Date of Award

1-1-2009

Degree Name

Master of Science

Department

Pharmacology

First Advisor

Elble, Randolph

Abstract

The calcium activated chloride regulator hCLCA2 (human, calcium activated chloride channel) is thought to be a breast tumor suppressor. Its mouse homolog mCLCA5 is upregulated during mammary involution and in vitro by withdrawal of growth factors, cell detachment, and contact inhibition. Cell to matrix adhesion is a key factor involved in cellular homeostasis and disruption of such interaction induces a special kind of apoptosis called anoikis (homelessness). The cell has to bypass anchorage dependent growth in particular during metastasis as well as wound healing. Detachment-induced apoptosis, or anoikis, is one of the primary obstacles to metastasis. We have observed that human CLCA2 is abundantly expressed in normal mammary epithelial cells but downregulated with tumor progression as cells become anoikis-resistant. We show that re-introduction of hCLCA2 into breast cancer cell lines induces apoptosis both in anoikis-sensitive and anoikis-resistant breast cancer cell lines. In immortalized cells, there are increases in p53 and the activated form of ERK (Extracellular Receptor Kinase) prior to cell death. Furthermore, hCLCA2 expression is up-regulated by serum withdrawal and cell detachment in mammary epithelial cell lines. Taken together, these results suggest an important role for hCLCA2 in anoikis. Cell detachment or serum withdrawal inactivates the PI3K-Akt pathway, allowing the Akt target FOXO transcription factors to enter the nucleus and transactivate genes responsible for cell cycle arrest and apoptosis. Here we show that inhibiting the PI3K-Akt pathway either with LY294002 or wortmannin induces hCLCA2 expression in MCF-7 and MDA-MB-231 breast cancer cells. We further show that adenoviruses encoding FOXO3A and dominant-negative Akt induce hCLCA2 transcription in breast cancer cell lines. hCLCA2 is upregulated only by FOXO3A not by FOXO1A as shown by luciferase reporter assays. The hCLCA2 promoter contains a potential binding site for FOXO3A within 300bp upstream of the transcription start site. Deletion analysis of the hCLCA2 promoter using luciferase fusions confirms that a FOXO3A-responsive site lies within that interval. Moreover, the upregulation of hCLCA2 by FOXO3A is dependent upon p53, a binding partner of FOXO3A. This was confirmed by luciferase reporter assays using a FOXO3A-DNA binding mutant and p53 knockdown. Finally, ectopic expression of hCLCA2 sensitized MCF7 cells to anoikis. These results demonstrate that hCLCA2 is upregulated upon cell detachment or serum withdrawal through the PI3K-Akt-FOXO3A pathway and plays a role in cell detachment mediated apoptosis.

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