Elsevier

Experimental Cell Research

Volume 326, Issue 2, 15 August 2014, Pages 315-325
Experimental Cell Research

Research Article
Functional role of the Ca2+-activated Cl channel DOG1/TMEM16A in gastrointestinal stromal tumor cells

https://doi.org/10.1016/j.yexcr.2014.05.003Get rights and content

Highlights

  • Subcellular DOG1 localization varies between GIST cells.

  • DOG1 in GIST is voltage- and Ca2+-activated.

  • Known TMEM16A modulators, like A01 and Eact, modulate DOG1.

  • DOG1 has small effects on cell viability and proliferation in vitro.

  • DOG1 impact early apoptotic GIST cells to undergo late apoptosis.

Abstract

DOG1, a Ca2+-activated Cl channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.

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

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