Research ArticleFunctional role of the Ca2+-activated Cl− channel DOG1/TMEM16A in gastrointestinal stromal tumor cells
Introduction
Ca2+-activated chloride (Cl−) channels (CaCCs) are omnipresent [1] and important regulators of normal physiological functions, including neuronal excitability, smooth muscle cell contraction, epithelial fluid secretion [2], and gastrointestinal motility [3], [4]. Clinically, they have been recognized as potential drug targets for treatment of asthma, secretory diarrheas, and hypertension [5]. The CaCCs are voltage- and Ca2+-activated Cl− channels and are typically situated in the plasma membrane and regulated by intracellular free Ca2+ levels (Ca2+i). The biological importance of CaCCs has been neglected for a long time, which is mainly related to poor understanding of Cl− conductance and its regulation. Today there is increasing evidence for the involvement of CaCC in tumorigenesis, cancer progression, metastasis, cell proliferation and migration [6], [7], [8], [9], [10]. Discovered on GIST-1 (DOG1) is a recent addition to the CaCC family, also known as the transmembrane protein 16A (TMEM16A) [11], [12] or anoctamin 1 (ANO1) [12]. Based on molecular plotting it has been shown that DOG1 has eight transmembrane domains and that the channel assembles as a dimer of two CaCC proteins [12], [13], [14]. Its large and non-selective channel pore, or the P-loop, is located between the fifth and sixth transmembrane domains, which allow also other negatively charged ions than Cl− to permeate [15]. DOG1 has been implicated in cancer development based on e.g. amplification in head and neck cancers [16] and esophageal cancers [17] as well as overexpression in gastrointestinal stromal tumors (GIST).
GISTs are mesenchymal tumors genetically characterized by oncogenic mutations in the stem cell factor receptor (KIT) or platelet-derived growth factor receptor α (PDGFRA) genes [18], [19]. Intriguingly, DOG1 is overexpressed in almost all GISTs [20], [21], but its function is still largely unknown. Moreover, based on high throughput screenings of ~110,000 compounds the specific pharmacological TMEM16A/DOG1 inhibitor T16Ainh-A01 [22] and activator Eact [23] have been identified.
These promising findings spurred us to investigate the functional role of DOG1 in GIST cells. In this study we determined the effects of pharmacological activation and inhibition of DOG1 on GIST cell viability, apoptosis, and proliferation in vitro.
Section snippets
Materials
To pharmacologically activate DOG1 we used N-aroylaminothiazole, referred to as Eact from here on, which has been shown to strongly activate and produce sustained DOG1 Cl− currents (EC50=3 µM) at zero Ca2+i levels [23]. Aminophenylthiazole T16Ainh-A01 was used to inhibit DOG1 CaCC (IC50=1.1 µM). This compound was shown to block salivary gland Cl− currents completely at 10 µM, without interfering with calcium signaling [22]. Both compounds were purchased from Merck Millipore (Billerica, MA, USA).
CD117 and DOG1 distribution in GIST882 and GIST48 cells
Expression of CD117 and DOG1 was verified by immunocytochemistry in both cell lines (Fig. 1), and confocal microscopy provided detailed information of their subcellular localizations (Fig. 2). CD117 showed cytoplasmic expression with a granular distribution in GIST882 cells and clustered distribution in GIST48 cells (Fig. 2A). DOG1 was predominantly seen at the cell periphery, including the plasma membrane, in GIST882 cells, whereas perinuclear localization was observed in GIST48 cells (Fig. 2
Survival and proliferation of GIST882 and GIST48 cells
To test whether the DOG1 function affects GIST cell survival or proliferation, DOG1 activity was modulated using varying concentrations of either T16Ainh-A01 or Eact. Proliferation analysis with WST-1 demonstrated that both cell lines tended to remain relatively stable throughout all time points. GIST882 cells are known to take longer to passage than GIST48 cells, which was verified by comparing the onset of cell growth in controls from both cell lines. Although 30 μM Eact and 1 μM and 10 μM T16A
Discussion
GISTs are the most common mesenchymal tumors in the gastrointestinal tract. Until the late 1990s GIST was poorly understood, commonly misclassified, and had a poor prognosis. Discoveries over the past two decades have revolutionized the understanding of GIST pathogenesis and lead to one of the first successful molecular targeted therapies [29]. In clinical practice, the diagnosis of GIST has been based partly on immunohistochemical evidence of CD34 and CD117 expression. However, CD34 turned out
Conflict of interest
The authors declare no conflict of interest.
Grant support
The study was financially supported by the Swedish Research Council Formas, the Novo Nordisk UK Research Foundation, the Swedish Cancer Society, funds from Karolinska Institutet, the Swedish Society of Medicine (Bengt Ihre grant), the Tore Nilsson Foundation, the Thuring Foundation, the Jeansson Foundation, Magn. Bergvall Foundation, and the Cancer Society in Stockholm. Financial support was also provided through the regional agreement on medical training and clinical research (ALF) between the
References (37)
- et al.
