Introduction
TMEM16A (also known as
ANO1,
DOG1,
ORAOV2 or
TAOS2) is a member of the Anoctamin family of membrane proteins, which consists of ten components (known as
TMEM16A-
K or
ANO1-
10) that share a highly conserved structure with eight transmembrane domains and cytosolic amino- and carboxy-termini domains. In 2008, three independent groups identified
TMEM16A as a bona fide Ca
2+-activated Cl
- channel (CaCC) essential for a variety of physiological functions including neuronal and cardiac excitation, olfactory and sensory signal transduction, vascular tone and pain perception [
1,
2].
TMEM16A is expressed in many tissues that are known to express CaCCs: bronchiolar epithelial cells, pancreatic acinar cells, proximal kidney tubule epithelium, retina, dorsal root ganglion sensory neurons, and sub- mandibular gland [
3‐
5]. In addition to epithelia,
TMEM16A is robustly expressed in interstitial cells of Cajal, which are responsible for generating pacemaker activity in smooth muscle of the gut [
6‐
8].
TMEM16A is a sensitive biomarker for the diagnosis of gastrointestinal stromal tumors (GISTs) and it is overexpressed in several cancers including urinary bladder cancer, esophageal cancer, prostate cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), parathyroid tumors and ovarian tumors [
9‐
13]. However, its role in tumor cell proliferation and migration is not completely understood. In cellular models,
TMEM16A has been reported to positively affect cell proliferation and/or migration [
9,
12‐
16]. Conversely, Wang et al. [
17] showed a negative effect of cell proliferation by preventing cell cycle transition from the G0/G1 phase to the S phase via inhibition of cyclin D1 and cyclin E expression [
17]. Several mechanisms have been reported that associate changes in Ca
2+-dependent channel activities with its tumorigenic potential are unknown: prevention of cell cycle transition from the G0/G1 phase to the S phase via inhibition of cyclin D1 and cyclin E expression [
17], activation of MAPK kinases with promotion of ERK1/2 and cyclin D1 activation [
9] and activation of EGFR and CAMK signalling [
13]. In general, similar to other channels such as Ca(2+)-activated K(+) channels and voltage-gated Cl(−) or K(+)channels,
TMEM16A might affect cell motility and proliferation though dynamic changes in the cellular volume [
18‐
21].
Alternative splicing of
TMEM16A can generate multiple protein isoforms with different electrophysiological properties affecting the voltage and Ca
2+-dependence of the channel [
22]. The most important AS occurring on
TMEM16A transcripts consist in skipping/ inclusion of three exons 6b, 13 and 15. Other minor splicing events affecting exon 1, 10, 14 and 18 have also been reported, leading to the formation of several TMEM16A transcripts [
23,
24]. We have previously reported the relative percentage of inclusion of each exon relative to the total amount of
TMEM16A transcripts showing that in several normal human tissues, transcripts that predominantly include exon 6b, also tend to lack exon 15 in the same mature transcripts and vice-versa [
22]. However, the relative proportion of each isoform that take into account the real association between the AS events on the same transcript is unknown.
In the
TMEM16A gene and thus on the nascent transcripts, the three exons, 6b, 13 and 15 are located relatively far apart (exons 6b and 15 are located at 50 kb). Comparison of data from transcript sequencing efforts, EST/cDNA sequences and microarray profiling experiments have provided evidence for AS coordination between multiple exons within a single gene [
25‐
28]. Indeed, distant alternative splicing events on the same gene can be correlated, through a mechanism known as intragenic Splicing Coordination (SC) [
27,
29,
30]. The presence of multiple splice variants on the same transcript, as found in
TMEM16A, can highly increase its potential to generate multiple transcripts. However, as AS events are generally analyzed separately through PCR amplification of short regions, this correlation is difficult to establish and have not been explored in tumors. The mechanism that regulates the coordinate selection of AS events in the same gene involves both
cis-acting regulatory elements present on the nascent transcripts and Polymerase II -dependent transcriptional elongation [
27].
