Developmental regulation of dermal adipose tissue by BCL11b.

The distinct anatomic environment in which adipose tissues arise during organogenesis is a principle determinant of their adult expansion capacity. Metabolic disease results from a deficiency in hyperplastic adipose expansion within the dermal/subcutaneous depot; thus, understanding the embryonic origins of dermal adipose is imperative. Using single-cell transcriptomics throughout murine embryogenesis, we characterized cell populations, including Bcl11b + cells, that regulate the development of dermal white adipose tissue (dWAT). We discovered that BCL11b expression modulates the Wnt signaling microenvironment to enable adipogenic differentiation in the dermal compartment. Subcutaneous and visceral adipose arises from a distinct population of Nefl + cells during embryonic organogenesis, whereas Pi16 + /Dpp4 + fibroadipogenic progenitors support obesity-stimulated hypertrophic expansion in the adult. Together, these results highlight the unique regulatory pathways used by anatomically distinct adipose depots, with important implications for human metabolic disease.

Important Role of Endogenous Nerve Growth Factor Receptor in the Pathogenesis of Hypoxia-Induced Pulmonary Hypertension in Mice.

Pulmonary arterial hypertension (PAH) remains a disease with poor prognosis; thus, a new mechanism for PAH treatment is necessary. Circulating nerve growth factor receptor (Ngfr)-positive cells in peripheral blood mononuclear cells are associated with disease severity and the prognosis of PAH patients; however, the role of Ngfr in PAH is unknown. In this study, we evaluated the function of Ngfr using Ngfr gene-deletion (Ngfr-/-) mice. To elucidate the role of Ngfr in pulmonary hypertension (PH), we used Ngfr-/- mice that were exposed to chronic hypoxic conditions (10% O2) for 3 weeks. The development of hypoxia-induced PH was accelerated in Ngfr-/- mice compared to littermate controls. In contrast, the reconstitution of bone marrow (BM) in Ngfr-/- mice transplanted with wild-type BM cells improved PH. Notably, the exacerbation of PH in Ngfr-/- mice was accompanied by the upregulation of pulmonary vascular remodeling-related genes in lung tissue. In a hypoxia-induced PH model, Ngfr gene deletion resulted in PH exacerbation. This suggests that Ngfr may be a key molecule involved in the pathogenesis of PAH.

Trametinib-Resistant Melanoma Cells Displaying MITFhigh/NGFRlow/IL-8low Phenotype Are Highly Responsive to Alternating Periods of Drug Withdrawal and Drug Rechallenge.

Despite significant advances in targeted therapies against the hyperactivated BRAFV600/MEK pathway for patients with unresectable metastatic melanoma, acquired resistance remains an unsolved clinical problem. In this study, we focused on melanoma cells resistant to trametinib, an agent broadly used in combination therapies. Molecular and cellular changes were assessed during alternating periods of trametinib withdrawal and rechallenge in trametinib-resistant cell lines displaying either a differentiation phenotype (MITFhigh/NGFRlow) or neural crest stem-like dedifferentiation phenotype (NGFRhigh/MITFlow). Neither drug withdrawal nor drug rechallenge induced cell death, and instead of loss of fitness, trametinib-resistant melanoma cells adapted to altered conditions by phenotype switching. In resistant cells displaying a differentiation phenotype, trametinib withdrawal markedly decreased MITF level and activity, which was associated with reduced cell proliferation capacity, and induced stemness assessed as NGFR-positive cells and senescence features, including IL-8 expression and secretion. All these changes could be reversed by trametinib re-exposure, which emphasizes melanoma cell plasticity. Trametinib-resistant cells displaying a dedifferentiation phenotype were less responsive presumably due to the already low level of MITF, a master regulator of the melanoma phenotype. Considering new directions of the development of anti-melanoma treatment, our study suggests that the phenotype of melanomas resistant to targeted therapy might be a crucial determinant of the selection of second-line therapy for melanoma patients.

The Nerve Growth Factor Receptor (NGFR/p75NTR): A Major Player in Alzheimer's Disease.

