Citation
- Authors: Lee C. et al.
- Year: 2023
- Journal: Nat Commun 14 439
- Applications: in vitro / DNA / jetOPTIMUS
- Cell types:
- Name: 4T1
Description: Malignant neoplasms of the mouse mammary gland cells, also known as 4T1-A - Name: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T - Name: MET-1
Description: Human skin squamous carcinoma cell line.
Known as: MET1; SCCT
- Name: 4T1
Method
Plasmid vectors and small interfering RNAs (siRNAs) were transiently transfected into target cells using jetOPTIMUS transfection reagent (Polyplus, #117-01) and G-Fectin (Genolution, Seoul, Korea), respectively, following the manufacturer’s instructions.
CRISPR/Cas9 genome editing was used to delete parts of Nrg1 enhancer elements in Eo771 breast cancer cells using the lentivirus delivery system. Single guide RNAs (sgRNAs) targeting Rbpj, Ap1, and Gata4-binding motifs, as well as the whole Nrg1 enhancer region were designed using the Benchling online tool (Supplementary Table 6). sgRNA pairs were annealed and inserted into the LentiCRISPRv2 plasmid vector. To produce lentiviruses, LentiCRISPRv2 constructs were co-transfected with VSVG and Δ8.9 enveloping/packaging vectors into H293T cells, and the conditioned medium was collected.
The Nrg1 enhancer or promoter region was inserted into a pGL4.23 reporter vector (Promega, Madison, WI, USA) while Rbpj cDNA was inserted into pCMV6–Myc/Flag (Origene). Met1 cancer cells were transfected with control or the Nrg1 enhancer reporter together with RBPJ expressing vectors. In addition, the control reporter, Nrg1 enhancer, Nrg1 promoter, or both reporter vectors were transfected into Met1 or 4T1 breast cancer cells, which were then treated with LG or HG.
Abstract
Hyperglycemia is a risk factor for breast cancer-related morbidity and mortality. Hyperglycemia induces Neuregulin 1 (Nrg1) overexpression in breast cancer, which subsequently promotes tumor progression. However, molecular mechanisms underlying hyperglycemia-induced Nrg1 overexpression remain poorly understood. Here, we show that hyperglycemia causes active histone modifications at the Nrg1 enhancer, forming enhanceosome complexes where recombination signal binding protein for immunoglobulin kappa J region (RBPJ), E1A binding protein p300 (P300), and SET domain containing 1 A (SETD1A) are recruited to upregulate Nrg1 expression. Deletions in RBPJ-binding sites causes hyperglycemia-controlled Nrg1 levels to be downregulated, resulting in decreased tumor growth in vitro and in vivo. Mice with modest-temporary hyperglycemia, induced by low-dose short-exposure streptozotocin, display accelerated tumor growth and lapatinib resistance, whereas combining lapatinib with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S42 phenylglycine t-butyl ester (DAPT) ameliorates tumor growth under these modest hyperglycemic conditions by inhibiting NOTCH and EGFR superfamilies. NOTCH activity is correlated with NRG1 levels, and high NRG1 levels predicts poor outcomes, particularly in HER2-positive breast cancer patients. Our findings highlight the hyperglycemia-linked epigenetic modulation of NRG1 as a potential therapeutic strategy for treating breast cancer patients with diabetes.