Gene expression profiles of FGF-treated prostate cancer cells
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ABSTRACT: Fibroblast growth factor 23 (FGF23) is a member of endocrine FGF subfamily, which includes FGF19, FGF21 and FGF23. We explored the question of whether different FGF ligands are activating the same transcriptional program in prostate cancer (PCa) cells or whether there are unique or partially overlapping activities as well.
Project description:Administration of FGF19 - a physiological regulator of bile acid homeostasis - to diabetic and diet-induced-obese mice can suppress plasma glucose concentration and improve insulin sensitivity. Repurposing FGF19 as a therapeutic agent for treating type 2 diabetes and cholestatic liver disease is therefore of significant interest. However, the tumorigenic risk associated with prolonged FGF19 administration is a major hurdle in realizing its full clinical potential. Here we show that non-mitogenic FGF19 variants that retain full beneficial glucose-lowering activity of wild-type FGF19 (FGF19WT) can be engineered by diminishing FGF19’s ability to induce dimerization of its cognate FGF receptors (FGFR). As proof-of-principle, we generated three such mutants, each with a partial defect in binding affinity to FGFR (FGF19ΔFGFR), and its co-receptors klotho (FGF19ΔKLB) or heparan sulfate (FGF19ΔHBS). Pharmacological assays in healthy and db/db mice confirmed that these variants incur a dramatic loss in mitogenic activity, yet are indistinguishable from FGF19WT in eliciting glycemic control. Our approach provides a simple framework for the development of safer and efficacious FGF19 analogs.
Project description:FGF21 belongs to the FGF superfamily and the highest expression of the FGF21 transcript was found in the liver. The initial studies identified FGF21 as an endocrine hepatokine with crucial roles in regulating lipid, glucose and energy metabolism. More recent studies have indicated a role for FGF21 in cardiovascular stress and diseases.The goal of our study is to analyze the effect of FGF21 on ischemic cardiomyocytes transcriptome using microarray. Total RNA was extracted from cultured neonatal mice cardiomyocytes after exposure to hypoxia (in 2% oxygen, 5% CO2 and 93% N2) for 12 h in the absence or presence of FGF21. Samples were processed for hybridization to the Mouse Gene 2.0 ST Array (Affymetrix). We sought to find out the differentially expressed genes regulated by FGF21 in hypoxic cardiomyocytes.
Project description:FGF21 belongs to the FGF superfamily and the highest expression of the FGF21 transcript was found in the liver. The initial studies identified FGF21 as an endocrine hepatokine with crucial roles in regulating lipid, glucose and energy metabolism. More recent studies have indicated a role for FGF21 in cardiovascular stress and diseases.The goal of our study is to analyze the effect of FGF21 on ischemic cardiomyocytes transcriptome using microarray.
Project description:Cell signaling molecules are essential drivers of gene expression patterns, which are crucial for the development of multicellular organisms. Although prior research has established the importance of the levels and distribution of these molecules for gene expression patterns during embryonic development, a complete understanding of the specific targets influenced by their activity remains elusive. This study investigates how the fibroblast growth factor (FGF), a key developmental signal, orchestrates gene expression during the organogenesis of the zebrafish lateral line. Our analysis provides a detailed catalog of genes mediated by FGF signaling and identified dose-dependent genes that consistently respond to varying levels of extracellular FGF signaling in vivo. Notably, we uncovered an unexpected group of target genes that show suppressed expression at increased levels of FGF ligand, independent of changes in extracellular signaling. Experiments utilizing mosaic mis-expression of FGF ligands demonstrate that this gene regulation occurs autonomously within individual cells. In embryos with endogenous FGF levels, these target genes are suppressed in FGF-producing cells that shows nuclear accumulation of the FGF ligand. Targeted degradation of nuclear FGF confirms its role in this suppression, while not interfering with the activity of extracellular FGF signaling in the neighboring cells. Thus, we propose a novel mechanism of gene expression patterning in which the FGF ligand is directed to the nucleus within source cells. This process autonomously regulates its signaling targets, which are induced by extracellular FGF, leading to a self-organized symmetry-breaking in FGF signaling targets. Our findings of consistent gene expression patterns across multiple tissues, together with the nuclear localization of other paracrine FGF ligands, indicate that the cell-autonomous gene regulation by nuclear-targeted ligands could be a more widespread mechanism.
