Epsins 1 and 2 promote NEMO linear ubiquitination via LUBAC to drive breast cancer development
Kai Song, … , Yibin Kang, Hong Chen
Kai Song, … , Yibin Kang, Hong Chen
Published September 22, 2020
Citation Information: J Clin Invest. 2021;131(1):e129374. https://doi.org/10.1172/JCI129374.
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Research Article Cell biology Oncology

Epsins 1 and 2 promote NEMO linear ubiquitination via LUBAC to drive breast cancer development

Abstract

Estrogen receptor–negative (ER-negative) breast cancer is thought to be more malignant and devastating than ER-positive breast cancer. ER-negative breast cancer exhibits elevated NF-κB activity, but how this abnormally high NF-κB activity is maintained is poorly understood. The importance of linear ubiquitination, which is generated by the linear ubiquitin chain assembly complex (LUBAC), is increasingly appreciated in NF-κB signaling, which regulates cell activation and death. Here, we showed that epsin proteins, a family of ubiquitin-binding endocytic adaptors, interacted with LUBAC via its ubiquitin-interacting motif and bound LUBAC’s bona fide substrate NEMO via its N-terminal homolog (ENTH) domain. Furthermore, epsins promoted NF-κB essential modulator (NEMO) linear ubiquitination and served as scaffolds for recruiting other components of the IκB kinase (IKK) complex, resulting in the heightened IKK activation and sustained NF-κB signaling essential for the development of ER-negative breast cancer. Heightened epsin levels in ER-negative human breast cancer are associated with poor relapse-free survival. We showed that transgenic and pharmacological approaches eliminating epsins potently impeded breast cancer development in both spontaneous and patient-derived xenograft breast cancer mouse models. Our findings established the pivotal role epsins played in promoting breast cancer. Thus, targeting epsins may represent a strategy to restrain NF-κB signaling and provide an important perspective into ER-negative breast cancer treatment.

Authors

Kai Song, Xiaofeng Cai, Yunzhou Dong, Hao Wu, Yong Wei, Uma T. Shankavaram, Kui Cui, Yang Lee, Bo Zhu, Sudarshan Bhattacharjee, Beibei Wang, Kun Zhang, Aiyun Wen, Scott Wong, Lili Yu, Lijun Xia, Alana L. Welm, Diane R. Bielenberg, Kevin A. Camphausen, Yibin Kang, Hong Chen

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Figure 2

Epsin is a potentially novel regulator of NF-κB signaling in ER-negative breast cancer.

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Epsin is a potentially novel regulator of NF-κB signaling in ER-negative...
(A) EMSA of NF-κB DNA binding activity (n = 3 independent experiments). Nuclear extracts (5 μg) were prepared from Ctrl and KD MDA231 cells and incubated with biotinylated probe (hot) or unlabeled as specific competitor (cold). Specific binding complex bands and free probes are indicated by arrows. (B) Western blot analyses of phosphorylated p65 (p-p65), total p65, phosphorylated IκB-α (p-IκB-α), total IκB-α, phosphorylated IKK-β/α (p-IKK-β/α), and total IKK-β in Ctrl and KD MDA231 cells (n = 3 independent experiments). (C and D) IHC analysis (C) and quantification (D) of epsin 1, p-p65, p-IκB-α, and cleaved caspase-3 in primary tumors of Ctrl and MEpC-DKO MMTV-PyMT mice. A minimum of 3 randomly chosen fields (×10 original magnification) of each tumor (n = 3 per genotype) were evaluated. Statistical values were calculated using unpaired, 2-tailed multiple t tests. Data are presented as the mean ± SEM. Scale bar: 100 μm. (E and F) Immunofluorescence analysis (E) and quantification (F) of p-p65 and epsin 1 in human normal (n = 2 biologically independent samples), benign (n = 8 biologically independent samples), or malignant (n = 8 biologically independent samples) breast tissues. Statistical values were calculated using 1-way ANOVA followed by Tukey’s multiple-comparisons test. Data are presented as the mean ± SD. Scale bar: 50 μm. (G) The Cancer Genome Atlas Breast Cancer Carcinoma data collection (TCGA BRCA) GSEA at the P value threshold of 0.01 unveiled positive correlation of NF-κB–related genes with combined epsin 1/2 expression. Gene expression data were converted to log2 scale. Notably, NEMO (IKBKG) is on top of the list. See also Supplemental Figure 2.