Marie Potier-Cartereau

Pivotal role of the lipid raft SK3-Orai1 complex in human cancer cell migration and bone metastases

The SK3 channel, a potassium channel, was recently shown to control cancer cell migration, a critical step in metastasis outgrowth. Here, we report that expression of the SK3 channel was markedly associated with bone metastasis. The SK3 channel was shown to control constitutive Ca(2+) entry and cancer cell migration through an interaction with the Ca(2+) channel Orai1. We found that the SK3 channel triggers an association with the Orai1 channel within lipid rafts. This localization of an SK3-Orai1 complex seemed essential to control cancer cell migration. This suggests that the formation of this complex in lipid rafts is a gain-of-function, because we showed that none of the individual proteins were able to promote the complete phenotype. We identified the alkyl-lipid Ohmline as a disrupting agent for SK3-Orai1 lipid raft localization. Upon Ohmline treatment, the SK3-Orai1 complex moved away from lipid rafts, and SK3-dependent Ca(2+) entry, migration, and bone metastases were subsequently impaired. The colocalization of SK3 and Orai1 in primary human tumors and bone metastases further emphasized the clinical relevance of our observations. Targeting SK3-Orai1 in lipid rafts may inaugurate innovative approaches to inhibit bone metastases.

KCa and Ca2 + channels: The complex thought ☆

Potassium channels belong to the largest and the most diverse super-families of ion channels. Among them, Ca(2+)-activated K(+) channels (KCa) comprise many members. Based on their single channel conductance they are divided into three subfamilies: big conductance (BKCa), intermediate conductance (IKCa) and small conductance (SKCa; SK1, SK2 and SK3). Ca(2+) channels are divided into two main families, voltage gated/voltage dependent Ca(2+) channels and non-voltage gated/voltage independent Ca(2+) channels. Based on their electrophysiological and pharmacological properties and on the tissue where there are expressed, voltage gated Ca(2+) channels (Cav) are divided into 5 families: T-type, L-type, N-type, P/Q-type and R-type Ca(2+). Non-voltage gated Ca(2+) channels comprise the TRP (TRPC, TRPV, TRPM, TRPA, TRPP, TRPML and TRPN) and Orai (Orai1 to Orai3) families and their partners STIM (STIM1 to STIM2). A depolarization is needed to activate voltage-gated Ca(2+) channels while non-voltage gated Ca(2+) channels are activated by Ca(2+) depletion of the endoplasmic reticulum stores (SOCs) or by receptors (ROCs). These two Ca(2+) channel families also control constitutive Ca(2+) entries. For reducing the energy consumption and for the fine regulation of Ca(2+), KCa and Ca(2+) channels appear associated as complexes in excitable and non-excitable cells. Interestingly, there is now evidence that KCa-Ca(2+) channel complexes are also found in cancer cells and contribute to cancer-associated functions such as cell proliferation, cell migration and the capacity to develop metastases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Targeting SKCa Channels in Cancer: Potential New Therapeutic Approaches

Many studies have reported changes in potassium channel expression in many cancers and the involvement of these channels in various stages of cancer progression. By contrast, data concerning SKCa channels (small conductance calcium-activated potassium channels) have only recently become available. This review aims i) to present the structure and physiology of SKCa channels, ii) to provide an overview of published data concerning the SKCa proteins produced in tumor cells, and, whenever possible, the biological function assigned to them and iii) to review previous and novel modulators of SKCa channels. SKCa channels are activated by low concentrations of intracellular calcium and consist of homo- or heteromeric assemblies of α-subunits named SK1, SK2 and SK3. SK2-3 channels are expressed in tumors and have been assigned a biological function in cancer cells: the enhancement of cell proliferation and cell migration by hijacking the functions of SK2 and SK3 channels, respectively. Two major classes of SKCa modulators have been described: toxins (apamin) and small synthetic molecules. Most SKCa blockers are pore blockers, but some modify the calcium sensitivity of SKCa channels without interacting with the apamin binding site. In this review, we present edelfosine and ohmline as atypical anticancer agents and novel SK3 inhibitors. Edelfosine and ohmline are synthetic alkyl-lipids with structures different from all previously described SKCa modulators. They should pave the way for the development of a new class of migration-targeted anticancer agents. We believe that such blockers have potential for use in the prevention or treatment of metastasis.

Lipid rafts, KCa/ClCa/Ca2+ channel complexes and EGFR signaling: Novel targets to reduce tumor development by lipids?

Membrane lipid rafts are distinct plasma membrane nanodomains that are enriched with cholesterol, sphingolipids and gangliosides, with occasional presence of saturated fatty acids and phospholipids containing saturated acyl chains. It is well known that they organize receptors (such as Epithelial Growth Factor Receptor), ion channels and their downstream acting molecules to regulate intracellular signaling pathways. Among them are Ca2+ signaling pathways, which are modified in tumor cells and inhibited upon membrane raft disruption. In addition to protein components, lipids from rafts also contribute to the organization and function of Ca2+ signaling microdomains. This article aims to focus on the lipid raft KCa/ClCa/Ca2+ channel complexes that regulate Ca2+ and EGFR signaling in cancer cells, and discusses the potential modification of these complexes by lipids as a novel therapeutic approach in tumor development. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell migration: a novel opportunity to modulate anti-EGFR mAb action by the alkyl-lipid Ohmline

Background Barely 10-20% of patients with metastatic colorectal cancer (mCRC) receive a clinical benefit from the use of anti-EGFR monoclonal antibodies (mAbs). We hypothesized that this could depends on their efficiency to reduce Store Operated Calcium Entry (SOCE) that are known to enhance cancer cells. Results In the present study, we demonstrate that SOCE promotes migration of colon cancer cell following the formation of a lipid raft ion channel complex composed of TRPC1/Orai1 and SK3 channels. Formation of this complex is stimulated by the phosphorylation of the reticular protein STIM1 by EGF and activation of the Akt pathway. Our data show that, in a positive feedback loop SOCE activates both Akt pathway and SK3 channel activity which lead to SOCE amplification. This amplification occurs through the activation of Rac1/Calpain mediated by Akt. We also show that Anti-EGFR mAbs can modulate SOCE and cancer cell migration through the Akt pathway. Interestingly, the alkyl-lipid Ohmline, which we previously showed to be an inhibitor of SK3 channel, can dissociated the lipid raft ion channel complex through decreased phosphorylation of Akt and modulation of mAbs action. Conclusions This study demonstrates that the inhibition of the SOCE-dependent colon cancer cell migration trough SK3/TRPC1/Orai1 channel complex by the alkyl-lipid Ohmline may be a novel strategy to modulate Anti-EGFR mAb action in mCRC.

NSC-34 Motor Neuron-Like Cells Are Unsuitable as Experimental Model for Glutamate-Mediated Excitotoxicity

Glutamate-induced excitotoxicity is a major contributor to motor neuron degeneration in the pathogenesis of amyotrophic lateral sclerosis (ALS). The spinal cord × Neuroblastoma hybrid cell line (NSC-34) is often used as a bona fide cellular model to investigate the physiopathological mechanisms of ALS. However, the physiological response of NSC-34 to glutamate remains insufficiently described. In this study, we evaluated the relevance of differentiated NSC-34 (NSC-34D) as an in vitro model for glutamate excitotoxicity studies. NSC-34D showed morphological and physiological properties of motor neuron-like cells and expressed glutamate receptor subunits GluA1–4, GluN1 and GluN2A/D. Despite these diverse characteristics, no specific effect of glutamate was observed on cultured NSC-34D survival and morphology, in contrast to what has been described in primary culture of motor neurons (MN). Moreover, a small non sustained increase in the concentration of intracellular calcium was observed in NSC-34D after exposure to glutamate compared to primary MN. Our findings, together with the inability to obtain cultures containing only differentiated cells, suggest that the motor neuron-like NSC-34 cell line is not a suitable in vitro model to study glutamate-induced excitotoxicity. We suggest that the use of primary cultures of MN is more suitable than NSC-34 cell line to explore the pathogenesis of glutamate-mediated excitotoxicity at the cellular level in ALS and other motor neuron diseases.

Inactivation of the Carney Complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase : a study using genetically-encoded FRET-based reporters

Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty skin pigmentation and endocrine overactivity. CNC is caused by inactivating mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit (RIα). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RIα in the spatiotemporal organization of the cAMP/PKA pathway. RIα knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E1 (PGE1)-stimulated total cellular PKA activity as reported by western blots of endogenous PKA targets and the FRET-based global PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular compartments, we identified similar increases in the response to PGE1 in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE1 was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE1-induced phosphorylation of membrane-associated vasodilator-stimulated phosphoprotein. Similar differences were observed between the cytoplasm and the plasma membrane in human adrenal cells carrying a RIα inactivating mutation. RIα inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial membrane and at the plasma membrane, as reported by targeted versions of the cAMP indicator Epac1-camps. These results show that RIα inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis.

Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cells.

Expression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects.

Functional cooperation between KCa3.1 and TRPC1 channels in human breast cancer: Role in cell proliferation and patient prognosis

Intracellular Ca2+ levels are important regulators of cell cycle and proliferation. We, and others, have previously reported the role of KCa3.1 (KCNN4) channels in regulating the membrane potential and the Ca2+ entry in association with cell proliferation. However, the relevance of KC3.1 channels in cancer prognosis as well as the molecular mechanism of Ca2+ entry triggered by their activation remain undetermined. Here, we show that RNAi-mediated knockdown of KCa3.1 and/or TRPC1 leads to a significant decrease in cell proliferation due to cell cycle arrest in the G1 phase. These results are consistent with the observed upregulation of both channels in synchronized cells at the end of G1 phase. Additionally, knockdown of TRPC1 suppressed the Ca2+ entry induced by 1-EBIO-mediated KCa3.1 activation, suggesting a functional cooperation between TRPC1 and KCa3.1 in the regulation of Ca2+ entry, possibly within lipid raft microdomains where these two channels seem to co-localize. We also show significant correlations between KCa3.1 mRNA expression and poor patient prognosis and unfavorable clinical breast cancer parameters by mining large datasets in the public domain. Together, these results highlight the importance of KCa3.1 in regulating the proliferative mechanisms in breast cancer cells as well as in providing a promising novel target in prognosis and therapy.

Singular Interaction between an Antimetastatic Agent and the Lipid Bilayer: The Ohmline Case

SK3 channels are abnormaly expressed in metastatic cells, and Ohmline (OHM), an ether lipid, has been shown to reduce the activity of SK3 channels and the migration capacity of cancer cells. OHM incorporation into the plasma membrane is proposed to dissociate the protein complex formed between SK3 and Orai1, a potassium and a calcium channel, respectively, and would lead to a modification in the lipid environment of both the proteins. Here, we report the synthesis of deuterated OHM that affords the determination, through solid-state NMR, of its entire partitioning into membranes mimicking the SK3 environment. Use of deuterated lipids affords the demonstration of an OHM-induced membrane disordering, which is dose-dependent and increases with increasing amounts of cholesterol (CHOL). Molecular dynamics simulations comfort the disordering action and show that OHM interacts with the carbonyl and phosphate groups of stearoylphosphatidylcholine and sphingomyelin and to a minor extent with CHOL. OHM is thus proposed to remove the CHOL OH moieties away from their main binding sites, forcing a new rearrangement with other lipid groups. Such an interaction takes its origin at the lipid–water interface, but it propagates toward the entire lipid molecules and leads to a cooperative destabilization of the lipid acyl chains, that is, membrane disordering. The consequences of this reorganization of the lipid phases are discussed in the context of the OHM-induced inhibition of SK3 channels.