In the present study, using a PCR-assay that amplifies transcripts across the three AS exons, we found that TMEM16A AS of exons 6b and 15 are coordinated in several normal tissues and that this coordination increases in breast tumors. AS of individual exons was not altered in breast tumor and overexpression of the TMEM16A isoforms in a controlled cellular system had no effect on proliferation and migration. These results suggest that TMEM16A expression and splicing does not confer a positive general effect on cell growth and motility. The unexpected increase of SC in breast tumors suggest that cancer cells maintain the regulatory mechanism that coordinates distant alternative spliced exons on the same transcript, a process that, by acting on multiple genes, may be important in tumor cell viability.
Discussion
In this paper, we have evaluated the potential role in breast cancer of the different
TMEM16A isoforms generated through alternative splicing. The analysis of the alternative splicing pattern
in vivo in breast tumors and the functional analysis of the isoforms overexpressed in a cellular model strongly suggest that the
TMEM16A Ca
2+-dependent Cl
- channel activities are not directly involved in tumorigenesis. More precisely, the lack of effect on HEK293 growth and motility indicates that, if
TMEM16A has a role on cancer progression, it is not due to a general effect on the regulation of cell cycle and migration, as previously proposed [
9,
12‐
15]. However, this does not exclude that
TMEM16A has a more cancer-specific role that is relevant to tumor growth
in vivo. On the other hand, in
TMEM16A transcripts derived from several normal tissues and breast tumors, inclusion/exclusion of the two distant alternatively spliced exon 6b and 15 is not independent but occur in a coordinated manner on a single transcript. As this splicing coordination is significantly increased in breast tumors, the regulatory mechanism that coordinates distant alternative spliced exons on the same transcript is maintained in these cells. We speculate that maintenance of the mechanisms that control SC is necessary for regulation of AS events in other genes directly involved in tumor cell viability.
Several studies have associated
TMEM16A expression with cancer [
9,
31‐
33]. However, as tumors may originate from cells that normally express
TMEM16A, like GIST, which is thought to derive from the interstitial cells of Cajal [
33,
34], it is not clear if this channel is just an associated marker or if it directly promotes tumorigenesis. In addition, the mechanism that associates its Ca
2+-dependent Cl
- activity to cellular proliferation is unclear, and several studies reported contradictory effects on proliferation and/or migration. In basilar smooth muscle cells, where
TMEM16A is abundantly expressed, its downregulation promotes proliferation and accordingly restoration of
TMEM16A activity was suggested to be beneficial on hypertension-associated cardiovascular disease such as stroke [
17]. On the contrary, other studies reported that
TMEM16A promotes cell migration alone [
35] or both proliferation and migration [
9,
12,
14,
15]. In xenografts, loss of
TMEM16A through siRNA-mediated silencing was reported to inhibit tumor growth [
9,
13]. As a result of this positive effect on proliferation, and in contrast to the effect on smooth muscle cells [
17], the therapeutic inhibition of
TMEM16A activity was proposed for the treatment of cancers [
9,
13]. In this study, the analysis of the different
TMEM16A isoforms generated through AS
in vivo in breast cancer and in a controlled cellular model, does not support a direct role of the major
TMEM16A isoforms in tumorigenesis. The evaluation of the different functional properties of isoforms derived from AS is not an easy task, in particular if referred to migration and proliferation. To avoid clonal variability that can affect transgene expression due to the numbers of copies integrated and site of integration, we used the inducible HEK293 Flp-In Tet-ON system and the results clearly show that the
TMEM16A isoforms per se are not sufficient to promote or inhibit proliferation and migration (Figure
4). Even if our result does not support a direct role of
TMEM16A, the different isoforms might indirectly affect proliferation and migration in a cell-type specific manner. Accordingly, to affect proliferation and/or migration,
TMEM16A expression would require expression of additional factors, and this association could be at the basis of the contradictory effects reported in different cell types. This cell type specificity might be critical for the development of therapeutic strategies, as different pathological conditions and target cells may differently respond to inhibition or restoration of the
TMEM16A activity, as previously reported [
9,
13,
17]. The HEK293 Flp-In Tet-ON system we have developed here, expressing the different isoforms in a regulated manner, can represent an interesting model to identify, with a high-throughput screening method, those factors that not only promote or inhibit proliferation in a TMEM-dependent manner but also potential AS-specific networks.
