Project description:Oncogenic PIK3CA mutations activate phosphoinositide 3-kinase (PI3K) and are among the commonest somatic mutations in cancer and mosaic, developmental overgrowth disorders. We recently demonstrated that the ‘hotspot’ variant PIK3CAH1047R exerts striking allele dose-dependent effects on stemness in human induced pluripotent stem cells (iPSCs), and moreover demonstrated multiple oncogenic PIK3CA copies in a substantial subset of human cancers. To identify the molecular mechanism underpinning PIK3CAH1047R allele dose-dependent stemness, we profiled isogenic wild-type, PIK3CAWT/H1047R and PIK3CAH1047R/H1047R iPSCs by high-depth transcriptomics, proteomics and reverse-phase protein arrays (RPPA). PIK3CAH1047R/H1047R iPSCs exhibited altered expression of 5644 genes and 248 proteins, whereas heterozygous hPSCs showed 492 and 54 differentially-expressed genes and proteins, respectively, confirming a nearly deterministic phenotypic effect of homozygosity for PIK3CAH1047R. Pathway and network-based analyses predicted a strong association between self-sustained TGFb/NODAL signaling and the ‘locked’ stemness phenotype induced by homozygosity for PIK3CAH1047R. This stemness gene signature was maintained without exogenous NODAL in PIK3CAH1047R/H1047R iPSCs and was reversed by pharmacological inhibition of TGFb/NODAL signaling but not by PIK3CA-specific inhibition. Analysis of PIK3CA-associated human breast cancers revealed increased expression of the stemness markers NODAL and POU5F1 as a function of disease stage and PIK3CAH1047R allele dosage. Together with emerging realization of the link between NODAL re-expression and aggressive cancer behavior, our data suggest that TGFb/NODAL inhibitors warrant testing in advanced breast tumors with multiple oncogenic PIK3CA copies.
Project description:TGFβ activates a signal transduction cascade that results in the microRNAs and genes transcription. The objective of this study is to identify miRNAs which are regulated through TGFB signaling pathway Cultured NHLF cells at 30-50% confluence were treated with 5 ng/mL TGFB1 or DMSO.
Project description:TGFβ activates a signal transduction cascade that results in the microRNAs and genes transcription. The objective of this study is to identify miRNAs which are regulated through TGFB signaling pathway
Project description:Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer that was specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this enhancer driving green fluorescent protein (TIE:EGFP). EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of mature melanomas. Single cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response, while up-regulating a novel set of chronic TGFb target genes. AP-1 factor binding is required for activation of this chronic TGFb reporter. Overexpression of the chromatin remodeler, SATB2, which is associated with tumor spreading shows activation of TGFb signaling in melanoma precursor zones and early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages are recruited to EGFP positive regions and preferentially phagocytose TIE:EGFP+ cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.
Project description:Epidermal lineages and injury induced regeneration are controlled by transcriptional programs coordinating cellular signaling and epigenetic regulators, but the mechanism remains unclear. Previous studies showed that conditional deletion of the transcriptional coactivator Mediator 1 (Med1) changes epidermal lineages and accelerates wound re-epithelialization. Here, we studied a molecular mechanism by which Med1 facilitates these processes, in particular, by focusing on TGFb signaling through genome wide transcriptome analysis. The expression of the TGF ligands (Tgfb1/b2) and their downstream target genes is decreased in both normal and wounded Med1 null skin. Med1 silencing in cultured keratinocytes likewise reduces the expression of the ligands (TGFb1/b2) and diminishes activity of TGFb signaling as shown by decreased p-Smad2/3. Silencing Med1 increases keratinocyte proliferation and migration in vitro. Epigenetic studies using chromatin immuno-precipitation and next generation DNA sequencing reveals that Med1 regulates transcription of TGFb components by forming large clusters of enhancers called super-enhancers at the regulatory regions of the TGFb ligand and SMAD3 genes. These results demonstrate that Med1 is required for the maintenance of the TGFb signaling pathway. Finally, we show that pharmacological inhibition of TGFb signaling enhances epidermal lineages and accelerates wound re-epithelialization in skin similar to that seen in the Med1 null mice, providing new insights into epidermal regeneration.
Project description:Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer that was specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this enhancer driving green fluorescent protein (TIE:EGFP). EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of mature melanomas. Single cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response, while up-regulating a novel set of chronic TGFb target genes. AP-1 factor binding is required for activation of this chronic TGFb reporter. Overexpression of the chromatin remodeler, SATB2, which is associated with tumor spreading shows activation of TGFb signaling in melanoma precursor zones and early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages are recruited to EGFP positive regions and preferentially phagocytose TIE:EGFP+ cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.
Project description:Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia progenitor-expressed integrin beta 8 activates microglia-expressed TGFB1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary domains of developmentally arrested “dysmature” microglia and homeostatic microglia that persist into adulthood. In the absence of autocrine TGFB1 signaling, we find that microglia adopt a similar reactive microglial phenotype, leading to astrogliosis and neuromotor symptoms almost identical to Itgb8 mutant mice. By comparing mice with genetic deletions in critical components downstream of Itgb8, we show that non-canonical (Smad-independent) signaling partially suppresses the dysmature microglia phenotype, associated neuromotor dysfunction and expression of disease-associated genes, providing compelling evidence for the adoption of microglial develomental signaling pathways in the context of injury or disease.
Project description:Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer that was specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this enhancer driving green fluorescent protein (TIE:EGFP). EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of mature melanomas. Single cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response, while up-regulating a novel set of chronic TGFb target genes. AP-1 factor binding is required for activation of this chronic TGFb reporter. Overexpression of the chromatin remodeler, SATB2, which is associated with tumor spreading shows activation of TGFb signaling in melanoma precursor zones and early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages are recruited to EGFP positive regions and preferentially phagocytose TIE:EGFP+ cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.
Project description:Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer that was specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this enhancer driving green fluorescent protein (TIE:EGFP). EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of mature melanomas. Single cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response, while up-regulating a novel set of chronic TGFb target genes. AP-1 factor binding is required for activation of this chronic TGFb reporter. Overexpression of the chromatin remodeler, SATB2, which is associated with tumor spreading shows activation of TGFb signaling in melanoma precursor zones and early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages are recruited to EGFP positive regions and preferentially phagocytose TIE:EGFP+ cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.