RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells.

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

Differential metabolic activity and discovery of therapeutic targets using summarized metabolic pathway models.

In spite of the increasing availability of genomic and transcriptomic data, there is still a gap between the detection of perturbations in gene expression and the understanding of their contribution to the molecular mechanisms that ultimately account for the phenotype studied. Alterations in the metabolism are behind the initiation and progression of many diseases, including cancer. The wealth of available knowledge on metabolic processes can therefore be used to derive mechanistic models that link gene expression perturbations to changes in metabolic activity that provide relevant clues on molecular mechanisms of disease and drug modes of action (MoA). In particular, pathway modules, which recapitulate the main aspects of metabolism, are especially suitable for this type of modeling. We present Metabolizer, a web-based application that offers an intuitive, easy-to-use interactive interface to analyze differences in pathway metabolic module activities that can also be used for class prediction and in silico prediction of knock-out (KO) effects. Moreover, Metabolizer can automatically predict the optimal KO intervention for restoring a diseased phenotype. We provide different types of validations of some of the predictions made by Metabolizer. Metabolizer is a web tool that allows understanding molecular mechanisms of disease or the MoA of drugs within the context of the metabolism by using gene expression measurements. In addition, this tool automatically suggests potential therapeutic targets for individualized therapeutic interventions.

Reactive oxygen species modulate macrophage immunosuppressive phenotype through the up-regulation of PD-L1.

The combination of immune checkpoint blockade with chemotherapy is currently under investigation as a promising strategy for the treatment of triple negative breast cancer (TNBC). Tumor-associated macrophages (TAMs) are the most prominent component of the breast cancer microenvironment because they influence tumor progression and the response to therapies. Here we show that macrophages acquire an immunosuppressive phenotype and increase the expression of programmed death ligand-1 (PD-L1) when treated with reactive oxygen species (ROS) inducers such as the glutathione synthesis inhibitor, buthionine sulphoximine (BSO), and paclitaxel. Mechanistically, these agents cause accumulation of ROS that in turn activate NF-κB signaling to promote PD-L1 transcription and the release of immunosuppressive chemokines. Systemic in vivo administration of paclitaxel promotes PD-L1 accumulation on the surface of TAMS in a mouse model of TNBC, consistent with in vitro results. Combinatorial treatment with paclitaxel and an anti-mouse PD-L1 blocking antibody significantly improved the therapeutic efficacy of paclitaxel by reducing tumor burden and increasing the number of tumor-associated cytotoxic T cells. Our results provide a strong rationale for the use of anti-PD-L1 blockade in the treatment of TNBC patients. Furthermore, interrogation of chemotherapy-induced PD-L1 expression in TAMs is warranted to define appropriate patient selection in the use of PD-L1 blockade.

AhR controls redox homeostasis and shapes the tumor microenvironment in BRCA1-associated breast cancer.

Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.

Ubiquitin ligase RNF8 suppresses Notch signaling to regulate mammary development and tumorigenesis.

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rnf8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function.

Gene Expression Integration into Pathway Modules Reveals a Pan-Cancer Metabolic Landscape.

Metabolic reprogramming plays an important role in cancer development and progression and is a well-established hallmark of cancer. Despite its inherent complexity, cellular metabolism can be decomposed into functional modules that represent fundamental metabolic processes. Here, we performed a pan-cancer study involving 9,428 samples from 25 cancer types to reveal metabolic modules whose individual or coordinated activity predict cancer type and outcome, in turn highlighting novel therapeutic opportunities. Integration of gene expression levels into metabolic modules suggests that the activity of specific modules differs between cancers and the corresponding tissues of origin. Some modules may cooperate, as indicated by the positive correlation of their activity across a range of tumors. The activity of many metabolic modules was significantly associated with prognosis at a stronger magnitude than any of their constituent genes. Thus, modules may be classified as tumor suppressors and oncomodules according to their potential impact on cancer progression. Using this modeling framework, we also propose novel potential therapeutic targets that constitute alternative ways of treating cancer by inhibiting their reprogrammed metabolism. Collectively, this study provides an extensive resource of predicted cancer metabolic profiles and dependencies.Significance: Combining gene expression with metabolic modules identifies molecular mechanisms of cancer undetected on an individual gene level and allows discovery of new potential therapeutic targets. Cancer Res; 78(21); 6059-72. ©2018 AACR.

