Hypoxia-induced CTCF promotes EMT in breast cancer

Description

Cancer cells experiencing hypoxic stress employ epithelial-mesenchymal transition (EMT) to undergo metastasis through rewiring of the chromatin landscape, epigenetics and, importantly, alternative splicing. Here, we showed that hypoxia modulates epigenetic landscape on CTCF promoter that upregulates its expression. Hypoxia driven epigenetic alteration, specifically DNA-demethylation mediated by TET2 is a prerequisite for CTCF induction. Mechanistically, in hypoxic conditions, HIF1α binds to the unmethylated CTCF promoter, causing transcriptional upregulation. Further, we uncover the pivotal role of CTCF in promoting EMT as loss of CTCF abrogated invasiveness of hypoxic breast cancer cells. These results validate the functional contribution of the HIF1α-CTCF axis in promoting EMT in hypoxic breast cancer cells. Lastly, CTCF expression is alleviated and the potential for EMT is diminished when the HIF1α binding is particularly disrupted through the dCas9-DNMT3A system mediated maintenance of DNA-methylation on the CTCF promoter. This may offer a unique therapeutic target in breast cancer.

Steps to reproduce

IHC: For IHC, slides were firstly fixed at 65°C for 2 hrs on the hot plate, deparaffinized and rehydrated. Subsequently, heat induced antigen retrieval was performed using 10 mM sodium citrate buffer (pH 6 in the laboratory microwave for 14 mins). Endogenous peroxidase was quenched with 1:10 dilution of 3% hydrogen peroxidase in methanol, followed by blocking with 3% bovine serum albumin (BSA). Primary antibodies against CAIX (1:50), and CTCF (1:200) were used. Further, HRP/DAB-chromogenic based Super Sensitive™* Polymer-HRP Detection System kit (BioGenex, Catalog no. QD430-XAKE) was used as per manufactures’ instructions. All slides were counterstained with Harris’ hematoxylin (Merck). The images were captured by Thermo Scientific™ Invitrogen™ EVOS™ FL Auto 2 Imaging System and at 10× and 40× magnifications. Images were then processed in Adobe Photoshop Version 7.0. Quantification was done using color deconvolution in ImageJ (Fiji) and mean gray values were then converted to optical density. Western blotting: Cells were washed with 1×PBS and protein was extracted by adding Urea lysis buffer (8 M urea, 2 M thiourea, 2% CHAPS, 1% DTT) supplemented with 1X Protease inhibitor (leupeptin 10μM, pepstatin 5 μM, EDTA 1mM, AEBSF 200 μM) and spun at 14,000 × g in 40C centrifuge. Protein concentration was determined and equal amounts of protein extract were separated by SDS/PAGE, electroblotted onto PVDF membranes, and were incubated with primary antibodies followed by secondary antibody. Protein bands were visualized using Odyssey Infrared imaging system (Licor). Quantification of the bands was done using ImageJ software.

Institutions

Categories

Funding

Licence