Alzheimer's disease (AD) represents the most prevalent type of dementia in elderly people, primarily characterized by brain accumulation of beta-amyloid (Aß) peptides, derived from Amyloid Precursor Protein (APP), in the extracellular space (amyloid plaques) and intracellular deposits of the hyperphosphorylated form of the protein tau (p-tau; tangles or neurofibrillary aggregates). The Nerve growth factor receptor (NGFR/p75NTR) represents a low-affinity receptor for all known mammalians neurotrophins (i.e., proNGF, NGF, BDNF, NT-3 e NT-4/5) and it is involved in pathways that determine both survival and death of neurons. Interestingly, also Aß peptides can blind to NGFR/p75NTR making it the "ideal" candidate in mediating Aß-induced neuropathology. In addition to pathogenesis and neuropathology, several data indicated that NGFR/p75NTR could play a key role in AD also from a genetic perspective. Other studies suggested that NGFR/p75NTR could represent a good diagnostic tool, as well as a promising therapeutic target for AD. Here, we comprehensively summarize and review the current experimental evidence on this topic.

Tracking of Melanoma Cell Plasticity by Transcriptional Reporters.

Clonal evolution and cellular plasticity are the genetic and non-genetic driving forces of tumor heterogeneity, which in turn determine tumor cell responses towards therapeutic drugs. Several lines of evidence suggest that therapeutic interventions foster the selection of drug-resistant neural crest stem-like cells (NCSCs) that establish minimal residual disease (MRD) in melanoma. Here, we establish a dual-reporter system, enabling the tracking of NGFR expression and mRNA stability and providing insights into the maintenance of NCSC states. We observed that a transcriptional reporter that contained a 1-kilobase fragment of the human NGFR promoter was activated only in a minor subset (0.72 ± 0.49%, range 0.3-1.5), and ~2-4% of A375 melanoma cells revealed stable NGFR mRNA. The combination of both reporters provides insights into phenotype switching and reveals that both cellular subsets gave rise to cellular heterogeneity. Moreover, whole transcriptome profiling and gene-set enrichment analysis (GSEA) of the minor cellular subset revealed hypoxia-associated programs that might serve as potential drivers of an in vitro switching of NGFR-associated phenotypes and relapse of post-BRAF inhibitor-treated tumors. Concordantly, we observed that the minor cellular subset increased in response to dabrafenib over time. In summary, our reporter-based approach provides insights into plasticity and identified a cellular subset that might be responsible for the establishment of MRD in melanoma.

A Single Injection of NTG-101 Reduces the Expression of Pain-Related Neurotrophins in a Canine Model of Degenerative Disc Disease.

BACKGROUND: Tissue sources of pain emanating from degenerative discs remains incompletely understood. Canine intervertebral discs (IVDs) were needle puncture injured, 4-weeks later injected with either phosphate-buffered saline (PBS) or NTG-101, harvested after an additional fourteen weeks and then histologically evaluated for the expression of NGFr, BDNF, TrkB and CALCRL proteins. Quantification was performed using the HALO automated cell-counting scoring platform. Immunohistochemical analysis was also performed on human IVD tissue samples obtained from spinal surgery. Immunohistochemical analysis and quantification of neurotrophins and neuropeptides was performed using an in vivo canine model of degenerative disc disease and human degenerative disc tissue sections. Discs injected with NTG-101 showed significantly lower levels of Nerve Growth Factor receptor (NGFr/TrkA, p = 0.0001), BDNF (p = 0.009), TrkB (p = 0.002) and CALCRL (p = 0.008) relative to PBS injections. Human IVD tissue obtained from spinal surgery due to painful DDD show robust expression of NGFr, BDNF, TrkB and CALCRL proteins. A single intradiscal injection of NTG-101 significantly inhibits the expression of NGFr, BDNF, TrkB and CALCRL proteins in degenerative canine IVDs. These results strongly suggest that NTG-101 inhibits the development of neurotrophins that are strongly associated with painful degenerative disc disease and may have profound effects upon the management of patients living with discogenic pain.

Implication of TrkC-miR2 in neurotrophin signalling pathway regulation through NGFR transcript targeting.

