TMEM16A alternative splicing coordination in breast cancer

Abstract

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.

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…Thus, Ubby et al overexpressed ANO1 in HEK293 cells and measured large ANO1-like currents, but no effect on migration was observed. 149 There are several reports highlighting the importance of ANO1 in cancer cell metastasis, i.e., Shi and coworkers demonstrated a correlation between ANO1 overexpression and lymph node metastasis of ESCC and advanced clinical stage of cancers 157 and Ayoub and collaborators found that increased ANO1 expression correlates with increased risk of developing metastases in oral and head and neck squamous cell carcinomas. 139 In contrast, Shiwarshi and coworkers find that reduced ANO1 expression slows proliferation and allow metastatic progression and they suggest that ANO1 expression is a switch between growth and metastasis.…”

“…148 However, as expression of these ANO1 isoforms does not affect proliferation or migration differently and does not associate with specific cancer tissues Ubby and coworkers suggested that the actual ANO1 channel activities are not directly involved in cell growth and motility. 149 …”

Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte

“…Ano1 primary transcript undergoes alternative splicing, thus resulting in the generation of multiple isoforms. Three alternative exons, 6b, 13, and 15, coding for segments of 22 (segment b), 4 (segment c), and 26 (segment c) amino acids, respectively, are differently spliced in human organs (9,40). Functional studies on ANO1 isoforms indicated that the inclusion/exclusion of specific exons influenced ANO1 channel properties (9).…”

“…Functional studies on ANO1 isoforms indicated that the inclusion/exclusion of specific exons influenced ANO1 channel properties (9). In human thyroid, the predominantly expressed ANO1 transcript contains exons 6b and 13 but lacks exon 15 (9,40). Here, we analyzed the expression of Ano1 in rat tissues as well as in the rat thyroid PCCl3 cell line by RT-PCR.…”

A novel exon in the human Ca2+-activated Cl− channel Ano1 imparts greater sensitivity to intracellular Ca2+

“…Outside of the GI tract, Ano1 is present in the vascular smooth muscle (3,36), salivary glands, thyroid, pancreas, prostate, and urinary and gall bladders (29). 11q13 chromosome amplification and subsequent Ano1 overexpression are indicators of poor prognosis for a variety of tumors (1,19,27,40).…”