Dietary Metabolite Inhibition of Breast Cancer Cell Survival

Institution: University of California, Berkeley
Investigator(s): Holly Nicastro, Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14GB-0142 Award: $72,508
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2008)

The goal of the proposed project is to determine the anti-cancer mechanism of 3,3í-diindolylmethane (DIM), an active metabolite of indole 3-carbinol (I3C), in breast cancer cells. Indole-3-carbinol is the focus of much research into its possible anti-carcinogenic and antioxidant effects. Consumption of indole-3-carbinol might influence cancer incidence due to its ability to alter estrogen metabolism and cell cycle regulation to reduce cell proliferation. This project focuses on the role of the PI3K/Akt cell signaling pathway, which mediates growth, survival, and chemotherapy resistance.

Components of this PI3K/Akt signaling pathway are frequently mutated or overexpressed in breast cancer. In fact, a recent review states that the gene encoding the catalytic subunit of PI3K is the most common mutation in breast cancer (Oncogene 2007;26:1338-45). In addition, Her-2, a growth factor receptor that activates the PI3K/Akt pathway, is amplified in 10-30% of breast cancers. Activation of the PI3K/Akt pathway also plays a role in resistance to current breast cancer therapies like trastuzimab (Herceptin) and tamoxifen. Compounds, like DIM, that inhibit this central pathway could be effective against breast cancers with certain genetic aberrations and could help to overcome resistance to current therapies.

DIMís effects on proliferation and cell cycle will be studied by direct cell counting, DNA staining and analysis by flow cytometry, cyclin D and myc expression, and NF-?B activation. Protein expression will be determined by Western blotting and protein activation (phosphorylation) will be determined by Western blotting using phospho-specific antibodies, or by immunoprecipitation of individual proteins followed by Western blotting for phosphotyrosine. Involvement of specific components of the PI3K/Akt pathway in DIMís cytostatic effects will be determined by overexpression of the specific proteins to potentially reverse DIMís effects. Finally, PI3K activation and activity will be determined by immunofluorescence and kinase assays, respectively.

By studying the mechanism whereby DIM inhibits this central breast cancer cell signaling pathway, we hope to identify a specific target for future investigation. Since breast cancer is a heterogeneous disease with specific genetic profile in each patient, our ultimate goal would be to identify specific mutations or overexpressed proteins that would predict those that can respond the best to DIM treatment.

Final Report (2010)

3,3í-Diindolylmethane (DIM), the major acid condensation product of Brassica-derived indole-3-carbinol, has anticancer effects in breast cancer. DIM inhibits Akt, a kinase whose signaling promotes proliferation, survival, and motility, in breast cancer cells in vitro. The goals of this project were to determine whether DIMís inhibition of Akt is partly responsible for DIMís anti-proliferative/pro-apoptotic effects and to determine a mechanism by which DIM inhibits Akt.

We have found that DIM inhibits proliferation, cell cycle progression and motility, and induces apoptosis in MDA-MB-231 breast cancer cells. These results are consistent with Akt inhibition. We have also shown that DIM inhibits Akt downstream of hepatocyte growth factor (HGF) but not of epidermal growth factor or insulin-like growth factor-1. HGF and the HGF receptor, c-Met, are frequently overexpressed in breast cancers, and c-Met overexpression is a strong and independent predictor of poor prognosis. We found that DIM decreases activation of c-Met at several tyrosine residues, indicating decreased activation of the receptor. When cells are pre-treated with cyclosporin A, a calcineurin inhibitor, DIM no longer inhibits c-Met activation. DIM also induces ligand-dependent degradation of c-Met, and this degradation can be reversed by pre-treating cells with inhibitors of endocytosis, lysosomes, or the proteasome.

In summary, we have determined a mechanism for DIM-induced inhibition of Akt and identified c-Met as a target of DIM, suggesting that DIM is a promising potential therapeutic option for breast cancers with aberrant HGF/c-Met/Akt signaling.