TrkC and NGFR neurotrophin receptors are associated with cell death, cancer and differentiation. TrkC-miR2, which is located in TrkC gene, is known to regulate Wnt signalling pathway, and its influence on other signalling pathways is under investigation. Here, through RT-qPCR, dual-luciferase assay and Western blotting we reveal that TrkC-miR2 targets NGFR. Overexpression of TrkC-miR2 also affected TrkA, TrkC, NFKB, BCL2 and Akt2 expressions involved in neurotrophin signalling pathway, and elevated survival rate of HEK293t and U87 cells was distinguished by flow cytometry and MTT assay. Consistently, an opposite expression correlation was obtained between TrkC-miR2 and NGFR or TrkC for the duration of NT2 differentiation. Meanwhile, TrkC-miR2 down-regulation attenuated NT2 differentiation into neural-like cells. Overall, here we present in silico and experimental evidence showing TrkC-miR2 as a new controller in regulation of neurotrophin signalling pathway.

Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment.

The p75 neurotrophin receptor (p75(NTR); also known as NGFR) can mediate neuronal apoptosis in disease or following trauma, and facilitate survival through interactions with Trk receptors. Here we tested the ability of a p75(NTR)-derived trophic cell-permeable peptide, c29, to inhibit p75(NTR)-mediated motor neuron death. Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29 treatment of SOD1(G93A) mice, a common model of amyotrophic lateral sclerosis, resulted in increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident with this, c29 treatment of these mice reduced the production of p75(NTR) cleavage products. Although c29 treatment inhibited mature- and pro-nerve-growth-factor-induced death of cultured motor neurons, and these ligands induced the cleavage of p75(NTR) in motor-neuron-like NSC-34 cells, there was no direct effect of c29 on p75(NTR) cleavage. Rather, c29 promoted motor neuron survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that low levels of brain-derived neurotrophic factor (BDNF) were present, an effect that was replicated in vivo in SOD1(G93A) mice. We conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in vivo.

Involvement of polymorphisms of the nerve growth factor and its receptor encoding genes in the etiopathogenesis of ischemic stroke.

BACKGROUND: Despite the important role of the nerve growth factor in the survival and maintenance of neurons in ischemic stroke, data regarding the relationships between variations in the encoding gene and stroke are lacking. In the present study, we evaluated the association of the functional polymorphisms in NGF (rs6330) and NGFR (rs2072446 and rs734194) genes with ischemic stroke in an Armenian population. METHODS: In total, 370 unrelated individuals of Armenian nationality were enrolled in this study. Genomic DNA samples of patients and healthy controls were genotyped using polymerase chain reaction with sequence-specific primers. RESULTS: The results obtained indicate that the minor allele of rs6330 (P corr = 2.4E-10) and rs2072446 (P corr = 0.02) are significantly overrepresented in stroke group, while the minor allele of rs734194 (P corr = 8.5E-10) was underrepresented in diseased subjects. Single nucleotide polymorphisms in NGF gene (rs6330) and NGFR gene (rs2072446 and rs734194) are associated with the disease. Furthermore, it was shown that the carriage of the NGF rs6330*T minor allele is associated with increased infarct volume and higher risk of recurrent stroke. CONCLUSIONS: In conclusion, our findings suggest that the NGF rs6330*T and NGFR rs2072446*T minor alleles might be nominated as a risk factor for developing ischemic stroke and NGFR rs734194*G minor allele as a protective against this disease at least in Armenian population.

IFT80 Improves Invasion Ability in Gastric Cancer Cell Line via ift80/p75NGFR/MMP9 Signaling.