Tumor suppression by a proapoptotic calcium-activated chloride channel in mammary epithelium
J. Biol. Chem.
(2001) - et al.
Eag1 and Bestrophin 1 are up-regulated in fast-growing colonic cancer cells
J. Biol. Chem.
(2008) - et al.
Expression cloning of TMEM16A as a calcium-activated chloride channel subunit
Cell
(2008) - et al.
The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status
Am. J. Pathol.
(2004) - et al.
TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells
J. Biol. Chem.
(2011) - et al.
Evidence for Ca(2+)-regulated ATP release in gastrointestinal stromal tumors
Exp. Cell Res.
(2013) - et al.
Effect of farnesyl transferase inhibitor R115777 on the growth of fresh and cloned myeloma cells in vitro
Blood
(2003) - et al.
Immunoblot analysis of CD34 expression in histologically diverse neoplasms
Am. J. Pathol.
(2000) - et al.
Neuronal Ca2+ -activated Cl− channels—homing in on an elusive channel species
Prog. Neurobiol.
(2000) - et al.
Calcium-activated chloride channels
Ann. Rev. Physiol.
(2005)
TMEM16A protein: a new identity for Ca(2+)-dependent Cl(-) channels
Physiology
Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles
J. Physiol.
Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract
Am. J. Physiol. Gastroint. Liver Physiol.
Chloride channels as drug targets
Nat. Rev. Drug Discov.
TMEM16A induces MAPK and contributes directly to tumorigenesis and cancer progression
Cancer Res.
Chloride accumulation drives volume dynamics underlying cell proliferation and migration
J. Neurophysiol.
An unexpected role for ion channels in brain tumor metastasis
Exp. Biol. Med.
FLJ10261 gene, located within the CCND1-EMS1 locus on human chromosome 11q13, encodes the eight-transmembrane protein homologous to C12orf3, C11orf25 and FLJ34272 gene products
Int. J. Oncol.
Cited by (46)
TMEM16A ion channel: A novel target for cancer treatment
2023, Life SciencesDOG1 expression is common in human tumors: A tissue microarray study on more than 15,000 tissue samples
2021, Pathology Research and PracticeCitation Excerpt :Calcium-activated chloride channel blocking drugs like niflumic acid have been shown to block slow waves, which produce motility, in the human intestine [24]. In cancer cells, blockage of DOG1 by CaCCinh-A01, T16Ainh-A01, trastuzumab and/or cetuximab has led to decreased cell proliferation, cell cycle arrest in G1 phase, decreased cell viability and reduced pEGFR and HER2 expression, reduced whole-cell chloride conductance or higher apoptosis rates [3,5,11,15,32]. High levels of DOG1 expression are a diagnostic hallmark of gastrointestinal stromal tumors (GIST), a tumor derived from interstitial cells of Cajal [30,43,56,67].
Direct interaction of the ATP-sensitive K<sup>+</sup> channel by the tyrosine kinase inhibitors imatinib, sunitinib and nilotinib
2021, Biochemical and Biophysical Research CommunicationsTransmembrane (TMEM) protein family members: Poorly characterized even if essential for the metastatic process
2020, Seminars in Cancer BiologyCitation Excerpt :Moreover, several compounds have been identified by high-throughput screening to act, on calcium-activated chloride channel functions, as activators (N-aroylaminothiazole “activators” compounds (Eact)), modulators (tetrazolylbenzamide “potentiator” compounds (Fact)) or as inhibitors (tannic acid, gallotannins and arylaminothiophene CaCCinh-A01) [141,142]. Most of these molecules are nonselective inhibitors of this protein family [143,144]. However, in 2011, Namkung et al. have discovered selective inhibitors (aminophenylthiazole compounds) including T16Ainh-A01 targeting TMEM16A activity [145,146].
The multifaceted role of TMEM16A in cancer
2019, Cell CalciumCitation Excerpt :Moreover, from the list of TMEM16A-interacting partners obtained by the proteomics approach, only radixin and EGFR have also been observed as TMEM16A-interacting partners in other investigations conducted in cancer cells [22,52], suggesting that great effort will be required to validate all of these putative partners of TMEM16A and to characterize their interaction and function in cancer cells. As discussed above, the pharmacological inhibition of TMEM16A using various molecules inhibits several cancer cell functions such as proliferation, migration or apoptosis resistance [13,25,33,37,52,54,58–60,63–66,68,70,72,103]. This suggests that TMEM16A channel activity contributes to the multifaceted role of TMEM16A in cancer.
High level of ANO1 promotes pancreatic cancer growth in concert with oncogenic KRAS
2023, Molecular Biology Reports