Orthoxenografts of Testicular Germ Cell Tumors Demonstrate Genomic Changes Associated with Cisplatin Resistance and Identify PDMP as a Resensitizing Agent.

Purpose: To investigate the genetic basis of cisplatin resistance as efficacy of cisplatin-based chemotherapy in the treatment of distinct malignancies is often hampered by intrinsic or acquired drug resistance of tumor cells.Experimental Design: We produced 14 orthoxenograft transplanting human nonseminomatous testicular germ cell tumors (TGCT) in mice, keeping the primary tumor features in terms of genotype, phenotype, and sensitivity to cisplatin. Chromosomal and genetic alterations were evaluated in matched cisplatin-sensitive and their counterpart orthoxenografts that developed resistance to cisplatin in nude mice.Results: Comparative genomic hybridization analyses of four matched orthoxenografts identified recurrent chromosomal rearrangements across cisplatin-resistant tumors in three of them, showing gains at 9q32-q33.1 region. We found a clinical correlation between the presence of 9q32-q33.1 gains in cisplatin-refractory patients and poorer overall survival (OS) in metastatic germ cell tumors. We studied the expression profile of the 60 genes located at that genomic region. POLE3 and AKNA were the only two genes deregulated in resistant tumors harboring the 9q32-q33.1 gain. Moreover, other four genes (GCS, ZNF883, CTR1, and FLJ31713) were deregulated in all five resistant tumors independently of the 9q32-q33.1 amplification. RT-PCRs in tumors and functional analyses in Caenorhabditis elegans (C. elegans) indicate that the influence of 9q32-q33.1 genes in cisplatin resistance can be driven by either up- or downregulation. We focused on glucosylceramide synthase (GCS) to demonstrate that the GCS inhibitor DL-threo-PDMP resensitizes cisplatin-resistant germline-derived orthoxenografts to cisplatin.Conclusions: Orthoxenografts can be used preclinically not only to test the efficiency of drugs but also to identify prognosis markers and gene alterations acting as drivers of the acquired cisplatin resistance. Clin Cancer Res; 24(15); 3755-66. ©2018 AACR.

DNA Methylomes Reveal Biological Networks Involved in Human Eye Development, Functions and Associated Disorders.

This work provides a comprehensive CpG methylation landscape of the different layers of the human eye that unveils the gene networks associated with their biological functions and how these are disrupted in common visual disorders. Herein, we firstly determined the role of CpG methylation in the regulation of ocular tissue-specification and described hypermethylation of retinal transcription factors (i.e., PAX6, RAX, SIX6) in a tissue-dependent manner. Second, we have characterized the DNA methylome of visual disorders linked to internal and external environmental factors. Main conclusions allow certifying that crucial pathways related to Wnt-MAPK signaling pathways or neuroinflammation are epigenetically controlled in the fibrotic disorders involved in retinal detachment, but results also reinforced the contribution of neurovascularization (ETS1, HES5, PRDM16) in diabetic retinopathy. Finally, we had studied the methylome in the most frequent intraocular tumors in adults and children (uveal melanoma and retinoblastoma, respectively). We observed that hypermethylation of tumor suppressor genes is a frequent event in ocular tumors, but also unmethylation is associated with tumorogenesis. Interestingly, unmethylation of the proto-oncogen RAB31 was a predictor of metastasis risk in uveal melanoma. Loss of methylation of the oncogenic mir-17-92 cluster was detected in primary tissues but also in blood from patients.

RANKL/RANK control Brca1 mutation- .

Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients.

Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance.

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.

A validated gene regulatory network and GWAS identifies early regulators of T cell-associated diseases.