The assembly and maintenance of cilia depend on intraflagellar transport (IFT) proteins, which play an important role in development and homeostasis. IFT80 is a newly defined IFT protein and partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III, both characterized by abnormal skeletal development. However, the role and mechanism of IFT80 in the invasion of gastric cancer is unknown. We established SGC-7901 and MKN-45 gastric cancer cell lines that stably overexpressed IFT80, as verified by quantitative reverse transcription-PCR, Western blot, and immunofluorescence. Matrix metalloproteinase-9 (MMP9) plays an important role in tumor invasion, and its expression was assessed by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence. The invasion ability of IFT80 on SGC-7901 and MKN-45 cells was examined by the Matrigel invasion assay. The relationship between p75NGFR, and the p75NGFR antagonists, PD90780 and IFT80, were detected by quantitative reverse transcription-PCR and Western blotting. We first detected an IFT80 expression pattern, and found that IFT80 was highly expressed in gastric cancer clinical samples. Overexpression of IFT80 in the gastric cancer cell lines, SGC-7901 and MKN-45, led to lengthening cilia. Additionally, overexpression of IFT80 significantly improved proliferation and invasion, but inhibited apoptosis, in gastric cancer cells. We further found that overexpression of IFT80 increased p75NGFR and MMP9 mRNA and protein expression. Treatment with the p75NGFR antagonist PD90780 inhibited the increased invasion ability resulting from overexpression of IFT80 in SGC-7901 and MKN-45 gastric cancer cells. Thus, these results suggest that IFT80 plays an important role in invasion of gastric cancer through regulating the ift80/p75NGFR/MMP9 signal pathways.

Estrogen induced changes in uterine brain-derived neurotrophic factor and its receptors.

STUDY QUESTION: Are brain-derived neurotrophic factor (BDNF) and its receptors, NTRK2, NGFR and SORT1, regulated by ovarian steroids in the uterus? SUMMARY ANSWER: BDNF and its low affinity receptor, nerve growth factor receptor (NGFR), are regulated by estradiol in the uterus. WHAT IS KNOWN ALREADY: Recent studies have revealed a central role for neurotrophins in placental development, endometrial stem cell neurogenesis, endometrial carcinoma and endometriosis. Complex signaling pathways involving BDNF and its receptors are regulated by ovarian hormones in the brain, however their expression and regulation in the uterus is poorly defined. STUDY DESIGN, SIZE, DURATION: This experimental animal study involved a total of 80 mice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Female C57BL/6 mice (n = 50) were monitored daily for estrous cycle stage, and uterine horns were collected. A second group of mice (n = 30) were ovariectomized and given estradiol, progesterone, estradiol + progesterone, or saline for 4 days. Uterine expression of BDNF and its receptors were quantified by real-time PCR and western blot, and localized using immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: During the estrous cycle, expression of BDNF, NTRK2 and SORT1 remained constant, while NGFR declined 11-fold from pro-estrus through to diestrus (P = 0.005). In ovariectomized mice, estradiol treatment increased uterine expression of mature BDNF greater than 6-fold (P = 0.013, 25 kDa; P = 0.003, 27 kDa), pro-BDNF 5-fold (P = 0.041, 37 kDa band; P = 0.046, 40 kDa band), and NGFR 5-fold (P < 0.001) when compared with other treatments. NTRK2 and SORT1 were unaffected by ovarian hormones. NGFR was primarily localized in epithelial cells in mice in diestrus or in ovariectomized mice treated with progesterone (P ≤ 0.001; P ≤ 0.001, respectively). In contrast, NGFR switched to a stromal localization in ovariectomized mice administered estradiol (P = 0.002). LIMITATIONS, REASONS FOR CAUTION: This study was performed in one only species. WIDER IMPLICATIONS OF THE FINDINGS: Results of this study demonstrate the uterine regulation of BDNF and NGFR by estradiol, and highlight the striking difference between hormone exposure during the estrous cycle and daily estradiol exposure after ovariectomy on neurotrophin expression in the uterus. The results also show the spatial regulation of NGFR in the uterus in response to ovarian hormones. Sustained estrogen exposure, as seen in estrogen-dependent disease, may alter the delicate neurotrophin balance and inappropriately activate potent BDNF-NTRK2 pathways which are capable of contributing to endometrial pathology. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Canadian Institutes of Health Research (CIHR) (W.G.F.), a NSERC Discovery Grant (W.G.F.), and a Vanier Canada Graduate Scholarship-CIHR (J.M.W.). J.M.W. is a member of the CIHR sponsored Reproduction and Early Development in Health training program. The authors declare no conflicts of interest.

Neurotrophin receptors and perineural invasion in desmoplastic melanoma.

