Browsing by Subject "RETINOIC ACID"
Now showing items 1-8 of 8
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(2015)Oncolytic viruses that selectively replicate in tumor cells can be used for treatment of cancer. Accumulating data suggests that virus induced oncolysis can enhance anti-tumor immunity and break immune tolerance. To capitalize on the immunogenic nature of oncolysis, we generated a quadruple modified oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (GMCSF). Ad5/3-E2F-Delta 24-GMCSF (CGTG-602) was engineered to contain a tumor specific E2F1 promoter driving an E1 gene deleted at the retinoblastoma protein binding site ("Delta 24"). The fiber features a knob from serotype 3 for enhanced gene delivery to tumor cells. The virus was tested preclinically in vitro and in vivo and then 13 patients with solid tumors refractory to standard therapies were treated. Treatments were well tolerated and frequent tumor-and adenovirus-specific T-cell immune responses were seen. Overall, with regard to tumor marker or radiological responses, signs of antitumor efficacy were seen in 9/12 evaluable patients (75%). The radiological disease control rate with positron emission tomography was 83% while the response rate (including minor responses) was 50%. Tumor biopsies indicated accumulation of immunological cells, especially T-cells, to tumors after treatment. RNA expression analyses of tumors indicated immunological activation and metabolic changes secondary to virus replication.
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(2022)Simple Summary We investigated the increased prevalence of Barrett's esophagus in adults with esophageal atresia. A higher polygenic risk score and disturbances in inflammatory, stress response and oncological pathways upon acid exposure suggest a genetic susceptibility and increased induction of inflammatory processes. Although further research is required to explore this hypothesis, this could be a first-step into selecting patients that are more at risk to develop Barrett's esophagus and/or esophageal carcinoma. Currently, an endoscopic screening and surveillance program is in practice in our institution for patients born with esophageal atresia, to early detect (pre)malignant lesions. Since recurrent endoscopies can be a burden for the patient, selecting patients by for example genetic susceptibility would allow to only include those at risk in future practice. The prevalence of Barrett's esophagus (BE) in adults born with esophageal atresia (EA) is four times higher than in the general population and presents at a younger age (34 vs. 60 years). This is (partly) a consequence of chronic gastroesophageal reflux. Given the overlap between genes and pathways involved in foregut and BE development, we hypothesized that EA patients have an intrinsic predisposition to develop BE. Transcriptomes of Esophageal biopsies of EA patients with BE (n = 19, EA/BE); EA patients without BE (n = 44, EA-only) and BE patients without EA (n = 10, BE-only) were compared by RNA expression profiling. Subsequently, we simulated a reflux episode by exposing fibroblasts of 3 EA patients and 3 controls to acidic conditions. Transcriptome responses were compared to the differential expressed transcripts in the biopsies. Predisposing single nucleotide polymorphisms, associated with BE, were slightly increased in EA/BE versus BE-only patients. RNA expression profiling and pathway enrichment analysis revealed differences in retinoic acid metabolism and downstream signaling pathways and inflammatory, stress response and oncological processes. There was a similar effect on retinoic acid signaling and immune response in EA patients upon acid exposure. These results indicate that epithelial tissue homeostasis in EA patients is more prone to acidic disturbances.
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(2019)Neural crest cells have broad migratory and differentiative ability that differs according to their axial level of origin. However, their transient nature has limited understanding of their stem cell and self-renewal properties. While an in vitro culture method has made it possible to maintain cranial neural crest cells as self-renewing multipotent crestospheres (Kerosuo et al., 2015), these same conditions failed to preserve trunk neural crest in a stem-like state. Here we optimize culture conditions for maintenance of avian trunk crestospheres, comprised of both neural crest stem and progenitor cells. Our trunk-derived crestospheres are multipotent and display self-renewal capacity over several weeks. Trunk crestospheres display elevated expression of neural crest cell markers as compared to those characteristic of ventrolateral neural tube or mesodermal fates. Moreover, trunk crestospheres express increased levels of trunk neural crest-enriched markers as compared to cranial crestospheres. Finally, we use lentiviral transduction as a tool to manipulate gene expression in trunk crestospheres. Taken together, this method enables long-term in vitro maintenance and manipulation of multipotent trunk neural crest cells in a premigratory stem or early progenitor state. Trunk crestospheres are a valuable resource for probing mechanisms underlying neural crest sternness and lineage decisions as well as accompanying diseases.