Early regulators of disease may increase understanding of disease mechanisms and serve as markers for presymptomatic diagnosis and treatment. However, early regulators are difficult to identify because patients generally present after they are symptomatic. We hypothesized that early regulators of T cell-associated diseases could be found by identifying upstream transcription factors (TFs) in T cell differentiation and by prioritizing hub TFs that were enriched for disease-associated polymorphisms. A gene regulatory network (GRN) was constructed by time series profiling of the transcriptomes and methylomes of human CD4(+) T cells during in vitro differentiation into four helper T cell lineages, in combination with sequence-based TF binding predictions. The TFs GATA3, MAF, and MYB were identified as early regulators and validated by ChIP-seq (chromatin immunoprecipitation sequencing) and small interfering RNA knockdowns. Differential mRNA expression of the TFs and their targets in T cell-associated diseases supports their clinical relevance. To directly test if the TFs were altered early in disease, T cells from patients with two T cell-mediated diseases, multiple sclerosis and seasonal allergic rhinitis, were analyzed. Strikingly, the TFs were differentially expressed during asymptomatic stages of both diseases, whereas their targets showed altered expression during symptomatic stages. This analytical strategy to identify early regulators of disease by combining GRNs with genome-wide association studies may be generally applicable for functional and clinical studies of early disease development.

DNA methylation plasticity of human adipose-derived stem cells in lineage commitment.

Adult stem cells have an enormous potential for clinical use in regenerative medicine that avoids many of the drawbacks characteristic of embryonic stem cells and induced pluripotent stem cells. In this context, easily obtainable human adipose-derived stem cells offer an interesting option for future strategies in regenerative medicine. However, little is known about their repertoire of differentiation capacities, how closely they resemble the target primary tissues, and the potential safety issues associated with their use. DNA methylation is one of the most widely recognized epigenetic factors involved in cellular identity, prompting us to consider how the analyses of 27,578 CpG sites in the genome of these cells under different conditions reflect their different natural history. We show that human adipose-derived stem cells generate myogenic and osteogenic lineages that share much of the DNA methylation landscape characteristic of primary myocytes and osteocytes. Most important, adult stem cells and in vitro-generated myocytes and osteocytes display a significantly different DNA methylome from that observed in transformed cells from these tissue types, such as rhabdomyosarcoma and osteosarcoma. These results suggest that the plasticity of the DNA methylation patterns plays an important role in lineage commitment of adult stem cells and that it could be used for clinical purposes as a biomarker of efficient and safely differentiated cells.

Lurbinectedin (PM01183), a new DNA minor groove binder, inhibits growth of orthotopic primary graft of cisplatin-resistant epithelial ovarian cancer.

PURPOSE: Epithelial ovarian cancer (EOC) is the fifth leading cause of death in women diagnosed with gynecologic malignancies. The low survival rate is because of its advanced-stage diagnosis and either intrinsic or acquired resistance to standard platinum-based chemotherapy. So, the development of effective innovative therapeutic strategies to overcome cisplatin resistance remains a high priority. EXPERIMENTAL DESIGN: To investigate new treatments in in vivo models reproducing EOCs tumor growth, we generated a preclinical model of ovarian cancer after orthotopic implantation of a primary serous tumor in nude mice. Further, matched model of acquired cisplatin-resistant tumor version was successfully derived in mice. Effectiveness of lurbinectedin (PM01183) treatment, a novel marine-derived DNA minor groove covalent binder, was assessed in both preclinical models as a single and a combined-cisplatin agent. RESULTS: Orthotopically perpetuated tumor grafts mimic the histopathological characteristics of primary patients' tumors and they also recapitulate in mice characteristic features of tumor response to cisplatin treatments. We showed that single lurbinectedin or cisplatin-combined therapies were effective in treating cisplatin-sensitive and cisplatin-resistant preclinical ovarian tumor models. Furthermore, the strongest in vivo synergistic effect was observed for combined treatments, especially in cisplatin-resistant tumors. Lurbinectedin tumor growth inhibition was associated with reduced proliferation, increased rate of aberrant mitosis, and subsequent induced apoptosis. CONCLUSIONS: Taken together, preclinical orthotopic ovarian tumor grafts are useful tools for drug development, providing hard evidence that lurbinectedin might be a useful therapy in the treatment of EOC by overcoming cisplatin resistance.