BACKGROUND: Perineural invasion (PNI) in desmoplastic melanoma is associated with increased local recurrence and reduced disease-free survival. The biological mechanisms underlying PNI remain unclear although several lines of evidence implicate neurotrophins and their receptors. OBJECTIVES: We investigated the expression of p75NGFR and TrkA, and the presence of functional RET polymorphism (RETp) as they relate to PNI in desmoplastic melanoma. METHODS: In all, 43 cases of desmoplastic melanoma were immunohistochemically evaluated for TrkA and p75NGFR expression and RETp was detected by direct DNA sequencing. RESULTS: PNI was present in 67% of cases. On univariate analysis, p75NGFR was associated with PNI (expression detected in 79% of PNI-positive cases compared with 36% of PNI-negative cases, P = .005), increased Breslow depth (P = .007), and greater Clark level (P = .01). RETp was noted in 28% of cases but was not significantly associated with PNI (P = .27) or other histopathologic variables. TrkA expression was absent in all cases. PNI was associated with increased Breslow depth and Clark level (P = .01 and P = .009, respectively). Controlling for the association between p75NGFR and depth, p75NGFR remained associated with an increased propensity for PNI (odds ratio 4.68, P = .04). LIMITATIONS: The sample size was limited. CONCLUSION: In desmoplastic melanoma, p75NGFR expression is significantly associated with PNI and a more locally aggressive phenotype.

Inhibition of circRNA NGFR promotes ferroptosis in gallbladder carcinoma cells.

Background: Gallbladder carcinoma (GBC) is a formidably aggressive malignancy. Circular RNAs (circRNAs) play crucial regulatory roles in cancer. NGFR is a novel circRNA implicated in various types of cancers. The primary goal of this study was to elucidate the role of NGFR in GBC. Methods: NGFR variants exhibiting discernible discrepancies were identified using RNA sequencing and validated using real-time PCR. Cell proliferation was assessed using 5-ethynyl-2'-deoxyuridine and Cell Counting Kit-8 assays. The ferroptotic phenotype was characterized by assessing the reactive oxygen species and Fe2+ levels. Western blotting was used to analyze ferroptosis-associated proteins. Superoxide dismutase, malondialdehyde, and glutathione levels were measured using commercially available reagent kits. The severity of mitochondrial damage was evaluated by assessing JC-1, MitoSOX, and ATP activities. Results: NGFR was upregulated, and its suppression inhibited cell proliferation and increased Fe2+ levels in GBC cells. Furthermore, NGFR downregulation disrupted mitochondrial function. Conclusion: Circular RNA NGFR can impede the advancement of GBC by modulating the ferroptotic phenotype, thereby potentially offering a novel avenue for the clinical diagnosis and treatment strategies of GBC.

Effect of amisulpride on the expression of serotonin receptors, neurotrophic factor BDNF and its receptors in mice with overexpression of the aggregation-prone [R406W] mutant tau protein.

Serotonin 5-HT7 receptors (5-HT7R) are attracting increasing attention as important participants in the mechanisms of Alzheimer's disease and as a possible target for the treatment of various tau pathologies. In this study, we investigated the effects of amisulpride (5-HT7R inverse agonist) in C57BL/6J mice with experimentally induced expression of the gene encoding the aggregation-prone human Tau[R406W] protein in the prefrontal cortex. In these animals we examined short-term memory and the expression of genes involved in the development of tauopathy (Htr7 and Cdk5), as well as biomarkers of neurodegenerative processes - the Bdnf gene and its receptors TrkB (the Ntrk2 gene) and p75NTR (the Ngfr gene). In a short-term memory test, there was no difference in the discrimination index between mice treated with AAV-Tau[R406W] and mice treated with AAV-EGFP. Amisulpride did not affect this parameter. Administration of AAV-Tau[R406W] resulted in increased expression of the Htr7, Htr1a, and Cdk5 genes in the prefrontal cortex compared to AAV-EGFP animals. At the same time, amisulpride at the dose of 10 mg/kg in animals from the AAV-Tau[R406W] group caused a decrease in the Htr7, Htr1a genes mRNA levels compared to animals from the AAV-Tau[R406W] group treated with saline. A decrease in the expression of the Bdnf and Ntrk2 genes in the prefrontal cortex was revealed after administration of AAV-Tau[R406W]. Moreover, amisulpride at various doses (3 and 10 mg/kg) caused the same decrease in the transcription of these genes in mice without tauopathy. It is also interesting that in mice of the AAV-EGFP group, administration of amisulpride at the dose of 10 mg/kg increased the Ngfr gene mRNA level. The data obtained allow us to propose the use of amisulpride in restoring normal tau protein function. However, it should be noted that prolonged administration may result in adverse effects such as an increase in Ngfr expression and a decrease in Bdnf and Ntrk2 expression, which is probably indicative of an increase in neurodegenerative processes.

