Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Elble, Randolph

Abstract

hCLCA2 and hCLCA4 are chloride channel regulators that are expressed in normal breast epithelial cells and frequently downregulated in breast cancers. Recent investigations revealed that these two proteins may have a role in suppressing breast cancer progression. In this thesis, I will address their role in maintaining epithelial differentiating and inhibiting cell proliferation of breast epithelial cells. The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells downregulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In breast cancer, EMT facilitates invasion of surrounding tissues and correlates closely with cancer metastasis and relapse. We found previously that the candidate tumor suppressor hCLCA2 is a p53-inducible proliferation-inhibitor that is frequently lost in breast cancer. We show here that another member of the CLCA gene family, hCLCA4, is expressed in mammary epithelial cells and is similarly downregulated in breast tumors and in breast cancer cell lines. Like CLCA2, the gene is stress-inducible, and ectopic expression inhibits colony formation. Transcriptional profiling studies revealed that hCLCA4 and hCLCA2 together are markers for mammary epithelial differentiation, and both are downregulated by TGF beta. Moreover, knockdown of either on in immortalized cells by shRNAs caused downregulation of epithelial marker E-cadherin, while mesenchymal markers N-cadherin, vimentin, and fibronectin were upregulated, indicating an EMT program. Double knockdown of hCLCA2 and hCLCA4 enhanced the mesenchymal profile. These findings suggest that hCLCA4 and hCLCA2 play complementary but distinct roles in epithelial differentiation. Clinically, low expression of hCLCA2 and hCLCA4 signaled lower relapse-free survival in breast cancers. Cellular senescence is a program of irreversible cell cycle arrest in response to stressors such as DNA damage, ROS, telomere erosion, or oncogene activation. It is one of the primary tumor suppression mechanisms mediated by p53 and is often disabled in cancer cells. However, the downstream signaling pathway whereby p53 induces cellular senescence remains incomplete. We reported previously that hCLCA2 was a p53 inducible gene that is downregulated with breast cancer progression. We and other group noticed that hCLCA2 was induced in parallel with several types of senescence. Lentiviral transduction of CLCA2 into MCF7 cells inhibited cell proliferation and cells showed senescence phenotype. To investigate the mechanism biochemically, we used pAd-Easy to express hCLCA2 in the model breast cancer cell line CA1d. A protein expression profile of these cells over a 6 day period revealed induction of p21, p53, and the DNA damage-response pathway. To test whether hCLCA2 is required for the cellular senescence process, hCLCA2 was knocked down in HMLE. The knockdown cells (KD) and negative control were treated with a low concentration of doxorubicin, and cell proliferation was measured. The KD cells were more resistant to growth inhibition by doxorubicin. Moreover, a time course experiment showed that induction of SA beta-galactosidase, DNA damage response, and lysosomal markers IFI30 and CTSS was delayed in the knockdown cells. These results suggest that hCLCA2 plays an important role in DNA damage response and the senescence program.

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