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Ewing sarcoma (ES) is characterized by chimeric fusion proteins, which act as oncogenes. Over the last decade, patient survival has not increased, especially for high risk patients. Knowing that ion channels are studied for their implication in tumorigenesis, the aim of this work is to study the involvement of the SK1 potassium channels in ES. RNA-Seq analyses showed a high restricted expression ofKCNN1, the gene encoding SK1, only in ES patients, and its expression is inversely correlated with patient survival. EWS-FLI1 silencing demonstrated the regulation ofKCNN1by these fusion proteins, which bind at GGAA microsatellites nearKCNN1promoter. In addition,KCNN1has been shown to be involved in the regulation of ES cell proliferation, its silencing being associated with a slowing of the cell cycle. Finally,KCNN1expression modulates membrane potential and calcium flux suggesting the role of calcium inKCNN1driving cell proliferation. These results highlight thatKCNN1is a direct EWS-FLI1 and EWS-ERG target, and is involved in the regulation of ES cell proliferation, making it an interesting therapeutic target in ES.

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PDF file - 579K, S1. Validation of the MDA-MB-435s cell system expressing luciferase gene and expressing or not KCNN3 gene. S2. SK3 knockdown reduced the development of metastases in cancer cell xenograft models. A) SK3 knockdown reduced the metastatic score in cancer cell xenograft model. S3.Role of Orai1 in the constitutive SK3-dependent calcium entry and SK3+ cell migration. S4. Effect of Ohmline on Orai1 and SK3 expression and on constitutive calcium entry. S5. SK3 protein was expressed in smooth muscle cells of urinary bladder detrusor and myometrium and in primary tumour form mice grafted with SK3+ cells 16 weeks post grafted.

[Image: see text] Peptide-based therapy against cancer is a field of great interest for biomedical developments. Since it was shown that SK3 channels promote cancer cell migration and metastatic development, we started using these channels as targets for the development of antimetastatic drugs. Particularly, tamapin (a peptide found in the venom of the scorpion Mesobuthus tamulus) is the most specific toxin against the SK2 channel currently known. Considering this fact, we designed diverse tamapin mutants based on three different hypotheses to discover a new potent molecule to block SK3 channels. We performed in vitro studies to evaluate this new toxin derivative inhibitor of cancer cell migration. Our results can be used to generate a new tamapin-based therapy against cancer cells that express SK3 channels.

International audience; The Transient Receptor Potential Vanilloid type 2 (TRPV2) channel is highly selective for Ca2+ and can be activated by lipids, such as LysoPhosphatidylCholine (LPC). LPC analogues, such as the synthetic alkyl-ether-lipid edelfosine or the endogenous alkyl-ether-lipid Platelet Activating Factor (PAF), modulates ion channels in cancer cells. This opens the way to develop alkyl-ether-lipids for the modulation of TRPV2 in cancer. Here, we investigated the role of 2-Acetamido-2-Deoxy-l-O-Hexadecyl-rac-Glycero-3-PhosphatidylCholine (AD-HGPC), a new alkyl-ether-lipid (LPC analogue), on TRPV2 trafficking and its impact on Ca2+ -dependent cell migration. The effect of AD-HGPC on the TRPV2 channel and tumour process was further investigated using calcium imaging and an in vivo mouse model. Using molecular and pharmacological approaches, we dissected the mechanism implicated in alkyl-ether-lipids sensitive TRPV2 trafficking. We found that TRPV2 promotes constitutive Ca2+ entry, leading to migration of highly metastatic breast cancer cell lines through the PI3K/Akt-Girdin axis. AD-HGPC addresses the functional TRPV2 channel in the plasma membrane through Golgi stimulation and PI3K/Akt/Rac-dependent cytoskeletal reorganization, leading to constitutive Ca2+ entry and breast cancer cell migration (without affecting the development of metastasis), in a mouse model. We describe, for the first time, the biological role of a new alkyl-ether-lipid on TRPV2 channel trafficking in breast cancer cells and highlight the potential modulation of TRPV2 by alkyl-ether-lipids as a novel avenue for research in the treatment of metastatic cancer.

