Abstract
BackgroundThe aim of this study is to explore the molecular mechanism of the LIM protein Ajuba and the transcription factor SP1 in the pathogenesis and progression of PDAC. Ajuba is a newly defined transcriptional co-regulator and plays important role in various cancer development, while SP1 is a classic transcription factor, and is closely related with a variety of gene expression and cancer development including PDAC.MethodsThe expression of Ajuba and SP1 in PDAC tissues was detected by immunohistochemistry (IHC), and the correlation between expression level and clinical prognosis of Ajuba and SP1 was extensively analyzed using online tools. The interaction between Ajuba and SP1 was examined by co-immunoprecipitation (co-IP) and GST-pulldown assays. Stable cell lines were established via lentiviral infection, and was examined by qRT-PCR and western blot assays. The effects of Ajuba/SP1 on PDAC cell proliferation were examined using CCK8 and colony formation assays. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were employed to examine the transcription activity.ResultsThe expression level (protein and mRNA) of Ajuba and SP1 was elevated in PDAC tissues and was positively correlated; patients with high Ajuba and SP1 expression had a poor prognosis. Mechanistically, Ajuba binds to the C-terminus of SP1 and functions as a co-activator to enhance SP1 gene expression and promote cell proliferation; the promoter of Ajuba contains functional SP1 responsive elements and Ajuba itself is a target gene of SP1.ConclusionAjuba functions as a co-activator of SP1 to induce its target gene, and that Ajuba itself is a target genes of SP1. Ajuba/SP1 complex could form a feed forward loop to drive SP1 target gene transcription and promote cell proliferation of pancreatic cancer cells. Ajuba and SP1 might be biomarkers for PDAC diagnostics, prognosis and targets for new therapeutics.
Highlights
The aim of this study is to explore the molecular mechanism of the LIM protein Ajuba and the transcription factor SP1 in the pathogenesis and progression of Pancreatic ductal adenocarcinoma (PDAC)
Ajuba acts as an obligate co-repressor of Snail and recruits histone modifying enzymes protein arginine methyltransferase 5 (Prmt5) [10], histone deacetylase (Hdac) and zeste gene enhancer homolog 2 (Ezh2) to directly repress gene expression [11]; Ajuba can interact with multiple nuclear hormone receptors to either repress or activate gene expression [12,13,14]
Epidermal growth factor receptor (EGFR), Insulin-like growth factor 1 receptor (IGF1R), vascular endothelial growth factor (VEGF) and COX-2 are among the list of the best known SP1 target genes that are critical regulators of both normal development and malignancies [20,21,22,23,24]. We discover that both Ajuba and SP1 are highly expressed in PDAC tissue and their expression is positively correlated; mechanistically, Ajuba functions as a novel co-activator of SP1 to induce EGFR and IGF1R expression and to promote pancreatic cancer cell growth; Ajuba itself is a direct target gene of SP1
Summary
The aim of this study is to explore the molecular mechanism of the LIM protein Ajuba and the transcription factor SP1 in the pathogenesis and progression of PDAC. Ajuba belongs to the LIM protein family characterized by the C-terminal tandem LIM motifs, a double zinc finger in structure and a module to mediate protein-protein interaction [4] Ajuba contains both nuclear localization signal (NLS) and nuclear exporting signal (NES) and can shuttle between nucleus and cytoplasm. This property enables Ajuba a versatile scaffold participating in the assembly of numerous protein complexes to regulate a variety of cellular processes including cell adhesion, cell migration, cell proliferation, apoptosis, mitosis and gene transcription [4,5,6,7,8,9,10]. Ajuba acts as an obligate co-repressor of Snail and recruits histone modifying enzymes protein arginine methyltransferase 5 (Prmt5) [10], histone deacetylase (Hdac) and zeste gene enhancer homolog 2 (Ezh2) to directly repress gene expression [11]; Ajuba can interact with multiple nuclear hormone receptors to either repress or activate gene expression [12,13,14]
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