zakusov Research Institute of Pharmacology. There are respectively bis-(N-monosuccinyl-L-methionyl-L-serine) heptamethylenediamide(GSB-214), bis-(N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide (GSB-106) and bis-(N-monosuccinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2). All of the ob-tained compounds activated a corresponding specific NGF or BDNF tyrosine kinase receptor (TrkA or TrkB), but had different postreceptor signaling patterns. GSB-106 activated the ERK and AKT, whereas GSB-214 and GK-2 only activated the AKT kinase. Here we report a comparative analysis of neuroprotective activity of these dipeptides in a model of ischemic stroke induced by transient middle cerebral artery occlusion (MCAO). The all three dimeric dipeptides showed a statistically significant decrease of infarct volumes with the treatment beginning 4 hour after surgery. In the experiment with BDNF mimetics, GSB-106 reduced this volume by 66% and GSB-214 by 26%. NGF GK-2 reduced the cerebral infarct volume by 45%. Thus, BDNF mimetic, which activated both the ERK and AKT, and NGF mimetic, which selectively activated PI3K/AKT, showed high neuroprotective efficacy. In addition, we studied neuroprotective effects of GK-2 at the beginning of the treatment 6, 8 and 24 hours after reperfusion. The neuroprotective effect of GK-2 persisted in all these conditions. The effectiveness of GK-2 at a delayed start of administration suggests that the dipeptide has neuroregenerative properties. The results obtained suggest a potential role for the dimeric dipeptide NGF and BDNF mimetics as therapeutic agents useful in the treatment of a stroke.

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.

Therapeutic Potentials of BDNF/TrkB in Breast Cancer; Current Status and Perspectives.

Brain-derived neurotrophic factor (BDNF) is a potent neurotrophic factor that has been shown to stimulate breast cancer cell growth and metastasis via tyrosine kinase receptors TrkA, TrkB, and the p75NTR death receptor. The aberrant activation of BDNF/TrkB pathways can modulate several signaling pathways, including Akt/PI3K, Jak/STAT, NF-kB, UPAR/UPA, Wnt/β-catenin, and VEGF pathways as well as the ER receptor. Several microRNAs have been identified that are involved in the modulation of BDNF/TrkB pathways. These include miR-206, miR-204, MiR-200a/c, MiR-210, MiR-134, and MiR-191; and these may be of value as prognostic and predictive biomarkers for detecting patients at high risk of developing breast cancer. It has been also been demonstrated that a high expression of genes involved in the BDNF pathway in breast cancer is associated with poor clinical outcome and reduced survival of patients. Several approaches have been developed for targeting this pathway, for example TKr inhibitors (AZD6918, CEP-701) and RNA interference. The aim of the current review was to provide an overview of the role of BDNF/TrkB pathways in the pathogenesis of breast cancer and its value as a potential therapeutic target. J. Cell. Biochem. 118: 2502-2515, 2017. © 2017 Wiley Periodicals, Inc.

Unravelling the Mechanism of TrkA-Induced Cell Death by Macropinocytosis in Medulloblastoma Daoy Cells.

Macropinocytosis is a normal cellular process by which cells internalize extracellular fluids and nutrients from their environment and is one strategy that Ras-transformed pancreatic cancer cells use to increase uptake of amino acids to meet the needs of rapid growth. Paradoxically, in non-Ras transformed medulloblastoma brain tumors, we have shown that expression and activation of the receptor tyrosine kinase TrkA overactivates macropinocytosis, resulting in the catastrophic disintegration of the cell membrane and in tumor cell death. The molecular basis of this uncontrolled form of macropinocytosis has not been previously understood. Here, we demonstrate that the overactivation of macropinocytosis is caused by the simultaneous activation of two TrkA-mediated pathways: (i) inhibition of RhoB via phosphorylation at Ser(185) by casein kinase 1, which relieves actin stress fibers, and (ii) FRS2-scaffolded Src and H-Ras activation of RhoA, which stimulate actin reorganization and the formation of lamellipodia. Since catastrophic macropinocytosis results in brain tumor cell death, improved understanding of the mechanisms involved will facilitate future efforts to reprogram tumors, even those resistant to apoptosis, to die.