UC Berkeley

ABSTRACT

Phytochemical Disruption of Hormone Receptor Expression and Intracellular Signaling in Human Reproductive Cancer Cells

By

Crystal Nicole Marconett

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Gary Firestone, Chair

ER-alpha is a critical target of therapeutic strategies to control the proliferation of hormone dependent breast cancers. Preferred clinical options have significant adverse side effects that can lead to treatment resistance due to the persistence of active estrogen receptors. This thesis details the molecular mechanism indole-3-carbinol (I3C) initiates to ablate ER-alpha; expression and subsequent downstream targets of ER-alpha transcriptional activity; IRS1, IGF1R, and hTERT and the ability of an aqueous mixture from the Scutellaria Barbata plant, BZL101, to arrest the proliferation of human reproductive cancer cells, regardless of hormonal status. I3C dependent activation of the aryl hydrocarbon receptor (AhR) initiates Rbx-1 E3 ligase-mediated ubiquitination and proteasomal degradation of ER-alpha protein. I3C disrupts endogenous GATA3 interactions with the ER-alpha promoter, leading to a loss of GATA3 and ER-alpha expression. Ectopic expression of GATA3 has no effect on I3C induced ER-alpha protein degradation but does prevent I3C inhibition of ER-alpha promoter activity, demonstrating the importance of GATA3 in this I3C triggered cascade. Our preclinical results implicate I3C as a novel anti-cancer agent in human cancers that co-express ER-alpha, GATA3 and AhR, a combination found in a large percentage of breast cancers but not in other critical ER-alpha target tissues essential to patient health. Estrogen signaling stimulates growth and proliferation of these cells by activation of key downstream targets, such as Insulin-like Growth Factor Receptor-1 (IGF1R) and Insulin Receptor Substrate-1 (IRS1) of the Insulin-like Growth Factor (IGF1) signaling pathway. We show that I3C downregulates both IGF1R and IRS1 RNA and protein expression. We established that this decrease in expression was contingent upon ER-alpha ablation by I3C. I3C is able to block hTERT expression through a loss of transcriptional activation. I3C mediated loss of hTERT expression was attributed to disrupted endogenous binding to a composite ER-alpha-Sp1 site. The block in hTERT expression disrupted telomerase activity, and ectopic expression of hTERT was able to restore telomerase activity. We have uncovered a critical role I3C employs to block the proproliferative activities of IGF signaling and telomerase activity in hormone sensitive breast cancer cells. Aqueous extracts from BZL101 were able to disrupt estrogen expression in the hormone MCF7 cell line. Specifically, BZL101 induced a G1 cell cycle arrest and ablation of Cyclin D, CDK2, and CDK4 expression in MCF7 cells. MDA-MB-231 and PC3 cells arrested in S phase with corresponding ablations in Cyclin A2 and CDK2. LNCaP cells arrested in G2/M phase upon exposure to BZL101 with a corresponding decrease in Cyclin B1 and CDK1. Our results indicate that BZL101 exerts cell type specific molecular changes that lead to arrest of reproductive cancer proliferation and the general disruption of ER-alpha by phytochemicals is a potent disruptor of hormone sensitive cancer growth and proliferation.

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Gary Firestone, PhD