An interesting and novel aspect of our study is the identification in several normal tissues of
TMEM16A SC and the observation that this coordination increases in breast tumors (Figure
6). In common with ~ 25% of human genes,
TMEM16A has more than one alternative splicing possibility and accordingly can generate several isoforms. Through a mechanism known as intragenic splicing coordination, [
27,
28,
36] the association between alternative spliced events is not random, as we have found for exon 6b and 15 in
TMEM16A. In normal breast, approximately 50% of samples showed SC, which increased to 84% in tumors. Normal mammary glands express
TMEM16A[
37‐
39] and the individual hormonal status might have an important effect on its AS pattern and SC thus explaining the variability observed in normal tissues. In several genes, hormones modulate AS [
40,
41] but the effect on splicing coordination is unknown. Through histological evaluation, we do not have evidence of the presence of tumor cells in normal samples, but normal glands showed a variable relative abundance of different cell types (like epithelial, stromal and adipocytes) and this might also contribute to the individual variability.
The fact that the expression of the two AS exons 6b and 15 is coordinated in the majority of
TMEM16A transcripts derived from tumors (84%), suggest that cancer progression is not associated with a relaxation of this phenomenon. The mechanisms underlying splicing coordination are largely unknown. Fibronectin (FN) [
27] and
slo-
1 BK potassium channel [
30] are well-studied examples of genes with intragenic AS coordination. In these cases, mutations that affect one AS exon had a profound effect on the other AS events, with both 5’ to 3’ polarity or bi-directionality for
FN and
Slo-
1, respectively. In addition, proper coordination of intragenic alternative splicing has been found essential for normal physiology of the
slo-
1 gene in vivo in
Caenorhabditis elegans[
30]. Based on this evidences a mechanism that lead to preferential expression of given alternate exon combination was engaged. Recruitment of splicing factors with direct interactions between the RNA-protein complexes from distinct splice sites, RNA secondary structure determinants and changes in Pol II elongation or processivity have been suggested to be involved [
27,
30]. Another interesting hypothesis to explain intragenic AS coordination might involve the formation of chromatin loops. Chromatin loops have been reported to physically link promoters to the end of the gene in order to facilitate Pol II-dependent transcription [
42‐
45]. These loops can also occur in introns, as demonstrated for the 85-Kb long
BRCA1 gene in human cell lines and in mouse mammary tissues [
31]. Interestingly, these loops change upon estrogen stimulation and during lactational development [
31]. Thus it is possible that loops that put in contact distant alternatively spliced regions on the same gene might have a role in splicing coordination and possibly regulated by hormonal status. In this manner, Pol II engaged in transcription at different AS exons might physically communicate with reciprocal influence on the corresponding splicing decisions. This event might be still operative in tumor cells in order to preserve cancer viability. More deep molecular studies are required to unravel the basic mechanism of intragenic splicing coordination and understand their role in cancer.
What could be the advantage of a tumor cell to maintain an intragenic splicing coordination? As none of the
TMEM16A isoforms directly affect proliferation or migration (Figure
4), it is possible that during tumorigenesis the cell would have to maintain active and efficient the mechanism involved in splicing coordination. This might not be specifically useful for the
TMEM16A expression but for coordinating splicing of other genes directly involved in proliferation or apoptosis such as
CD44 gene,
Ron gene or
FGFR2 gene [
46‐
49]
.
In conclusion, this study has improved our understanding of TMEM16A splicing coordination with the identification and characterization of a non-random distribution of the mRNA isoforms in normal adult human breast tissues and tumor.
In this context, the maintenance of splicing coordination will be required for preventing a massive transcript alteration that will lead to cell death and thus the study of the basic mechanism involved might be useful for the identification of novel targets for therapeutic intervention.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
IU and FP conceived the idea and designed the experiments; IU, EB, PS performed the experiments; AC, GS and ML provide human breast and breast cancer tissues and performed histological analysis; IU, LG and FP wrote the manuscript. All authors read and approved the final manuscript.