Project description:We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts regulating Fgf23 production and mineralization in Hyp mice. We discovered that activation of Fgf receptor- and Wnt-pathways contribute to increased Ffg23 gene transcription in Hyp bone. We found evidence in Hyp bone for increased expression of Fgf1, Fgf7, and Egr2 in the Fgf-signaling pathway and decrements in Sost and Cpz and increments in Sfrp1 and 4 in the Wnt-signaling pathway. Moreover, activation of Fgf and Wnt-signaling stimulated, whereas Tgf β inhibited Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the Fgf23 regulatory extracellular matrix protein Dmp1. These findings suggest that elevation of Fgf23 expression in osteocytes is regulated by interactions between cell surface expression of Phex, extracellular matrix proteins and paracrine effects of Fgf and Wnt. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced post-translational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regards to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone. We isolated total RNAs from long bones of both WT and Hyp mice at 12 days of age. Since the RNA yields from the long bones are very low, we combined 2 bone samples with same genotype (WT or Hyp) for one RNA extraction. We will compare the difference of the gene expressions between Hyp and WT. We will use 4 samples in each animal condition.
Project description:Purpose. Despite having excellent prognosis when detected early, colorectal cancer (CRC) remains a leading cause of cancer-related deaths globally. Barriers to screening reduces compliance and negatively impacts patient outcomes, necessitating alternatives. A blood-based approach provides a more convenient and accessible modality, but serum markers are lacking. Using a meta-transcriptomics approach, we identified Fibroblast Growth Factor 19 (FGF19), an enteroendocrine FGF responsible for bile acid (BA) homeostasis, as an attractive CRC marker. However, whether FGF19 is aberrantly secreted into blood by colorectal tumors or induces endocrine-like paraneoplastic effects is unknown. Methods. To test if colorectal tumors contribute FGF19 into blood, subcutaneous xenografts of HCT116 and Colo201 cells were established in four male and female NSG mice. Tumor volume, as well as serum and urine FGF19 were assessed over 36 days. Following euthanasia, murine liver was processed for downstream mRNA and bile acid quantification. To determine global paraneoplastic effects of malignant FGF19 on its primary target organ (liver), hepatic mRNA was isolated and subjected to RNA sequencing on an Illumina NovaSeq 6000 system. Reads were processed, aligned, mapped, and analyzed for differential expression and functional enrichment using an integrated informatics pipeline implemented in R. Results. Circulating FGF19 levels derived from subcutaneous xenografts of Colo201, but not HCT116, cells exerted paraneoplastic effects on liver including suppression of BA synthesis, dysregulation of cholesterol metabolism, and induction of pre-neoplasia. Conclusion. Here we report novel paraneoplastic effects of tumor derived FGF19 on liver tissue. Study limitations include in vivo experimentation using two different cell lines, and significant gender-related batch effects in RNA sequencing data.
Project description:Single cell technologies hold promise for resolving complex early developmental phenotypes. Here we define a novel Hedgehog (Hh)-Fibroblast Growth Factor (FGF) signaling axis for the formation of anterior mesoderm lineages during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations—culminating in defects to anterior embryonic structures including the pharyngeal arches, heart, and anterior somites. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and FGF signaling for mesoderm migration. Mesoderm-specific Fgf4/Fgf8 double mutants recapitulated anterior mesoderm defects and Hh-dependent GLI transcription factors modulated enhancers at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic node and executed by FGF signals in nascent mesoderm to control anterior mesoderm patterning.
Project description:We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts regulating Fgf23 production and mineralization in Hyp mice. We discovered that activation of Fgf receptor- and Wnt-pathways contribute to increased Ffg23 gene transcription in Hyp bone. We found evidence in Hyp bone for increased expression of Fgf1, Fgf7, and Egr2 in the Fgf-signaling pathway and decrements in Sost and Cpz and increments in Sfrp1 and 4 in the Wnt-signaling pathway. Moreover, activation of Fgf and Wnt-signaling stimulated, whereas Tgf β inhibited Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the Fgf23 regulatory extracellular matrix protein Dmp1. These findings suggest that elevation of Fgf23 expression in osteocytes is regulated by interactions between cell surface expression of Phex, extracellular matrix proteins and paracrine effects of Fgf and Wnt. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced post-translational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regards to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone.
Project description:The mesodermal precursor populations for different internal organ systems are specified during gastrulation by the combined activity of extracellular signaling systems such as BMP, Wnt, Nodal, and FGF. The BMP, Wnt and Nodal signaling requirements for the differentiation of specific mesoderm subtypes in mammals have been mapped in detail, but which mesodermal cell types depend on FGF signaling is not precisely known. It is also not clear how FGF signaling modulates the activity of orthogonal signaling systems involved in mesoderm differentiation. Here, we address these questions by analyzing the effects of targeted signaling manipulations in differentiating stem cell populations with single cell resolution. We identify opposing functions of BMP and FGF, and map the boundary between FGF-dependent and -independent mesodermal lineages. Stimulation with exogenous FGF boosts the expression of endogenous Fgfs while repressing Bmp ligands. This intercellular positive autoregulation of FGF signaling coupled to the repression of BMP signaling may contribute to the specification of reproducible and coherent cohorts of cells with the same identity via a community effect, both in the embryo and in synthetic embryo-like systems.