Distinct DNA methylomes of newborns and centenarians.

Human aging cannot be fully understood in terms of the constrained genetic setting. Epigenetic drift is an alternative means of explaining age-associated alterations. To address this issue, we performed whole-genome bisulfite sequencing (WGBS) of newborn and centenarian genomes. The centenarian DNA had a lower DNA methylation content and a reduced correlation in the methylation status of neighboring cytosine--phosphate--guanine (CpGs) throughout the genome in comparison with the more homogeneously methylated newborn DNA. The more hypomethylated CpGs observed in the centenarian DNA compared with the neonate covered all genomic compartments, such as promoters, exonic, intronic, and intergenic regions. For regulatory regions, the most hypomethylated sequences in the centenarian DNA were present mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. We extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray that reinforced the observation of more hypomethylated DNA sequences in the advanced age group. WGBS and 450,000 analyses of middle-age individuals demonstrated DNA methylomes in the crossroad between the newborn and the nonagenarian/centenarian groups. Our study constitutes a unique DNA methylation analysis of the extreme points of human life at a single-nucleotide resolution level.

Gene expression differences between colon and rectum tumors.

PURPOSE: Colorectal cancer studies typically include both colon and rectum tumors as a common entity, though this assumption is controversial and only minor differences have been reported at the molecular and epidemiologic level. We conducted a molecular study based on gene expression data of tumors from colon and rectum to assess the degree of similarity between these cancer sites at transcriptomic level. EXPERIMENTAL DESIGN: A pooled analysis of 460 colon tumors and 100 rectum tumors from four data sets belonging to three independent studies was conducted. Microsatellite instable tumors were excluded as these are known to have a different expression profile and have a preferential proximal colon location. Expression differences were assessed with linear models, and significant genes were identified using adjustment for multiple comparisons. RESULTS: Minor differences at a gene expression level were found between tumors arising in the proximal colon, distal colon, or rectum. Only several HOX genes were found to be associated with tumor location. More differences were found between proximal and distal colon than between distal colon and rectum. CONCLUSIONS: Microsatellite stable colorectal cancers do not show major transcriptomic differences for tumors arising in the colon or rectum. The small but consistent differences observed are largely driven by the HOX genes. These results may have important implications in the design and interpretation of studies in colorectal cancer.

Validation of a DNA methylation microarray for 450,000 CpG sites in the human genome.

DNA methylation is the most studied epigenetic mark and CpG methylation is central to many biological processes and human diseases. Since cancer has highlighted the contribution to disease of aberrant DNA methylation patterns, such as the presence of promoter CpG island hypermethylation-associated silencing of tumor suppressor genes and global DNA hypomethylation defects, their importance will surely become apparent in other pathologies. However, advances in obtaining comprehensive DNA methylomes are hampered by the high cost and time-consuming aspects of the single nucleotide methods currently available for whole genome DNA methylation analyses. Following the success of the standard CpG methylation microarrays for 1,505 CpG sites and 27,000 CpG sites, we have validated in vivo the newly developed 450,000 (450K) cytosine microarray (Illumina). The 450K microarray includes CpG and CNG sites, CpG islands/shores/shelves/open sea, non-coding RNA (microRNAs and long non-coding RNAs) and sites surrounding the transcription start sites (-200 bp to -1,500 bp, 5'-UTRs and exons 1) for coding genes, but also for the corresponding gene bodies and 3'-UTRs, in addition to intergenic regions derived from GWAS studies. Herein, we demonstrate that the 450K DNA methylation array can consistently and significantly detect CpG methylation changes in the HCT-116 colorectal cancer cell line in comparison with normal colon mucosa or HCT-116 cells with defective DNA methyltransferases (DKO). The provided validation highlights the potential use of the 450K DNA methylation microarray as a useful tool for ongoing and newly designed epigenome projects.

Genetic variants in apoptosis and immunoregulation-related genes are associated with risk of chronic lymphocytic leukemia.

