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Supplementary Materials and Methods from Phospholipase D1 Acts through Akt/TopBP1 and RB1 to Regulate the E2F1-Dependent Apoptotic Program in Cancer Cells
journal contribution
posted on 2023-03-31, 00:30 authored by Dong Woo Kang, Shin Wha Lee, Won Chan Hwang, Bo Hui Lee, Yong-Seok Choi, Young-Ah Suh, Kang-Yell Choi, Do Sik Min1. Plasmids, shRNAs and miRNA. 2. Promoter reporter constructs. 3. 3Â'UTR reporter constructs and site-directed mutagenesis. 4. Viral production and infection. 5. Reagents 6. Transient transfection and reporter gene assay. 7. Real-time quantitative PCR. 8. Quantification of mature miRNA. 9. ChIP assay. 10. mRNA microarray analysis. 11. EpiTect Methyl q-PCR Assay. 12. Mice genotype. 13. PLD activity assay. 14. In vitro limiting dilution assays (LDAs). 15. Additive statistical analysis.
Funding
National Research Foundation of Korea
Translational Research Center for Protein Function Control
Ministry for Health, Welfare, and Family Affairs
Korean Health Technology R&D Project
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ARTICLE ABSTRACT
The RB1/E2F1 signaling pathway is frequently deregulated in colorectal cancer and has been suggested to intersect with Wnt/β-catenin and PI3K/Akt pathways, but molecular evidence for this link is lacking. In this study, we demonstrate that phospholipase D1 (PLD1), a transcriptional target of β-catenin/TCF4, orchestrates functional interactions between these pathways during intestinal tumor development. Overexpression of PLD1 in intestinal epithelial cells protected cells from apoptosis induced by PLD1 ablation in the Apcmin/+ mouse model of intestinal tumorigenesis. Mechanistic investigations revealed that genetic and pharmacologic targeting of PLD1 promote the E2F1-dependent apoptotic program via both miR-192/4465–mediated downregulation of RB1 and inhibition of Akt–TopBP1 pathways. Moreover, the miRNA–RB1 axis and Akt pathway also contributed to the PLD1-mediated self-renewal capacity of colon cancer–initiating cells. Finally, PLD1-driven E2F1 target gene expression positively correlated with tumor stage in patients with colorectal cancer. Overall, our findings suggest that PLD1 mediates cross-talk between multiple major signaling pathways to promote the survival and malignancy of colon cancer cells and may therefore represent an ideal signaling node for therapeutic targeting. Cancer Res; 77(1); 142–52. ©2016 AACR.Usage metrics
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Keywords
Cancer PreventionAnimal models of preventionCell CycleSignal transduction pathwaysCell Death And SenescenceTranscriptional control of apoptosisDrug MechanismsCellular responses to anticancer drugsDrug ResistanceRegulation of gene expression in drug resistanceGastrointestinal CancersColorectal cancerGene RegulationPosttranscriptional and translational controlOncogenes & Tumor SuppressorsTumor suppressor genesPreclinical ModelsAnimal models of cancer
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