-activated Cl Ϫ channels (CaCCs) 2 play a major physiological role in various types of cells and tissues (1-4). In epithelial cells, opening of CaCCs in the apical membrane generates a flux of Cl Ϫ that drives transepithelial water transport. The resulting CaCC-dependent electrolyte/fluid secretion is one of the mechanisms responsible for exocrine secretion in many types of glands and for hydration of the airway surface (2). In particular, the activity of CaCC and of the cystic fibrosis transmembrane conductance regulator chloride channel controls the thickness of the periciliary fluid that is important for optimal mucociliary clearance (5, 6). Deficit in cystic fibrosis transmembrane conductance regulator activity, which occurs in cystic fibrosis, favors bacterial colonization of the airways. Under such conditions, activity of CaCCs may be important in compensating, at least partially, the defect in Cl Ϫ transport. In smooth muscle cells, activation of CaCCs is important in the process of contraction (1,7,8). CaCCs are also present in olfactory receptors, in dorsal root ganglion neurones, and in oocytes (3, 4, 9, 10). The biophysical properties of CaCCs are not homogeneous among different cells and tissues. In many cases, CaCCs are activated by Ca 2ϩ in a wide range of concentrations between 0.06 and 1 M (11-14) and are also voltage-dependent, with membrane depolarization increasing the activity (7, 10 -16). However, CaCCs that need much higher Ca 2ϩ concentration (9, 17, 18) and that are devoid of voltage dependence are also known (9,17,19). Moreover, the mechanism of regulation by Ca 2ϩ is unclear. In some studies, Ca 2ϩ seems to activate the channels through calmodulin (20). In others, activation requires the intervention of a Ca 2ϩ /calmodulin-dependent kinase (19,21). Finally, there are cell types where phosphorylation has actually an inhibitory effect on CaCCs (7,8,14). The differences may be due to heterogeneity of the proteins that actually constitute the CaCCs.Until recently, the molecular identity of CaCCs was a controversial issue, with ClCA proteins, ClC3, and bestrophins being postulated as possible candidates (1,22). Three recent studies, including one from our laboratory, have identified the TMEM16A protein (also known as ANO-1) as a probable . TMEM16A is a membrane protein with eight putative transmembrane segments belonging to a family including other nine members (TMEM16B-K). In our previous study (23), we identified several TMEM16A transcripts probably generated by selection of alternative splice sites. The alternative sequences coded for protein segments that we named a (116 residues), b (22 residues), c (4 residues), and d (26 residues). The former two segments are localized in the N terminus, whereas the latter two segments are localized in the first intracellular loop.A TMEM16A mouse knock-out model has been also generated. The phenotype of these animals is severe and characterized by altered formation of tracheal cartilage rings (26). This alteration, causing airway collapse, may be resp...

BackgroundTMEM16A, also known as Anoctamin-1, is a calcium-activated chloride channel gene overexpressed in many tumors. The role of TMEM16A in cancer is not completely understood and no data are available regarding the potential tumorigenic properties of the multiple isoforms generated by alternative splicing (AS).MethodsWe evaluated TMEM16A AS pattern, isoforms distribution and Splicing Coordination (SC), in normal tissues and breast cancers, through a semi-quantitative PCR-assay that amplifies transcripts across three AS exons, 6b, 13 and 15.ResultsIn breast cancer, we did not observe an association either to AS of individual exons or to specific TMEM16A isoforms, and induced expression of the most common isoforms present in tumors in the HEK293 Flp-In Tet-ON system had no effect on cellular proliferation and migration. The analysis of splicing coordination, a mechanism that regulates AS of distant exons, showed a preferential association of exon 6b and 15 in several normal tissues and tumors: isoforms that predominantly include exon 6b tend to exclude exon 15 and vice versa. Interestingly, we found an increase in SC in breast tumors compared to matched normal tissues.ConclusionsAs the different TMEM16A isoforms do not affect proliferation or migration and do not associate with tumors, our results suggest that the resulting channel activities are not directly involved in cell growth and motility. Conversely, the observed increase in SC in breast tumors suggests that the maintenance of the regulatory mechanism that coordinates distant alternative spliced exons in multiple genes other than TMEM16A is necessary for cancer cell viability.

Mutations in Tp53 compromise therapeutic response, due either to the dominant-negative effect over the functional wild-type allele; or as a result of the survival advantage conferred by mutant p53 to which cancer cells become addicted. Thus, targeting mutant p53 represents an effective therapeutic strategy to treat over half of all cancers. We have therefore generated a series of small-interfering-RNAs, capable of targeting four p53 hot-spot mutants which represent ~20% of all p53 mutations. These mutant–p53-specific siRNAs (MupSi) are highly specific in silencing the expression of the intended mutants without affecting wild-type p53. Functionally, these MupSis induce cell death by abrogating both the addiction to mutant p53 and the dominant-negative effect; and retard tumor growth in xenografts when administered in a therapeutic setting. These data together demonstrate the possibility of targeting mutant p53 specifically to improve clinical outcome.