Sulforaphane (SFN), an isothiocyanate derived from broccoli, has recently been shown to be effective in reducing UVB-induced non-melanoma skin cancer (NMSC) in mice. Although the role of SFN in the activation of the phase II response is under close scrutiny, inhibition of the Activator Protein-1 (AP-1) transcription factor by SFN may also be critical for the chemopreventive effects of this compound after UVB (Zhu et al., Mol. Carcinogen. vol 41, 2004). AP-1, a dimer of Jun/Jun or Jun/Fos family members, is activated by UVB stimulation in vitro and in vivo. Transgenic inhibition of AP-1 blocks UVB-induced NMSC in mice (Cooper et al., Mol. Cancer Res., vol 1, 2003). Although we have previously shown that SFN inhibits AP-1 luciferase in keratinocytes in culture, we now show that SFN is also effective in reducing UVB-induced AP-1 luciferase signals in the skin of reporter mice. In addition, SFN affects UVB-induced AP-1 DNA binding in the cell as demonstrated by chromatin immunoprecipitation (ChIP) assays. Previous reports indicate that a specific cysteine in the DNA binding domain of cFos and cJun may be crucial for the response of AP-1 to redox regulation; this cysteine (a.a. 154 in Fos and 272 in Jun) must be in the reduced state in order for AP-1 to bind DNA. SFN is known to bind to cysteines, causing oxidation which could therefore block DNA binding. In fact, diamide, another cysteine oxidizing agent, is also effective at inhibiting AP-1 DNA binding in EMSA assays. To address whether this cysteine interacts with SFN to affect AP-1 DNA binding we created recombinant truncated forms of cFos and cJun (tFos and tJun) which contain only the beta-zip domain of each protein. The purified, renatured tFos/tJun dimers bind very efficiently to DNA as shown by EMSA assays, and are competed from binding to labeled oligos by pre-treatment with cold competitors, but not mutant competitors. These tFos/tJun dimers are also sensitive to SFN inhibition in a dose-dependant manner. Significantly, mutating the redox-sensitive cysteine of tFos and tJun to serine resulted in loss of sensitivity to inhibition by both SFN and diamide. This work is the first to pinpoint the redox-sensitive DNA binding cysteine of AP-1 as critical for the response of this transcription factor to SFN. The inhibition of AP-1 by SFN may therefore be a critical factor in the ability of this compound to inhibit NMSC and other cancers. This work was supported by P01 CA27502 and R25 CA78447.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 22.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO