Contribution of Anoctamins to Cell Survival and Cell Death

Abstract

Before anoctamins (TMEM16 proteins) were identified as a family of Ca²⁺-activated chloride channels and phospholipid scramblases, the founding member anoctamin 1 (ANO1, TMEM16A) was known as DOG1, a marker protein for gastrointestinal stromal tumors (GIST). Meanwhile, ANO1 has been examined in more detail, and the role of ANO1 in cell proliferation and the development of different types of malignomas is now well established. While ANO5, ANO7, and ANO9 may also be relevant for growth of cancers, evidence has been provided for a role of ANO6 (TMEM16F) in regulated cell death. The cellular mechanisms by which anoctamins control cell proliferation and cell death, respectively, are just emerging; however, the pronounced effects of anoctamins on intracellular Ca²⁺ levels are likely to play a significant role. Recent results suggest that some anoctamins control membrane exocytosis by setting Ca²⁺_i levels near the plasma membrane, and/or by controlling the intracellular Cl^- concentration. Exocytosis and increased membrane trafficking induced by ANO1 and ANO6 may enhance membrane expression of other chloride channels, such as CFTR and volume activated chloride channels (VRAC). Notably, ANO6-induced phospholipid scrambling with exposure of phosphatidylserine is pivotal for the sheddase function of disintegrin and metalloproteinase (ADAM). This may support cell death and tumorigenic activity of IL-6 by inducing IL-6 trans-signaling. The reported anticancer effects of the anthelminthic drug niclosamide are probably related to the potent inhibitory effect on ANO1, apart from inducing cell cycle arrest through the Let-7d/CDC34 axis. On the contrary, pronounced activation of ANO6 due to a large increase in intracellular calcium, activation of phospholipase A2 or lipid peroxidation, can lead to ferroptotic death of cancer cells. It therefore appears reasonable to search for both inhibitors and potent activators of TMEM16 in order to interfere with cancer growth and metastasis.

Keywords: ANO1; ANO6; Ca2+ signaling; TMEM16A; TMEM16F; anoctamin; apoptosis; cancer; inflammation; proliferation.

Figure 1

Proliferation-dependent expression of ANO10 in FRT cells. (A) FRT cells were grown in FCS-containing media at 70% density (Proliferating), as confluent monolayer in FCS-free media (Monolayer), or as polarized monolayer on permeable supports and in FCS-free media (Polarized). Expression of endogenous ANO10 (green fluorescence) was intracellular in dividing cells (Proliferating), but was detected in the plasma membrane and in the primary cilium in densely grown cells (Monolayer). ANO10 was more prominent in plasma membrane and primary cilium in polarized cells (Polarized). For further details and references, see main text. (B) Hypothetical model proposing variable cellular locations of ANO10 depending on cell proliferation or cell polarization. ANO10 is found primarily intracellularly, but is also in the plasma membrane during cell cycle. Reduced expression of ANO10 and translocation into the primary cilium is observed once cells move into G₀. Bar, 20 µm [108].

Figure 2

Upregulation and redistribution of ANO1 during proliferation and cancer. Scheme summarizing reported factors and signaling pathways that lead to upregulation of expression of ANO1 and cellular redistribution during proliferation and cancer growth (Table 1). For further details and references, see main text.

Figure 3

Mechanisms for ANO1-induced cell proliferation and cancer development. Scheme summarizing reported mechanisms for ANO1-induced cell proliferation and development of cancer. All pathways are inhibited by niclosamide and other inhibitors of anoctamins (Table 1). For further details and references, see main text.

Figure 4

Compartmentalized Ca²⁺ signaling by anoctamins. Scheme illustrating the effects of anoctamins on Ca²⁺ signaling. ANO1 tethers ER Ca²⁺ stores close to the plasma membrane, which leads to improved ATP-induced apical Ca²⁺ signaling. Activation of both ANO1 and ANO6 induce plasma membrane depolarization, supporting release of Ca²⁺ from ER stores via inositol trisphosphate receptors (IP₃R) and ryanodine receptors (RyR). In addition, Ca²⁺ store content was found to be enhanced by ANO1. ANO6 is permeable for Ca²⁺ and therefore supports Ca²⁺ entry. ANO4 localized in the ER interacts with Orai1 [110]. For further details and references, see main text.

Figure 5

Potential action of anoctamins on exocytosis, growth and microvesicular signaling. ANO1 and ANO6 determine the extent of membrane protrusions and membrane blebbing in macrophages and other cell types, and support cell migration, diapedesis and cancer metastasis. Exosome release and paracrine signaling by epithelial cells is probably anoctamin-dependent. Support of membrane unfolding, cell swelling and subsequent activation of VRAC could be a general property of anoctamins. Mucus secretion and release of inflammatory mediators such as autacoids and cytokines was shown to be ANO1-dependent. Exocytosis leads to enhanced expression of membrane proteins, cell growth, and extensions such as motile cilia and the primary cilium, as proposed for ANO1. For further details and references, see main text.

Figure 6

ANO6-induced cell death. Scheme summarizing the contribution of ANO6 to different regulated cell death pathways such as apoptosis, necroptosis, pyroptosis, and ferroptosis. Anoctamins may contribute to regulated cell death by cell shrinkage (apoptosis), increase in compartmentalized intracellular Ca²⁺ (all cell death pathways), or cell swelling, scrambling, blebbing, and membrane disintegration (ferroptosis, pyroptosis). For further details and references, see main text.