Indole (I3C) Control of Breast Cancer by ER Downregulation

Institution: University of California, Berkeley
Investigator(s): Crystal Marconett, B.S. -
Award Cycle: 2007 (Cycle 13) Grant #: 13GB-0162 Award: $76,000
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2007)

Reproductive cancers are characterized by their responsiveness to hormonal stimuli. Breast cancer classically evolves from an estrogen-responsive to unresponsive state. These hormonal responsive changes are validated by cell lines excised at discrete points in the disease progression sequence. Estrogen-responsive tumors have the ability to activate the IGF (insulin growth factor) and VEGF (vascular endothelial growth factor) pathways through the ability of ERalpha (ERα) to bind and increase the gene activity for these and other proteins required for key cell growth signaling pathways. Previously, the dietary indole, indole-3-carbinol (I3C) found in cruciferous vegetables, has been shown to decrease ERα levels in the estrogen-sensitive cell line, MCF7, but the mechanism underlying this effect has not been determined.

The purpose of the proposed research is to elucidate the mechanism by which I3C influences ERα levels. This will be accomplished by isolating the factor (gene transcription factor protein) that is directly responsible for ERα downregulation (i.e. decrease in protein expression), followed by studying the associated signaling pathways. By this strategy we hope to establish how I3C inhibits ERα-responsive breast cancers, possibly leading to alternative chemotherapeutics with reduced side effects compared to current drugs. Our approach will be to isolate fragments from the ERα gene-regulatory promoter, then insert them into a luciferase-based (i.e., fluorescent) assay system to test which portions of the ERα??promoter are sensitive to I3C. Then, we will use the I3C-responsive piece of the ERα promoter to identify and isolate the particular transcription factors that bind to this region of DNA. After separation, the proteins will be analyzed by mass spectrometry. The eventual goal is to identify the proteins and the signaling pathways involved in the repression of ERα by I3C. This new information could potentially connect the other critical breast cancer cell pathways known to be affected by I3C, such as elastase and Sp1. Alternately, any newly identified factor could be part of a novel ERα pathway not previously known to be affected by I3C.

Exploration into the upstream signaling mechanism regulated by I3C that breast cells utilize to inhibit ERα could open up a new realm of anti-cancer treatments.

Final Report (2009)

Indole-3-Carbinol (I3C), a phytochemical found in cruciferous vegetables such as broccoli, has been shown to arrest the growth and proliferation of human breast cancer cells. I3C is able to accompolish this activity by affecting the expression of critical cell cycle regulators and stimulii, one of which is Estrogen Receptor-alpha (ERα). The goal of this project was to establish the molecular mechanism I3C elicits in human breast cancer cells to ablate the expression of ERα and what the functional consequences of the resulting ablation are on cancer growth and proliferation.

We were able to establish the precise molecular mechanism 13C induces to ablate ERα expression (Aim 1). I3C activation of the aryl hydrocarbon receptor (AhR) and resulting recruitment of the E3 ubiquitin ligase Rbx-1 resulted in the targeted ubiquitination and subsequent degradation of ERα. This in turn resulted in a loss of ERα downstream target gene expression, one of which was determined to be GATA3. Loss of GATA3 expression resulted in transcriptional ablation of ERα mRNA production, thus enforcing the loss of ERα expression in hormone sensitive breast cancer cells. We determined that ERα-dependent loss of other downstream gene targets accounted for the loss of proliferation (Aim 2). These genes were identified as IGF1R and IRS1, critical regulators of growth factor signaling in breast cancer. We were unable to characterize the effect I3C has on angiogenesis, and specifically VEGF (Aim 3) in the period of this study.

In summary, we were able to determine how a dietary component is able to block the expression of a protein involved in 70% of early stage breast cancer, thus implicating I3C as a novel selective estrogen receptor down-regulator (SERD) and a potential therapeutic option.

Symposium Abstract (2007)

The vast majority of breast cancers are classified as estrogen-sensitive, with the ability to respond and grow in the presence of estrogen. The two estrogen receptor (ER) subtypes, ERalpha and ERbeta, are expressed in these cancers, are able to bind to their estrogen ligand, dimerize, and act as transcription factors within the nucleus. ER-alpha can serve as an activating transcription factor for classical target genes, such as Progesterone Receptor (PR), and more recently as an activator of genes such as Insulin-like Growth Factor Receptor 1 (IGF1R) or Insulin Receptor Substrate-1 (IRS-1), which are involved in tumor metastasis.

The ability of breast cancer cells to respond to estrogen is critical to their proliferative capacity. Gulcobrassicin is a naturally occurring compound in vegetables of the Brassica genus, a plant group, which includes broccoli and brussels sprouts. Indole-3-Carbinol (I3C) is a dietary indole, which is released from the compound glucobrassicin during ingestion.

Our lab has previously shown that I3C transcriptionally downregulates ER-alpha expression in the estrogen responsive breast cancer cell line MCF7, having an effect on both the proliferation and growth of hormone sensitive breast cancers.

We are currently isolating the transcription factor(s) responsible for the downregulation of ERalpha in order to elucidate the mechanism by which I3C can inhibit hormone sensitive breast cancer.

Indole-3-Carbinol Triggers Aryl Hydrocarbon Receptor-dependent Estrogen Receptor (ER){alpha} Protein Degradation in Breast Cancer Cells Disrupting an ER{alpha}-GATA3 Transcriptional Cross-Regulatory Loop.
Periodical:Molecular and Cellular Biology
Index Medicus: Mol Cell Biol
Authors: Marconett CN, Sundar SN, Poindexter KM, Stueve TR, Bjeldanes LF, Firestone GL.
Yr: 2010 Vol: 21 Nbr: 7 Abs: Pg:1166-77