Simple Summary Currently, neuroendocrine prostate cancers remain fatal, so it is crucial to better understand mechanisms of resistance to hormone therapy driving this phenotype. We have shown that Enza, a new generation hormone therapy, promotes prostate cancer cells neurodifferentiation by activating a positive feedback loop between the key transcription factor of epithelial to mesenchymal transition Zeb1 and the calcium-sensitive potassium channel SK3. These two actors are overexpressed in patients with neuroendocrine castration-resistant prostate cancer. Targeting SK3 channel by Ohmline, a synthetic ether lipid, inhibits neuroendocrine differentiation of prostate cancer cells, which opens new therapeutic prospects for neuroendocrine prostate cancers. Abstract Therapeutic strategies for metastatic castration-resistant prostate cancer aim to target androgen receptor signaling. Despite initial survival benefits, treatment resistance invariably occurs, leading to lethal disease. Therapies targeting the androgen receptor can induce the emergence of a neuroendocrine phenotype and reactivate embryonic programs associated with epithelial to mesenchymal transition. We recently reported that dysregulation of the calcium signal can induce the transcription factor Zeb1, a key determinant of cell plasticity during tumor progression. The aim of this study was to determine whether the androgen receptor-targeted treatment Enzalutamide could induce dysregulation of the calcium signal involved in the progression toward epithelial to mesenchymal transition and neuroendocrine differentiation, contributing to therapeutic escape. Our results show that Zeb1 and the SK3 potassium channel are overexpressed in vivo in neuroendocrine castration-resistant prostate cancer and in vitro in LNCaP cells neurodifferentiated after Enzalutamide treatment. Moreover, the neuroendocrine phenotype is associated with a deregulation of the expression of Orai calcium channels. We showed that Zeb1 and SK3 are critical drivers of neuroendocrine differentiation. Interestingly, Ohmline, an SK3 inhibitor, can prevent the expression of Zeb1 and neuroendocrine markers induced by Enzalutamide. This study offers new perspectives to increase hormone therapy efficacy and improve clinical outcomes.

International audience; The modulation of SK3 ion channels can be efficiently and selectively achieved by using the amphiphilic compound Ohmline (a glyco-glycero-ether-lipid). We report herein a series of Ohmline analogues featuring the replacement of one ether function by a thioether function located at the same position or shifted close to its initial position. The variation of the lipid chain length and the preparation of two analogues featuring either one sulfoxide or one sulfone moiety complete this series. Patch clamp measurements indicate that the presence of the thioether function (compounds 7 and 17a) produces strong activators of SK3 channels, whereas the introduction of a sulfoxide or a sulfone function at the same place produces amphiphiles devoid of an effect on SK3 channels. Compounds 7 and 17a are the first amphiphilic compounds featuring strong activation of SK3 channels (close to 200% activation). The cytosolic calcium concentration determined from fluorescence at 3 different times for compound 7b (13 min, 1 h, 24 h) revealed that the effect is different suggesting that the compound could be metabolized over time. This compound could be used as a strong SK3 activator for a short time. The capacity of 7b to activate SK3 was then used to induce vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. For the first time, we report that an amphiphilic compound can affect the endothelium dependent vasorelaxation.; Des analogues de l’Ohmline, contenant une fonction soufrée, sont décrits et testés comme modulateur des canaux ioniques SK3. Deux puissants activateurs sont identifiés. Néanmoins leur effet est limité dans le temps. Il est suggéré que ces composés, dotés d’une fonction thioéther, soient oxydés en sulfoxide et sulfone au cours du temps conduisant à une modification de leur activité. Un compose inhibiteur est également identifié mais son manque de sélectivité pourrait expliquer l’absence d’effet sur les entrées calciques associé à l’inhibition des canaux SK3. Finalement, il est montré pour la première fois qu’un composé amphiphile (l’un des activateurs des canaux SK3) présente un effet de vasorelaxation.

We report the synthesis of three bioactive pyrene-based fluorescent analogues of Ohmline which is the most efficient and selective inhibitor of SK3 ion channel. The interaction of these Ohmline-pyrene (OP1-3) with liposomes of different composition reveals that only OP2 and OP3 are readily integrated into liposomes. Fluorescence measurements indicate that, depending on their concentration, OP2 and OP3 exist either as monomer or as a mixture of monomer and excimers within the liposome bilayer. Among the three Ohmline Pyrene compounds (OP1-3) only OP2 is able to reduce SK3 currents and is the first efficient fluorescent modulator of SK3 channel as revealed by patch clamp measurements (- 71.3 ± 13.3% at 10 mM) and by its inhibition of SK3-dependent cancer cell migration at (32.5% ± 4.8% at 1 mM). We also report the first fluorescence study on living breast cancer cells (MDA-MB-231) showing that OP2 is rapidly integrated in bio-membranes followed by cell internalization., Nous décrivons la synthèse d’analogues de l’Ohmline comportant au niveau de la partie lipidique une sonde fluorescente de type pyrène. Nous montrons que l’un d’entre eux (le composé OP2) est capable de réduire les courants potassiques SK3 de 71% et de réduire la migration cellulaire SK3-dépendante de 32%. Il s’agit, à notre connaissance, du premier inhibiteur des canaux ioniques SK3 de nature amphiphile et fluorescent.

Data suggest a role of the potassium channel SK3 (KCNN3 gene) in oxaliplatin-induced neurotoxicity (OIN). Length variations in the polymorphic CAG repeat of the KCNN3 gene may be associated with the risk of OIN. We performed patch-clamp experiments on HEK293 cell lines, expressing SK3 channel isoforms with short (11) or long (24) CAG repetitions, to measure intracellular calcium concentrations to test the effects of oxaliplatin on current density. A retrospective study was carried out on patients with colorectal cancer who had received oxaliplatin-based chemotherapy. DNA for KCNN3 genotyping was extracted from leukocytes. The region containing the CAG repeats was amplified by PCR and the products separated by capillary electrophoresis for length analysis. The patients were divided into three groups depending on whether they carried two short alleles, one short allele and one long allele, or two long alleles. The primary endpoint was the onset of grade 2 or 3 neuropathy to oxaliplatin. There was no difference in current density, but oxaliplatin induced a differential effect on apamin-sensitive current density between the two isoforms expressed in the HEK cell lines. There was a significant reduction of store-operated calcium entry into cells expressing the short and more active isoform only after high concentration of oxaliplatin exposition. Eighty-six patients were included in the clinical study. There was no significant association between OIN and KCNN3 polymorphism for the three groups. We observed a slight association between OIN and CAG repeat polymorphisms of the KCNN3 gene in a preclinical model, but not a clinical study.

The intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research.

International audience; Small Conductance Calcium (Ca 2-)-activated potassium (K-) channels (SKCa) are now proved to be involved in many cancer cell behaviors such as proliferation or migration. The SIG channel isoform was particularly described in breast cancer where it can be associated with the Orai 1 Ca 2-channel to form a complex that regulates the ca 2-homeostasis during tumor development and acts as a potent mediator of bone metastases development in vivo. Until now, very few specific blockers of Orail and/or SIG have been developed as potential anti-metastatic compounds. ln this study, we illustrated the synthesis of new families of lipophilic pyridine and tetrahydropyridine derivatives designed as potential modulators of SK3 channel. The toxicity of the newly synthesized compounds and their migration effects were evaluated on the breast cancer cell line MDA-MB-435s. Two molecules (7a and toc) demonstrated a significant decrease in the SK3 channel-dependent migration as well as the SK3/Orai1-related Ca 2-entry. Current measurements showed that these compounds are more likely SK3-selective. Taken all together these results suggest that such molecules could be considered as promising anti-metastatic drugs in breast cancer.

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International audience; Epithelial Ovarian cancer (EOC) is the deadliest gynecologic malignancy and represents the fifth leading cause of all cancer-related deaths in women. The majority of patients are diagnosed at an advanced stage of the disease that has spread beyond the ovaries to the peritoneum or to distant organs (stage FIGO III-IV) with a 5-year overall survival of about 29%. Consequently, it is necessary to understand the pathogenesis of this disease. Among the factors that contribute to cancer development, lipids and ion channels have been described to be associated to cancerous diseases particularly in breast, colorectal and prostate cancers. Here, we reviewed the literature data to determine how lipids or lipid metabolites may influence EOC risk or progression. We also highlighted the role and the expression of the calcium (Ca2+) and calcium-activated potassium (KCa) channels in EOC and how lipids might regulate them. Although lipids and some subclasses of nutritional lipids may be associated to EOC risk, lipid metabolism of LPA (lysophosphatidic acid) and AA (arachidonic acid) emerges as an important signaling network in EOC. Clinical data showed that they are found at high concentrations in EOC patients and in vitro and in vivo studies referred to them as triggers of the Ca2+entry in the cancer cells inducing their proliferation, migration or drug resistance. The cross-talk between lipid mediators and Ca2+ and/or KCa channels needs to be elucidated in EOC in order to facilitate the understanding of its outcomes and potentially suggest novel therapeutic strategies including treatment and prevention.

Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl chains of variable length are not available. These compounds are, however, necessary as standards for analytical methods. Here, we investigated different reported procedures and we adapted some of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0 (20–24%), 16:0 (42–54%) and 18:1 (6–16%) and, to a lesser extent, (0.2–2%) for each of the following: 16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours and compare their composition to those of non-tumour tissues.

Purpose: Taxane-induced peripheral neuropathy is a common side effect induced by anticancer agents, and no drug capable of preventing its occurrence or ameliorating its long-term course has been identified. The physiology of taxane neuropathy is not clear, and diverse mechanisms have been suggested, with ion channels regulating Ca2+ homeostasis appearing good candidates. The calcium-activated potassium channel SK3 is encoded by the KCNN3 gene, which is characterized by a length polymorphism due to variable number of CAG repeats. Experimental Design: To study the influence of the polymorphism of CAG motif repeat of KCNN3 on the development of taxane-induced neuropathy, we evaluated 176 patients treated with taxanes for breast cancer. In parallel, we measured Ca2+ entry using Fura2-AM dye in HEK cells expressing short versus long CAG alleles of KCNN3. Results: In the current study, we report that in the presence of docetaxel, Ca2+ entry was significantly increased in cells expressing short versus long CAG alleles of SK3 and that a SK3-lipid blocker inhibits this effect. We found that patients carrying a short KCNN3 allele exhibited significantly increased incidence of taxane neuropathy compared with those carrying longer alleles. Conclusions: The clinical implication of these findings is that KCNN3 polymorphism may increase patient susceptibility to taxane neurotoxicity and that the use of SK3 blockers during taxanes’ administration may represent an interesting approach for the prevention of this neurotoxicity. Clin Cancer Res; 24(21); 5313–20. ©2018 AACR.

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 solidstate NMR, of its entire partitioning into membranes mimicking the SK3 environment. Use of deuterated lipids affords the demonstration of an OHMinduced 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. Fil: Herrera, Fernando Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Sevrain, Charlotte M.. Université de Brest; Francia. Network and Cancer-Canceropole Grand Ouest; Francia Fil: Jaffrès, Paul Alain. Université de Brest; Francia. Network and Cancer-Canceropole Grand Ouest; Francia Fil: Couthon, Hélène. Université de Brest; Francia. Network and Cancer-Canceropole Grand Ouest; Francia Fil: Grélard, Axelle. Universite de Bordeaux; Francia Fil: Dufourc, Erick J.. Universite de Bordeaux; Francia Fil: Chantome, Aurélie. Network and Cancer-Canceropole Grand Ouest; Francia. Université François Rabelais de Tour; Francia Fil: Potier Cartereau, Marie. Network and Cancer-Canceropole Grand Ouest; Francia. Université François Rabelais de Tour; Francia Fil: Vandier, Christophe. Network and Cancer-Canceropole Grand Ouest; Francia. Université François Rabelais de Tour; Francia Fil: Bouchet, Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Network and Cancer-Canceropole Grand Ouest; Francia. Université François Rabelais de Tour; Francia

The remodeling of calcium homeostasis contributes to the cancer hallmarks and the molecular mechanisms involved in calcium channel regulation in tumors remain to be characterized. Here, we report that SigmaR1, a stress-activated chaperone, is required to increase calcium influx by triggering the coupling between SK3, a Ca2+-activated K+ channel (KCNN3) and the voltage-independent calcium channel Orai1. We show that SigmaR1 physically binds SK3 in BC cells. Inhibition of SigmaR1 activity, either by molecular silencing or by the use of sigma ligand (igmesine), decreased SK3 current and Ca2+ entry in breast cancer (BC) and colorectal cancer (CRC) cells. Interestingly, SigmaR1 inhibition diminished SK3 and/or Orai1 levels in lipid nanodomains isolated from BC cells. Analyses of tissue microarray from CRC patients showed higher SigmaR1 expression levels in cancer samples and a correlation with tumor grade. Moreover, the exploration of a cohort of 4937 BC patients indicated that high expression of SigmaR1 and Orai1 channels was significantly correlated to a lower overall survival. As the SK3/Orai1 tandem drives invasive process in CRC and bone metastasis progression in BC, our results may inaugurate innovative therapeutic approaches targeting SigmaR1 to control the remodeling of Ca2+ homeostasis in epithelial cancers.

SK3 channel mediates the migration of various cancer cells. When expressed in breast cancer cells, SK3 channel forms a complex with Orai1, a voltage-independent Ca(2+) channel. This SK3-Orai1 complex associates within lipid rafts where it controls a constitutive Ca(2+) entry leading to cancer cell migration and bone metastases development. Since cAMP was found to modulate breast cancer cell migration, we hypothesized that this could be explained by a modulation of SK3 channel activity. Herein, we study the regulation of SK3 channel by the cAMP-PKA pathway and the consequences for SK3-dependent Ca(2+) entry and cancer cell migration. We established that the beta-adrenergic receptor agonist, isoprenaline, or the direct adenylyl cyclase activator forskolin alone or in combination with the PDE4 inhibitor, CI-1044, decreased SK3 channel activity without modifying the expression of SK3 protein at the plasma membrane. Forskolin and CI-1044 reduced the SK3-dependent constitutive Ca(2+) entry and the SK3-dependent migration of MDA-MB-435s cells. PKA inhibition with KT 5720 reduced: (1) the effect of forskolin and CI-1044 by 50 % on Ca(2+) entry and (2) SK3 activity by inhibiting the serine phosphorylation of SK3. These cAMP-elevating agents displaced Orai1 protein outside lipid rafts in contrast to SK3, which remained in the lipid rafts fractions. All together, these results show that activation of the cAMP-PKA pathway decreases SK3 channel and SK3-Orai1 complex activities, leading to a decrease in both Ca(2+) entry and cancer cell migration. This work supports the potential use of cAMP-elevating agents to reduce cancer cell migration and may provide novel opportunities to address/prevent bone metastasis.

Despite the recent advances in the treatment of tumors with intrinsic chemotherapy resistance, such as melanoma and renal cancers, their prognosis remains poor and new chemical agents with promising activity against these cancers are urgently needed. Sphaeropsidin A, a fungal metabolite whose anticancer potential had previously received little attention, was isolated from Diplodia cupressi and found to display specific anticancer activity in vitro against melanoma and kidney cancer subpanels in the National Cancer Institute (NCI) 60-cell line screen. The NCI data revealed a mean LC50 of ca. 10 µM and a cellular sensitivity profile that did not match that of any other agent in the 765,000 compound database. Subsequent mechanistic studies in melanoma and other multidrug-resistant in vitro cancer models showed that sphaeropsidin A can overcome apoptosis as well as multidrug resistance by inducing a marked and rapid cellular shrinkage related to the loss of intracellular Cl− and the decreased HCO3 − concentration in the culture supernatant. These changes in ion homeostasis and the absence of effects on the plasma membrane potential were attributed to the sphaeropsidin A-induced impairment of regulatory volume increase (RVI). Preliminary results also indicate that depending on the type of cancer, the sphaeropsidin A effects on RVI could be related to Na–K–2Cl electroneutral cotransporter or Cl−/HCO3 − anion exchanger(s) targeting. This study underscores the modulation of ion-transporter activity as a promising therapeutic strategy to combat drug-resistant cancers and identifies the fungal metabolite, sphaeropsidin A, as a lead to develop anticancer agents targeting RVI in cancer cells.

The SK3 potassium channel is involved in the development of bone metastasis and in the settlement of cancer cells in Ca(2+) -rich environments. Ohmline, which is a lactose-based glycero-ether lipid, is a lead compound that decreases SK3 channel activity and consequently limits the migration of SK3-expressing cells. Herein we report the synthesis of three new ohmline analogues in which the connection of the disaccharide moieties (1→6 versus 1→4) and the stereochemistry of the glycosyl linkage was studied. Compound 2 [3-(hexadecyloxy)-2-methoxypropyl-6-O-α-d-glucopyranosyl-β-d-galactopyranoside], which possesses an α-glucopyranosyl-(1→6)-β-galactopyranosyl moiety, was found to decrease SK3 current amplitude (70 % inhibition at 10 μm), displace SK3 protein outside caveolae, and decrease constitutive Ca(2+) entry (50 % inhibition at 300 nm) and SK3-dependent cell migration (30 % at 300 nm) at a level close to that of the benchmark compound ohmline. Compound 2, which decreases the activity of SK3 channel (but not SK2 channel), is a new drug candidate to reduce cancer cell migration and to prevent bone metastasis.

// Maxime Gueguinou 1, 9 , Thomas Harnois 2, 9 , David Crottes 3 , Arnaud Uguen 6, 7 , Nadine Deliot 2, 9 , Audrey Gambade 1 , Aurelie Chantome 1, 9 , Jean Pierre Haelters 4, 9 , Paul Alain Jaffres 4, 9 , Marie Lise Jourdan 1, 10 , Gunther Weber 1 , Olivier Soriani 8 , Philippe Bougnoux 1, 9, 10 , Olivier Mignen 6, 9 , Nicolas Bourmeyster 2, 9 , Bruno Constantin 2, 9 , Thierry Lecomte 5, 9, 10 , Christophe Vandier 1, 9, * , Marie Potier-Cartereau 1, 9, * 1 INSERM UMR 1069, Universite de Tours, Tours, France 2 Equipe ERL 7368, CNRS, Universite de Poitiers, Poitiers, France 3 Department of Physiology, University of California, San Francisco, San Francisco, CA, USA 4 CNRS-UMR 6521-Universite de Brest, Brest, France 5 GICC-UMR 7292 Universite de Tours, Tours, France 6 INSERM-UMR 1078 Universite de Brest, Brest, France 7 CHRU Brest, Service d’Anatomie et Cytologie Pathologiques, Brest, France 8 CNRS UMR 7299, INSERM-UMR 1099, Universite de Nice Sophia-Antipolis, Nice, France 9 Ion Channels Network and Cancer-Canceropole Grand Ouest (IC-CGO), France 10 CHRU Tours, Tours, France * These authors have contributed equally to this work Correspondence to: Marie Potier-Cartereau, email: marie.potier-cartereau@univ-tours.fr Keywords: SOCE, Ohmline, lipid-raft channel complex, anti-EGFR mAbs, Akt signaling Received: September 24, 2015 Accepted: March 28, 2016 Published: April 18, 2016 ABSTRACT 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.