To identify low-penetrance susceptibility alleles for chronic lymphocytic leukemia (CLL), we performed a case-control study genotyping 768 single-nucleotide polymorphisms (SNP) in 692 cases of CLL and 738 controls. We investigated nonsynonymous SNPs, SNPs with potential functional effect, and tag SNPs in regulatory gene regions in a total of 172 genes involved in cancer biology. After adjustment for multiple testing, we found a strong association between CLL risk and six genetic variants: CCNH (rs2266690, V270A), APAF1 (rs17028658, 3'region), IL16 (rs4505265, first intron), CASP8 (rs1045485, D302H), NOS2A (rs2779251, promoter), and CCR7 (rs3136687, intron 1). We found association with CLL susceptibility and 22 haplotypes in APAF1, IL6, TNFRSF13B, IL16, CASP3, CCR7, LTA/TNF, BAX, BCL2, CXCL12, CASP10/CASP8, CASP1, CCL2, BAK1, and IL1A candidate genes. Finally, we evaluated using public data sets the potential functional effect on gene expression levels of the CLL associated genetic variants detected in regulatory regions. Minor alleles for APAF1 and IL16 were associated with lower mRNA levels; no expression differences were observed for CCR7, whereas NOS2A could not be assessed. This study suggests that common genetic variation in apoptosis- and immunoregulation-related genes is associated with the CLL risk.

Fas-activated serine/threonine phosphoprotein (FAST) is a regulator of alternative splicing.

Fas-activated serine/threonine phosphoprotein (FAST) is a survival protein that is tethered to the outer mitochondrial membrane. In cells subjected to environmental stress, FAST moves to stress granules, where it interacts with TIA1 to modulate the process of stress-induced translational silencing. Both FAST and TIA1 are also found in the nucleus, where TIA1 promotes the inclusion of exons flanked by weak splice recognition sites such as exon IIIb of the fibroblast growth factor receptor 2 (FGFR2) mRNA. Two-hybrid interaction screens and biochemical analysis reveal that FAST binds to several alternative and constitutive splicing regulators, suggesting that FAST might participate in this process. The finding that FAST is concentrated at nuclear speckles also supports this contention. We show that FAST, like TIA1, promotes the inclusion of exon IIIb of the FGFR2 mRNA. Both FAST and TIA1 target a U-rich intronic sequence (IAS1) adjacent the 5' splice site of exon IIIb. However, unlike TIA1, FAST does not bind to the IAS1 sequence. Surprisingly, knockdown experiments reveal that FAST and TIA1 act independently of one another to promote the inclusion of exon IIIb. Mutational analysis reveals that FAST-mediated alternative splicing is separable from the survival effects of FAST. Our data reveal that nuclear FAST can regulate the splicing of FGFR2 transcripts.

Transgenic mice overexpressing the full-length neurotrophin receptor TrkC exhibit increased catecholaminergic neuron density in specific brain areas and increased anxiety-like behavior and panic reaction.

Accumulating evidence has suggested that neurotrophins participate in the pathophysiology of mood disorders. We have developed transgenic mice overexpressing the full-length neurotrophin-3 receptor TrkC (TgNTRK3) in the central nervous system. TgNTRK3 mice show increased anxiety-like behavior and enhancement of panic reaction in the mouse defense test battery, along with an increase in the number and density of catecholaminergic (tyrosine hydroxylase positive) neurons in locus coeruleus and substantia nigra. Furthermore, treatment of TgNTRK3 mice with diazepam significantly attenuated the anxiety-like behaviors in the plus maze. These results provide evidence for the involvement of TrkC in the development of noradrenergic neurons in the central nervous system with consequences on anxiety-like behavior and panic reaction. Thus, changes in TrkC expression levels could contribute to the phenotypic expression of panic disorder through a trophic effect on noradrenergic neurons in the locus coeruleus. Our results demonstrate that the elevated NT3-TrkC tone via overexpression of TrkC in the brain may constitute a molecular mechanism for the expression of anxiety and anxiety.