Loss of CLCA4 Promotes Epithelial-to-Mesenchymal Transition in Breast Cancer 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 CLCA2 is expressed in differentiated, growth-arrested mammary epithelial cells but is downregulated during tumor progression and EMT. We further demonstrated that CLCA2 is a p53-inducible proliferation-inhibitor whose loss indicates an increased risk of metastasis. We show here that another member of the CLCA gene family, CLCA4, 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 CLCA4 and CLCA2 together are markers for mammary epithelial differentiation, and both are downregulated by TGF beta. Moreover, knockdown of CLCA4 in immortalized cells by shRNAs caused downregulation of epithelial marker E-cadherin and CLCA2, while mesenchymal markers N-cadherin, vimentin, and fibronectin were upregulated. Double knockdown of CLCA2 and CLCA4 enhanced the mesenchymal profile. These findings suggest that CLCA4 and CLCA2 play complementary but distinct roles in epithelial differentiation. Clinically, low expression of CLCA4 signaled lower relapse-free survival in basal and luminal B breast cancers.