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(2022)Aberrant epithelial differentiation and regeneration contribute to colon pathologies, including inflammatory bowel disease (iBD) and colitis-associated cancer (CAC). Myeloid translocation gene 16 (MTG16, also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear. Here, we identified MTG16 as a central mediator of epithelial differentiation, promoting goblet and restraining enteroendocrine cell development in homeostasis and enabling regeneration following dextran sulfate sodium-induced (DSS-induced) colitis. Transcriptomic analyses implicated increased Ephrussi box-binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a mouse model with a point mutation that attenuates MTG16:E protein interactions (Mtg16(P20ST)), we showed that MTG16 exerts control over colonic epithelial differentiation and regeneration by repressing E protein-mediated transcription. Mimicking murine colitis, MTG16 expression was increased in biopsies from patients with active IBD compared with unaffected controls. Finally, uncoupling MTG16:E protein interactions partially phenocopied the enhanced tumorigenicity of Mtg16(-/)(-) colon in the azoxymethane/DSS-induced model of CAC, indicating that MTG16 protects from tumorigenesis through additional mechanisms. Collectively, our results demonstrate that MTG16, via its repression of E protein targets. is a key regulator of cell fate decisions during colon homeostasis, colitis, and cancer.
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Orphan G protein-coupled receptor GPRC5A modulates integrin beta 1-mediated epithelial cell adhesion (2017)G-Protein Coupled Receptor (GPCR), Class C, Group 5, Member A (GPRC5A) has been implicated in several malignancies. The underlying mechanisms, however, remain poorly understood. Using a panel of human cell lines, we demonstrate that CRISPR/Cas9-mediated knockout and RNAi-mediated depletion of GPRC5A impairs cell adhesion to integrin substrates: collagens I and IV, fibronectin, as well as to extracellular matrix proteins derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Matrigel). Consistent with the phenotype, knock-out of GPRC5A correlated with a reduced integrin beta 1 (ITGB1) protein expression, impaired phosphorylation of the focal adhesion kinase (FAK), and lower activity of small GTPases RhoA and Rac1. Furthermore, we provide the first evidence for a direct interaction between GPRC5A and a receptor tyrosine kinase EphA2, an upstream regulator of FAK, although its contribution to the observed adhesion phenotype is unclear. Our findings reveal an unprecedented role for GPRC5A in regulation of the ITGB1-mediated cell adhesion and it's downstream signaling, thus indicating a potential novel role for GPRC5A in human epithelial cancers.
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(2020)The mammalian precerebellar pontine nucleus (PN) has a main role in relaying cortical information to the cerebellum. The molecular determinants establishing ordered connectivity patterns between cortical afferents and precerebellar neurons are largely unknown. We show that expression of Hox5 transcription factors is induced in specific subsets of postmitotic PN neurons at migration onset. Hox5 induction is achieved by response to retinoic acid signaling, resulting in Jmjd3-dependent derepression of Polycomb chromatin and 3D conformational changes. Hoxa5 drives neurons to settle posteriorly in the PN, where they are monosynaptically targeted by cortical neuron subsets mainly carrying limb somatosensation. Furthermore, Hoxa5 postmigratory ectopic expression in PN neurons is sufficient to attract cortical somatosensory inputs regardless of position and avoid visual afferents. Transcriptome analysis further suggests that Hoxa5 is involved in circuit formation. Thus, Hoxa5 coordinates postmitotic specification, migration, settling position, and subcircuit assembly of PN neuron subsets in the cortico-cerebellar pathway.
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(2019)A prerequisite for lifelong sperm production is that spermatogonial stem cells (SSCs) balance self-renewal and differentiation, yet factors required for this balance remain largely undefined. Using mouse genetics, we now demonstrate that the ubiquitously expressed transcription factor upstream stimulatory factor (USF)1 is critical for the maintenance of SSCs. We show that USF1 is not only detected in Sertoli cells as previously reported, but also in SSCs. Usf1-deficient mice display progressive spermatogenic decline as a result of age-dependent loss of SSCs. According to our data, the germ cell defect in Usf1(-/-) mice cannot be attributed to impairment of Sertoli cell development, maturation, or function, but instead is likely due to an inability of SSCs to maintain a quiescent state. SSCs of Usf1(-/-) mice undergo continuous proliferation, which provides an explanation for their age-dependent depletion. The proliferation-coupled exhaustion of SSCs in turn results in progressive degeneration of the seminiferous epithelium, gradual decrease in sperm production, and testicular atrophy. We conclude that the general transcription factor USF1 is indispensable for the proper maintenance of mammalian spermatogenesis.
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(2017)Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.
