Gene expression data from human prostate cancer cell line PC3 and PAR1 and PAR-2-knockout PC3 cells generated using CRISPR/Cas9 targeting
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PROVIDER: PRJNA872167 | ENA |
REPOSITORIES: ENA
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Project description:We found that PC3 cells secrete proteases that cleavae and activate PAR1 and PAR2. In order to understand gene regulation by this autocrine signaling loop, PAR1 and PAR2 knockout cell gene expression was compared to WT PC3 cell gene expression. Proteinase activated receptors (PARs) are G protein-coupled receptors (GPCRs) activated by limited N-terminal proteolysis. A variety of proteolytic enzymes derived from the coagulation cascade and inflammatory milieu activate PARs, however specific activators in different physiological and pathophysiological contexts remain poorly defined. PARs are highly expressed in many cancer cells and regulate various aspects of tumor growth and metastasis. Endogenous proteinases that regulate PARs in the setting of various tumors however remains unresolved. Prostate cancer (PCa) remains a major cause of mortality in men despite advances in early detection and clinical intervention. PAR expression has been reported in PCa, however, their role here remains poorly defined. In androgen independent PC3 cells, we find functional expression of PAR1 and PAR2 but not PAR4. Using genetically encoded PAR cleavage biosensors, we find that PCa cells secrete proteolytic enzymes that cleave PARs and trigger autocrine signaling. Deletion of PAR1 and PAR2 using CRISPR/Cas9 combined with microarray analysis revealed genes that are differentially regulated by this autocrine signalling mechanism. Interestingly, several genes that are known PCa prognostic factors or biomarker were differentially expressed in PAR1-KO and PAR2-KO PC3 cells. We also examined PAR1 and PAR2 regulation of PCa cell proliferation and migration using PAR1 and PAR2-KO PC3 cells, as well as PAR1 and PAR2 specific agonists and antagonists. We find that PAR1 and PAR2 have opposite effects on PC3 cell proliferation and migration. In summary, we have identified an autocrine signaling mechanism through PARs as a regulator of PCa cell function.
2022-08-24 | GSE211813 | GEO
Project description:Background: Senescent hepatocytes accumulate in parallel with fibrosis progression during NASH. The mechanisms that enable progressive expansion of nonreplicating cell populations and the significance of that process in determining NASH outcomes are unclear. Many types of senescing cells upregulate the THBD-PAR-1 signaling axis to remain viable. Vorapaxar, an FDA-approved PAR-1 inhibitor, blocks the activity of that pathway. We used vorapaxar to determine if and how THBD-PAR1 signaling promotes fibrosis progression in NASH. Methods: We evaluated the THBD-PAR1 pathway in liver biopsies from NAFLD patient cohorts with a spectrum of liver fibrosis. Chow fed mice were treated with viral vectors to over-express p16 specifically in hepatocytes and induce replicative senescence. Effects on the THBD-PAR-1 axis and regenerative capacity were assessed; the transcriptome of p16 over-expressing hepatocytes was characterized and we examined how conditioned medium from senescent but viable (dubbed ‘undead’) hepatocytes reprograms hepatic stellate cells. A genetically obese mouse model of NASH with little liver fibrosis, and a diet-induced mouse model of NASH with advanced fibrosis were treated with vorapaxar to determine effects on hepatocyte senescence and liver damage. Results: Inducing senescence up-regulates the THBD-PAR1 signaling axis in hepatocytes and induces their expression of fibrogenic factors, including hedgehog ligands. Hepatocyte THBD-PAR1 signaling increases in NAFLD and supports sustained hepatocyte senescence that limits effective liver regeneration and promotes maladaptive repair. Inhibiting PAR-1 signaling with vorapaxar interrupts this process, reduces the burden of ‘undead’ senescent cells, and safely improves NASH and fibrosis despite ongoing lipotoxic stress Conclusion: The THBD-PAR1 signaling axis is a novel therapeutic target for NASH because blocking this pathway prevents accumulation of senescing but viable hepatocytes that generate factors that promote maladaptive liver repair.
2023-03-01 | GSE204901 | GEO
Project description:Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation via cleavage of protease-activated receptors (PAR)1 and PAR2. KLK6 levels are elevated in multiple inflammatory and autoimmune conditions, but no definitive role in pathogenesis has been established. Here, we show that skin-targeted overexpression of KLK6 causes generalized, severe psoriasiform dermatitis with spontaneous development of debilitating psoriatic arthritis-like joint disease. The psoriatic skin and joint phenotypes are reversed by normalization of skin KLK6 levels and attenuated following genetic elimination of PAR1 but not PAR2. Conservation of this regulatory pathway was confirmed in human psoriasis using vorapaxar, an FDA-approved PAR1 antagonist, on explanted lesional skin from psoriasis patients. Beyond defining a critical role for KLK6-PAR1 signaling in promoting psoriasis, our results demonstrate that KLK6-PAR1-mediated inflammation in the skin alone is sufficient to drive inflammatory joint disease. Further, we identify PAR1 as a promising cytokine-independent target in therapy of psoriasis and psoriatic arthritis.
