Tumor Suppression by a Proapoptotic Calcium-activated Chloride Channel in Mammary Epithelium

Abstract

Little is known of the roles played by ion channels in cancer. Here we describe a pair of closely related calciumactivated chloride channels whose differential regulation in normal, apoptotic, and transformed mouse cells suggests that channel function is proapoptotic and antineoplastic. While mCLCA1 predominates over mCLCA2 under normal physiological conditions, this relationship is reversed by apoptotic stress both in developing mammary gland and in cultured HC11 mammary epithelial cells. Consistent with an apoptosis-promoting role, splicing of mCLCA2 is disrupted in apoptosis-resistant tumor cell lines and in HC11 cells selected for resistance to detachment-induced apoptosis (anoikis). Unexpectedly, mCLCA1 message is also down-regulated in these cells by at least 30-fold. These results suggest that both genes antagonize survival of mammary tumor cells by sensitizing them to anoikis. When MCF7 or HEK293 tumor cells were transfected with plasmids encoding either mCLCA1 or mCLCA2, colony formation was greatly reduced relative to a vector-transfected control, demonstrating that calcium-sensitive chloride channel (CLCA) expression is deleterious to tumor cell survival. Furthermore, mammary epithelial cells overexpressing mCLCA2 had twice the rate of apoptosis of normal cells when subjected to serum starvation and formed multinuclear giants at a high frequency in normal culture, suggesting that mCLCA2 can promote either apoptosis or senescence.

Re-expression of Detachment-inducible Chloride Channel mCLCA5 Suppresses Growth of Metastatic Breast Cancer Cells

“…At present, the best genetic evidence comes from studies of CLCA down-regulation in various cancers. In addition to the loss of hCLCA2 in human breast cancer and the data reported herein, we also detected down-regulation of other mouse CLCA family members, mCLCA1 and -2, in mammary tumor cell lines (21) and transformed endothelial cells. 2 Others have reported the near universal down-regulation of hCLCA1 and -4 in human colon cancer biopsies when compared with autologous normal tissues (54).…”

“…Equal numbers of cells were aliquoted into polypropylene 1.5-ml tubes (Costar) and rotated end-over-end at 37°C for 1, 2, 4, 8, and 24 h. RNA was harvested, reverse-transcribed, and subjected to PCR for mCLCA5 and GAPDH as described above. Bax primers were 5Ј-TCCGGGAGCA-GCTTGGGAGC-3Ј and 5Ј-CACAAAGATGGTCACTGTCTG-3Ј (94°C, 20 s; 55°C, 45 s; 72°C, 45 s; 35 cycles) (21).…”

“…To confirm and extend these results, the organs identified in the array and several others were isolated from a C57BL/6 mouse. RNA was reverse-transcribed and amplified as above, with GAPDH as an internal control for RNA amount (primers: 5Ј-ACGACCCCTTCATTGACCTC-3Ј and 5Ј-CTTTC-CAGAGGGGCCATCCAC-3Ј) (21).…”

“…mCLCA5 is not the only CLCA gene induced in mammary involution. We previously described a pair of genes, mCLCA1 and mCLCA2, that are nearly identical to each other but reciprocally regulated (21). mCLCA1 is repressed during mammary involution and by detachment in vitro, whereas mCLCA2 is induced by these conditions, implying opposing functions.…”

“…hCLCA2 belongs to a recently discovered family of proteins, currently comprising about a dozen members, that are widely expressed in mammals but apparently not beyond (19 -28). Various family members are found in epithelia, endothelia, and smooth muscle of multiple organs (21,24,25,27,28). CLCAs do not resemble any other known Cl Ϫ channel (i.e.…”