NGFR regulates stromal cell activation in germinal centers.

Nerve growth factor receptor (NGFR) is expressed by follicular dendritic cells (FDCs). However, the role of NGFR in the humoral response is not well defined. Here, we study the effect of Ngfr loss on lymph node organization and function, demonstrating that Ngfr depletion leads to spontaneous germinal center (GC) formation and an expansion of the GC B cell compartment. In accordance with this effect, stromal cells are altered in Ngfr-/- mice with a higher frequency of FDCs, characterized by CD21/35, MAdCAM-1, and VCAM-1 overexpression. GCs are located ectopically in Ngfr-/- mice, with lost polarization together with impaired high-affinity antibody production and an increase in circulating autoantibodies. We observe higher levels of autoantibodies in Bcl2 Tg/Ngfr-/- mice, concomitant with a higher incidence of autoimmunity and lower overall survival. Our work shows that NGFR is involved in maintaining GC structure and function, participating in GC activation, antibody production, and immune tolerance.

Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression.

Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vector production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFR protein. To eradicate the high background NGFR expression in future retroviral vector packaging cells, we here employed the CRISPR/Cas9 system to generate human suspension 293-F NGFR knockout cells. The expression of a fluorescent protein coupled via a 2A peptide motif to the NGFR targeting Cas9 endonuclease enabled the simultaneous depletion of cells expressing Cas9 and remaining NGFR-positive cells. Thus, a pure population of NGFR-negative 293-F cells lacking persistent Cas9 expression was obtained in a simple and easily applicable procedure.

Resveratrol Induces Autophagy and Apoptosis in Non-Small-Cell Lung Cancer Cells by Activating the NGFR-AMPK-mTOR Pathway.

Resveratrol (RSV) has been reported to induce autophagy and apoptosis in non-small-cell lung cancer A549 cells, and the nerve growth factor receptor (NGFR) regulates autophagy and apoptosis in many other cells. However, the effect of NGFR on autophagy and apoptosis induced by RSV in A549 cells remains unclear. Here, we found that RSV reduced the cell survival rate in time- and concentration-dependent manners, activating autophagy and apoptosis. Lethal autophagy was triggered by RSV higher than 55 µM. The relationship between autophagy and apoptosis depended on the type of autophagy. Specifically, mutual promotion was observed between apoptosis and lethal autophagy. Conversely, cytoprotective autophagy facilitated apoptosis but was unaffected by apoptosis. RSV enhanced NGFR by increasing mRNA expression and prolonging the lifespan of NGFR mRNA and proteins. RSV antagonized the enhanced autophagy and apoptosis caused by NGFR knockdown. As the downstream pathway of NGFR, AMPK-mTOR played a positive role in RSV-induced autophagy and apoptosis. Overall, RSV-induced autophagy and apoptosis in A549 cells are regulated by the NGFR-AMPK-mTOR signaling pathway.

LMO1 Plays an Oncogenic Role in Human Glioma Associated With NF-kB Pathway.

