Abstract P1-05-21: The role of epsin in promoting Epithelial-Mesenchymal Transition and metastasis by activating NF-kB signaling in breast cancer
Authors: Cai X , Brophy ML , Hahn S , McManus J , Chang B , Pasula S , Chen H 
Published: August 2014

Background: Breast Cancer progression and metastasis are multi-step processes that involve local tumor growth and invasion followed by tumor dissemination to and re-establishment at distant sites. The ability of a tumor to metastasize is the major determinant of the mortality of cancer patients. Thus, elucidating the molecular pathways essential for tumor metastasis is of high priority in cancer biology. We have previously demonstrated that deficiency of epsins, a family of endocytic clathrin adaptor proteins, decreases tumor growth by enhancing VEGF signaling in vascular endothelial cells and subsequently promoting dysfunctional tumor angiogenesis.

Research Objective: Our immediate aim in the current research is to determine the role of epsin in regulating breast cancer growth and metastasis by activating NF-κB signaling. Our ultimate goal is to seek better treatment for breast cancer patients.

Rationale: Our preliminary data demonstrate that epsins are overexpressed in human cancers, including breast cancer. Knockdown of epsins in human breast cancer cell line MDA-MB-231 inhibits in vitro cell proliferation and migration. Xenograft or tail-vein injection of epsin-deficient breast cancer cells reveals marked reduction in in vivo tumorigenesis and lung metastasis. Moreover, E-cadherin is increased and vimentin decreased in epsin-deficient MDA-MB-231 and in tumors derived from epsin-deficient MDA-MB-231 tumor models. Conversely, overexpression of epsin in breast epithelial cell line MCF10A and MDA-MB-231 results in decreased E-cadherin and increased vimentin expression.

Hypothesis: We hypothesize that epsin regulates EMT through modulating NF-κB signaling.

Methods: we use combined techniques of western blot, confocal immunofluorescence and RT-PCR to examine NF-κB activation.

Results: NF-κB phosphorylation, nuclear translocation and NF-κB target gene snail/slug expression is downregulated in epsin-deficient MDA-MB-231. Epsin overexpression in MDA-MB-231 increases NF-κB phosphorylation. Mechanistically, we show that epsin co-immunoprecipitates with components of TNF Receptor Signaling Complex (TNFR-SC) including TNFR1, TRADD, RIP, TRAF2 and NEMO in MDA-MB-231, which can be enhanced by TNFα stimulation. Conversely, recruitment of NEMO and TRAF2 to TNFR1 is impaired in epsin-deficient MDA-MB-231 compared to control upon TNFα treatment. Given that ubiquitin-interacting motifs (UIM) of epsin is important for its interaction with ubiquitinated proteins, we show that wild type (WT) but not a UIM-deficient mutant epsin (epsin ΔUIM) coprecipitates with TNFR1 in 293T cells. Overexpression of epsin ΔUIM inhibits NF-κB activation in MDA-MB-231 and 293T cells compared to WT. In addition, UIM of epsin and polyubquitination of RIP are required for the interaction of epsin with RIP in 293T cells response to TNFα.

Conclusion: epsin promotes breast cancer growth and metastasis by upregulating NF-kB signaling and EMT. Epsin serves as scaffolding protein through UIM-polyubiquitin chain interaction to stabilize TNFR1 signaling complex (TNFR-SC) and subsequently enhance NF-kB signaling in breast cancer cells. Our study provides a basis for novel therapeutic targets for the development of anti-metastatic cancer treatments.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-21.