Date of Award


Degree Name

Doctor of Philosophy



First Advisor

Elble, Randolph


The breast tumor suppressor hCLCA2 is a putative chloride regulator that is expressed in normal breast epithelial cells and frequently down-regulated in breast cancers. The first CLCA protein was described as a calcium-activated, plasma-membrane chloride channel having four or five transmembrane pass structure that could form a channel pore. However, CLCA topology is inconsistent with chloride channel function. We showed that hCLCA2 itself is unlikely to form a channel as it has only a single transmembrane segment with a short cytoplasmic tail and is mostly extracellular. Moreover, the N-terminal 109-kDa ectodomain is cleaved at the cell surface and shed into the medium while the 35-kDa C-terminal product is retained by the cell membrane. The general goal of my project was to study the function of this novel protein and its role in breast cancer. In addition to its role in chloride regulation, hCLCA2 behaves as a tumor suppressor gene that is frequently down-regulated in breast cancer. We previously demonstrated that murine homologs of hCLCA2 are transcriptionally induced during mammary involution, when the gland shuts down and 80% of the mammary epithelial cells die by apoptosis. In cell culture, conditions that cause G1 arrest such as contact inhibition and depriving cells of growth factors and anchorage induced these genes. Therefore, one of the goals of this project was to find if this is true of hCLCA2 in human breast epithelial cells. We found that hCLCA2 was induced by the above mentioned stresses and by pharmacological blockage of cell survival signaling. In addition, we found that DNA-damaging agents doxorubicin and aphidicolin potently induced hCLCA2 in p53-positive cell lines such as MCF-7 but not in p53-deficient cells such as MDA-MB231. An adenovirus encoding p53 induced hCLCA2 expression in a broad spectrum of breast cancer cell lines while a control virus did not, suggesting that hCLCA2 is a p53-inducible gene. To further test the hypothesis, we performed chromatin immunoprecipitation (ChIP) to determine whether p53 bound to the hCLCA2 promoter. This analysis showed that p53 binds directly to the hCLCA2 promoter between -157 and -359bp upstream of the translation initiation site. This segment was required for the p53-dependent expression of an hCLCA2-luciferase fusion gene. Point mutation of the p53 consensus binding motif abolished this induction. Induction of hCLCA2 in MCF-7 cells by doxorubicin was inhibited by p53 knockdown and by p53 inhibitor pifithrin, indicating that p53 activates the endogenous hCLCA2 promoter in response to DNA damage. An adenovirus encoding hCLCA2 induced a cell cycle lag in G0/G1 phase, decreased intracellular pH from 7.49 to 6.7, caused Bax and Bad translocation to the mitochondria, activated caspases, induced PARP cleavage, and promoted apoptosis. Conversely, hCLCA2 knockdown enhanced proliferation of epithelial MCF10A cells and reduced sensitivity to doxorubicin. These results reveal the molecular mechanism of hCLCA2 induction and downstream events that may provide protection from tumorigenesis. Epithelial cells acquire mesenchymal characteristics by undergoing phenotypic and genotypic changes during cancer progression. An early step in the epithelial to mesenchymal transition (EMT) is the disruption of intercellular connections due to loss of epithelial cadherins. We find that expression of tumor suppressor hCLCA2 is strongly associated with epithelial differentiation and that induction of EMT by mesenchymal transcription factors represses its expression. Moreover, we found that knockdown of hCLCA2 by RNA interference results in disruption of cell-cell junctions by downregulating E-cadherin. This also imparts invasiveness and anoikis-resistance to epithelial cells but is insufficient to induce full EMT. However, activation of Ras oncogene in combination with hCLCA2 knockdown is sufficient to induce full EMT in vitro. These findings indicate that, like E-cadherin, hCLCA2 is required for epithelial differentiation and that its loss during tumor progression may contribute to metastasis.




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