Background: LIM domain only protein1(LMO1), a nuclear transcription coregulator, is implicated in the pathogenesis of T-cell acute lymphoblastic leukemia and neuroblastoma. However, the clinical significance and potential mechanism of LMO1 in human gliomas remain to be determined. Methods: In this study, expression level data and clinical information were obtained via three databases. The Cox proportional hazards regression model was used to predict outcomes for glioma patients. In vitro and in vivo assays were used to explore the function of LMO1 in human glioma. Gene set enrichment analysis (GSEA), RNA-seq and western blot were used to explore the potential molecular mechanisms. A prognostic model was built for predicting the overall survival(OS) of human glioma patients. Results: High LMO1 expression was associated with a high tumor grade and a poor prognosis in patients. High levels of LMO1 mRNA were correlated with poor prognosis in patients with isocitrate dehydrogenase (IDH)-wild-type (wt) and 1p/19q non-codeletion gliomas. Gene silencing of LMO1 significantly inhibited tumor growth, invasion and migration in vitro. In contrast, LMO1 over-expression promoted tumor growth, invasion and migration. Mechanically, LMO1 may positively regulate the level of NGFR mRNA and protein. NGFR mediated the regulation between LMO1 and NF-kB activation. Consistently, the nude mice study further confirmed that knockdown of LMO1 blocked tumor growth via NGFR-NF-kB axis. Finally, The nomogram based on the LMO1 signature for overall survival (OS) prediction in human glioma patients exhibited good performance in the individual mortality risk. Conclusion: This study provides new insights and evidences that high level expression of LMO1 is significantly correlated with progression and prognosis in human gliomas. LMO1 played a critical role in tumorigenesis and progression. The present study first investigated the LMO1-NGFR-NF-kB axis regulate cell growth and invasion in human glioma cells, whereby targeting this pathway may be a therapeutic target for glioma.

Identification of Prognostic Genes in Gliomas Based on Increased Microenvironment Stiffness.

With a median survival time of 15 months, glioblastoma multiforme is one of the most aggressive primary brain cancers. The crucial roles played by the extracellular matrix (ECM) stiffness in glioma progression and treatment resistance have been reported in numerous studies. However, the association between ECM-stiffness-regulated genes and the prognosis of glioma patients remains to be explored. Thus, using bioinformatics analysis, we first identified 180 stiffness-dependent genes from an RNA-Seq dataset, and then evaluated their prognosis in The Cancer Genome Atlas (TCGA) glioma dataset. Our results showed that 11 stiffness-dependent genes common between low- and high-grade gliomas were prognostic. After validation using the Chinese Glioma Genome Atlas (CGGA) database, we further identified four stiffness-dependent prognostic genes: FN1, ITGA5, OSMR, and NGFR. In addition to high-grade glioma, overexpression of the four-gene signature also showed poor prognosis in low-grade glioma patients. Moreover, our analysis confirmed that the expression levels of stiffness-dependent prognostic genes in high-grade glioma were significantly higher than in low-grade glioma, suggesting that these genes were associated with glioma progression. Based on a pathophysiology-inspired approach, our findings illuminate the link between ECM stiffness and the prognosis of glioma patients and suggest a signature of four stiffness-dependent genes as potential therapeutic targets.

Optimized NGFR-derived hinges for rapid and efficient enrichment and detection of CAR T cells in vitro and in vivo.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated unprecedented success with high remission rates for heavily pretreated patients with hematological malignancies. The hinge connecting the extracellular antigen recognition unit to the transmembrane domain provides the length and flexibility of the CAR constructs and ensures that the CAR can reach the target antigen and mediate recognition and killing of target cells. The hinge can also include specific amino acid sequences to improve CAR expression, influence T cell proliferation, and facilitate CAR T cell detection, enrichment, and even elimination. Here, we report the generation of two novel hinge domains derived from the low-affinity p75 chain of the human nerve growth factor receptor (NGFR), termed N3 and N4, which, when incorporated into the CAR backbone, allow detection as well as high-grade enrichment of CAR T cells with GMP-compatible immunomagnetic reagents. After optimizing the MACS protocol for excellent CAR T cell purity and yield, we demonstrated that N3- and N4-hinged CAR T cells are as efficacious as their CD8-hinged counterparts in vitro against hematological blasts and also in vivo in the control of acute monocytic leukemia in an immunodeficient mouse xenograft model. Thus, both hinges could potentially be an integral part of future CAR designs and universally applicable in clinical applications.