International audience; Tight control of basal cytosolic Ca2+ concentration is essential for cell survival and to fine-tune Ca2+-dependent cell functions. A way to control this basal cytosolic Ca2+ concentration is to regulate membrane Ca2+ channels including store-operated Ca2+ channels and secondary messenger-operated channels linked to G-protein-coupled or tyrosine kinase receptor activation. Orai, with or without its reticular STIM partner and Transient Receptor Potential (TRP) proteins, were considered to be the main Ca2+ channels involved. It is well accepted that, in response to cell stimulation, opening of these Ca2+ channels contributes to Ca2+ entry and the transient increase in cytosolic Ca2+ concentration involved in intracellular signaling. However, in various experimental conditions, Ca2+ entry and/or Ca2+ currents can be recorded at rest, without application of any experimental stimulation. This led to the proposition that some plasma membrane Ca2+ channels are already open/activated in basal condition, contributing therefore to constitutive Ca2+ entry. This article focuses on direct and indirect observations supporting constitutive activity of channels belonging to the Orai and TRP families and on the mechanisms underlying their basal/constitutive activities.

BACKGROUND AND PURPOSE The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelfosine) is an ether-linked phospholipid with promising anti-cancer properties but some side effects that preclude its full clinical therapeutic exploitation. We hypothesized that this lipid could interact with plasma membrane ion channels and modulate their function. EXPERIMENTAL APPROACH Using cell migration-proliferation assays, patch clamp, spectrofluorimetry and 125I-Apamin binding experiments, we studied the effects of edelfosine on the migration of breast cancer MDA-MB-435s cells, mediated by the small conductance Ca2+-activated K+ channel, SK3/KCa2.3. KEY RESULTS Edelfosine (1 µM) caused plasma membrane depolarization by substantially inhibiting activity of SK3/KCa2.3 channels, which we had previously demonstrated to play an important role in cancer cell migration. Edelfosine did not inhibit 125I-Apamin binding to this SKCa channel; rather, it reduced the calcium sensitivity of SK3/KCa2.3 channel and dramatically decreased intracellular Ca2+ concentration, probably by insertion in the plasma membrane, as suggested by proteinase K experiments. Edelfosine reduced cell migration to the same extent as known SKCa channel blockers. In contrast, K+ channel openers prevented edelfosine-induced anti-migratory effects. SK3 protein knockdown decreased cell migration and totally abolished the effect of edelfosine on MDA-MB-435s cell migration. In contrast, transient expression of SK3/KCa2.3 protein in a SK3/KCa2.3-deficient cell line increased cell migration and made these cells responsive to edelfosine. CONCLUSIONS AND IMPLICATIONS Our data clearly establish edelfosine as an inhibitor of cancer cell migration by acting on SK3/KCa2.3 channels and provide insights into the future development of a new class of migration-targeted, anti-cancer agents.