2020-03-25 | GSE144312 | GEO
Project description:Transcription profiling of wild type and transgenic 35S:PAR1-GG seedlings. To characterize the transcriptional networks altered by constitutive overexpression of PAR1 in seedlings, using two-color long-oligonucleotide microarrays.
2007-11-03 | E-ATMX-29 | biostudies-arrayexpress
Project description:Label-free proteomics comparison of PC3 cells with versus without CRISPR/Cas9 knockout of RIPK2. The goal was to identify proteins downstream of RIPK2 in prostate cancer cells.
2022-02-09 | PXD018890 | Pride
Project description:shRNA against PAR1 and control haripin sh004 transduced PANC-1(Pancreatic cancer) cell line
2021-05-14 | GSE155010 | GEO
Project description:Objective: Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of protease-activated receptors (PARs). The predominant thrombin receptor is PAR1 and in endothelial cells (ECs) thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear if thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N-terminus have remained an unresolved issue.Approach and Results: Here, we used a quantitative phosphoproteomics approach to show that ‘classical’ and ‘peptide’ activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs.Conclusions: Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrates that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-TL peptide induce similar phosphoregulation and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology.
2017-08-22 | PXD004264 | Pride
Project description:BACKGROUND: Cancer stem-like cells are proposed to sustain solid tumors by virtue of their capacity for self-renewal and differentiation to cells that comprise the bulk of the tumor, and have been identified for a variety of cancers based on characteristic clonal morphologies and patterns of marker gene expression. METHODS: Single cell cloning and spheroid culture studies were used to identify a population of cancer stem-like cells in the androgen-independent human prostate cancer cell line PC3. RESULTS: We demonstrate that, under standard culture conditions, ~10% of PC3 cells form holoclones with cancer stem cell characteristics. These holoclones display high self-renewal capability in spheroid formation assays under low attachment and serum-free culture conditions, retain their holoclone morphology when passaged at high cell density, exhibit moderate drug resistance, and show high tumorigenicity in scid immunodeficient mice. PC3 holoclones readily form spheres, and PC3-derived spheres yield a high percentage of holoclones, further supporting their cancer stem cell-like nature. We identified one gene, FAM65B, whose expression is consistently up regulated in PC3 holoclones compared to paraclones, the major cell morphology in the parental PC3 cell population, and two genes, MFI2 and LEF1, that are consistently down regulated. This molecular profile, FAM65Bhigh/MFI2low/LEF1low, also characterizes spheres generated from parental PC3 cells. The PC3 holoclones did not show significant enriched expression of the putative prostate cancer stem cell markers CD44 and integrin α2β1. PC3 tumors seeded with holoclones showed dramatic down regulation of FAM65B and dramatic up regulation of MFI2 and LEF1, and unexpectedly, a marked increase in tumor vascularity compared to parental PC3 tumors, suggesting a role of cancer stem cells in tumor angiogenesis. CONCLUSIONS: These findings support the proposal that PC3 tumors are sustained by a small number of tumor-initiating cells with stem-like characteristics, including strong self-renewal and pro-angiogenic capability and marked by the expression pattern FAM65Bhigh/MFI2low/LEF1low. These markers may serve as targets for therapies designed to eliminate cancer stem cell populations associated with aggressive, androgen-independent prostate tumors such as PC3. (Mol Cancer. 2010 Dec 29;9:319. doi: 10.1186/1476-4598-9-319; PMID: 21190562; PMCID: PMC3024252).
2013-11-07 | GSE52161 | GEO
Project description:Sequence Confirmation and Recombination Evidence of a Human PAR1 Length Polymorphism
| PRJEB13480 | ENA
Project description:APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine to uracil deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3s can trigger the cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through the homology-directed repair (HDR) of Cas9-generated double-strand breaks . In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks can be further processed to yield mutations mainly involving insertions or deletions (indels). Mechanistically, both APOBEC3-mediated deamination and DNA repair proteins play important roles in the generation of these indels. Correspondingly, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can substantially repress these unwanted mutations in genomic DNA.
2017-11-10 | GSE105146 | GEO