Dimethyl Fumarate Inhibits the Nuclear Factor κB Pathway in Breast Cancer Cells by Covalent Modification of p65 Protein

Irida Kastrati,Marton I Siklos,Esther L Calderon-Gierszal,Lamiaa El-Shennawy,Gergana Georgieva,Emily N Thayer,Gregory R.J Thatcher,Jonna Frasor

doi:10.1074/jbc.m115.679704

Abstract

In breast tumors, activation of the nuclear factor κB (NFκB) pathway promotes survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy--all phenotypes of aggressive disease where therapy options remain limited. Adding an anti-inflammatory/anti-NFκB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a monotherapy or adjuvant therapy. To address this need, we examined whether dimethyl fumarate (DMF), an anti-inflammatory drug already in clinical use for multiple sclerosis, can inhibit the NFκB pathway. We found that DMF effectively blocks NFκB activity in multiple breast cancer cell lines and abrogates NFκB-dependent mammosphere formation, indicating that DMF has anti-cancer stem cell properties. In addition, DMF inhibits cell proliferation and significantly impairs xenograft tumor growth. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity but has no effect on upstream proteins in the NFκB pathway. Dimethyl succinate, the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFκB activity. Also, the cell-permeable thiol N-acetyl l-cysteine, reverses DMF inhibition of the NFκB pathway, supporting the notion that the electrophile, DMF, acts via covalent modification. To determine whether DMF interacts directly with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality and found that DMF covalently modifies p65, with cysteine 38 being essential for the activity of DMF. These results establish DMF as an NFκB inhibitor with anti-tumor activity that may add therapeutic value in the treatment of aggressive breast cancers.

Highlights

In the United States, breast cancer is the second most prevalent cancer among women and claims over 40,000 lives each year
The inhibitory effect of dimethyl fumarate (DMF) is shown in three different breast cancer cell lines (MCF-7, BT474, and MDA-MB-231), representing different breast cancer subtypes: estrogen receptor-positive, Her2-positive, and triple-negative subtype, respectively
Similar to cytokine-induced activation of nuclear factor ␬B (NF␬B), we found that adding DMF blocks Dox-induced gene expression in CA-IKK␤ cells in a dose-dependent manner (Fig. 1F)

Summary

The abbreviations used are

NF␬B, nuclear factor ␬B; CSC, cancer stem cell; IKK, I␬B kinase; DMF, dimethyl fumarate; MS, mammosphere(s); DMS, dimethyl succinate; NAC, N-acetyl L-cysteine; ER, estrogen receptor; CA, constitutionally active; Dox, doxycycline; QPCR, quantitative PCR; IHC, immunohistochemistry; IP, immunoprecipitation; 2D, monolayer twodimensional. The development of safe NF␬B inhibitors is even more challenging [22], especially for anti-cancer therapy where continued inhibitor use is required. This raises the issue of how to safely and effectively inhibit the NF␬B pathway. We found that DMF covalently modifies the NF␬B transcription factor p65 to block its nuclear translocation and DNA binding activity. These results provide proof-of-principle evidence that DMF can be used to inhibit NF␬B activity in breast cancer cells. Understanding the mechanism of action of DMF could provide the needed rationale to advance DMF into the clinic for aggressive breast cancer therapy

Experimental Procedures

Results

Discussion