Project description:The circadian clock regulates metabolism and other critical cellular functions. We used CRISPR-Cas9 to knockout the core clock gene Arntl in KPC cells
Project description:To gain mechanistic insight into how Setd2 loss promotes activation of AKT signaling, we performed CUT&Tag sequencing in PDAC cells (KPC1199) with Setd2-KO and Setd2-WT). Setd2-WT and -KO murine PDAC cells were cultured and freshly collected as soon as possible. Hyperactive pA-Tn5 Transposase for CUT&Tag kit from Vazyme (TD901), and antibodies against H3K36me3 and H3K27me3 were employed. Trueprep index kit v2 and v3 for illumina were used to establish DNA library.
Project description:TGFbeta promotes the bypass of KRAS* dependency in PDAC. To dissect the molecular mechanisms that regulated by TGFbeta in PDAC cells, we conducted RNA-seq analysis of iKPC PDAC cells with or without TGFbeta treatment.
Project description:Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.
Project description:WNT signaling promotes pancreatic ductal adenocarcinoma (PDAC) through diverse effects on proliferation, differentiation, survival, and stemness. A subset of PDAC with inactivating mutations in ring finger protein 43 (RNF43) have growth dependency on autocrine WNT ligand signaling, which renders them susceptible to porcupine inhibitors (PORCNi) that block WNT ligand acylation and secretion. For this study, non-targeted metabolomic analyses were performed to explore the therapeutic response of RNF43-mutant PDAC to the PORCNi LGK974. AsPC-1 (RNF43-mutant) PDAC cells were treated with 25 nM LGK974 to explore stable isotope-resolved metabolomics with uniform 1, D-glucose [U13-C6] labeling.
Project description:Pancreatic ductal adenocarcinoma is aggressive disease with a dismal five-year survival of 5%. Gene expression profiling has been instrumental for subtype classification in cancer, highlighting fundamental differences in tumors at the molecular level. Over the last years, multiple genomics studies have led to the classification of PDAC into two major subtypes: classical and basal-type. The classical subtype expresses higher levels of endodermal lineage specifiers, including HNF4A, GATA6, FOXA2, FOXA3 than the basal-type. The basal-type confers a worse prognosis, raising the possibility that loss of these lineage specifiers might enhance the malignant potential of PDAC. We found that the lineage specifier HNF4a plays a key role in maintaining a transcriptional network that characterizes the classical subtype, restraining growth in different PDAC models. Additionally, we demonstrated that HNF4a controls PDAC cell identity and proliferation, and represses the expression of SIX family members, two mesodermal lineage specifiers highly expressed in basal-type.
Project description:To gain mechanistic insight into how Setd2 loss reprogramming tumor cell metabolism, we performed CUT&Tag sequencing in PDAC cells with Setd2-KO and Setd2-WT sorted from orthotopic PDAC tumor. Setd2-WT and -KO murine PDAC cells were sorted with DAPI-CD45.2-Pdpn-Epcam+ or cultured, and freshly collected as soon as possible. Hyperactive pA-Tn5 Transposase for CUT&Tag kit from Vazyme (TD901), and antibodies against H3K36me3, H3K27Ac and H3K27me3 were employed. Trueprep index kit v2 and v3 for illumina were used to establish DNA library.
Project description:Lysine (K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in pancreatic ductal adenocarcinoma (PDAC). However, how KDM6A loss impacts PDAC tumor immune microenvironment is not known. Tumor-associated neutrophils (TANs) and neutrophil extracellular traps (NETs) in the tumor microenvironment contribute to PDAC progression. This study used genetically engineered pancreas-specific Kdm6a-knockout PDAC mouse model and human PDAC tissue samples to demonstrate that KDM6A loss correlates with increased TANs and NETs formation. Genome-wide Bru-seq analysis showed that the expression of many chemotactic cytokines, especially CXC motif chemokine ligand 1 (CXCL1), were upregulated in KDM6A-knockout PDAC cells. We confirmed that KDM6A-deficient PDAC cells secreted higher levels of CXCL1 protein, which in turn recruits neutrophils. Furthermore, the CXCL1 neutralizing antibody blocked the chemotactic and NETs-promoting property of KDM6A-deficient PDAC cells and tumor growth in a syngeneic orthotopic xenograft mouse model, confirming that CXCL1 was the main mediator of chemotaxis and PDAC growth in this model. These findings shed light on how KDM6A regulates tumor immune microenvironment and PDAC progression and suggest that the CXCL1-CXCR2 axis may be a candidate target for treating PDACs with KDM6A loss.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Currently, there is no effective therapy to treat PDAC, and thus detailed molecular and functional evaluation of PDAC is needed to identify and develop better therapeutic strategies. Here we show that the transcription factor Kruppel-like factor 7 (KLF7) is overexpressed in PDAC and that inhibition of KLF7 blocks PDAC tumor growth and metastasis in cell culture and in mice. KLF7 expression in PDAC can be upregulated due to activation of a MAP kinase pathway or inactivation of the tumor suppressor p53, two alterations that occur in a large majority of PDACs. ShRNA-mediated knockdown of KLF7 inhibits the expression of interferon-stimulated genes (ISGs), which are necessary for KLF7-mediated PDAC tumor growth and metastasis. KLF7 knockdown also results in the downregulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), resulting in Golgi complex fragmentation, and reduced protein glycosylation leading to reduced secretion of cancer-promoting growth factors such as chemokines. Genetic or pharmacological activation of Golgi complex fragmentation blocks PDAC growth and metastasis similar to KLF7 inhibition. Our results demonstrate a therapeutically amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex integrity.