64 p.-6 fig., Synthetic alkyl lipids, such as the ether lipids edelfosine (1-O-octadecyl-2-O-methyl-rac- glycero-3-phosphocholine) and ohmline (1-O-hexadecyl-2-O-methyl-rac-glycero-3-β- lactose), are forming a class of antitumor agents that target cell membranes to induce apoptosis and to decrease cell migration/invasion, leading to the inhibition of tumor and metastasis development. In this review, we present the structure-activity relationship of edelfosine and ohmline, and we point out differences and similarities between these two amphiphilic compounds. We also discuss the mechanisms of action of these synthetic alkyl ether lipids (involving, among other structures and molecules, membrane domains, Fas/CD95 death receptor signaling, and ion channels), and highlight a key role for lipid rafts in the underlying process. The reorganization of lipid raft membrane domains induced by these alkyl lipids affects the function of death receptors and ion channels, thus leading to apoptosis and inhibition of cancer cell migration. The possible therapeutic use of these alkyl lipids and the clinical perspectives for these lipids in prevention or/and treatment of tumor development and metastasis are also discussed., This work was supported by the Agence Nationale de la Recherche N°ANR-08-EBIO-020-01, la Ligue Contre le Cancer, Region Centre (LIPIDS project of ARD2020-Biomédicaments), INSERM, CNRS, Cancéropôle Grand Ouest, the association “CANCEN”, Tours’ Hospital oncology association ACORT", grants from Spanish Ministerio de Economia y Competitividad (SAF2011-30518, SAF2014-59716-R, and RD12/0036/0065 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union), and European Community’s Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011- 256986, PANACREAS). Ana Bouchet holds a post-doc mobility grant from PRESTIGE-European FP7 / University of Tours.

GRAPHICAL ABSTRACTAbstract. In this short review paper we reports our efforts to design new inhibitors of SK3 ion channel that are involved in cancer cell migration and metastases formation. The originality of the new SK3 inhibitors comes from their amphiphilic nature and contrast form others inhibitors that are peptides or heterocyclic compounds. We found that ether lipids and ether phospho-glyco-lipids are indeed efficient to modulate SK3 ion channel that consequently reduced cancer cell migration (in vitro assays) and abolished the formation of bone metastases in a murine model of breast cancer.

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.

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.

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 Ca2+ entry and cancer cell migration through an interaction with the Ca2+ 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 Ca2+ 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. Cancer Res; 73(15); 4852–61. ©2013 AACR.

International audience; The recent discoveries of the involvement of SK3 channel in some cell motility mechanisms occurring in cancer disease have opened up the way to the synthesis of inhibitors that could reduce metastasis formation. On the basis of our recent previous works showing that both lactose-glycero-ether lipid () and some phosphate analogues () were efficient compounds to modulate SK3 channel activity, the present study, which found its inspiration in the structure of the natural glycolipid DiGalactosylDiacylGlycerol (DGDG), reports the incorporation of a digalactosyl moiety (α-galactopyranosyl-(1→6)-β-galactopyranosyl-) as the polar head of a glycero ether lipid. For the construction of the digalactosyl fragment, two synthetic approaches were compared. The standard strategy which is based on the use of the benzyl protecting group to produce 1→6 disaccharide unit, was compared with a second method that made use of the trimethylsilyl moiety as a protecting group. This second strategy, which is applied for the first time to the synthesis of (1→6)-disaccharide unit, presents a net advantage in terms of efficacy (better global yield) and cost. Finally, compound , which is characterized by a (1→6) DiGalactosyl unit (DG) as the polar head of the amphiphilic structure, was tested as a modulator of the SK3 channel activity. Patch-clamp experiments have shown that compound reduced SK3 currents (-28.2 ± 2.0% at 5 μM) and cell migration assays performed at 300 nM have shown a reduction of cell migration (SK3 + HEK293T) by 19.6 ± 2.7%.

The human inducible nitric oxide synthase (iNOS or NOSII) gene is regulated through an extended and complex promoter. In this study, the transcriptional regulation of human NOSII is investigated in the human colon cell line HCT-8R. Stimulation with a cytokine mix (interferon-gamma, interleukin 1-beta, and tumor necrosis factor alpha) induces NOSII mRNA accumulation, as well as promoter activity in these cells. Several random deletions were performed within the proximal 7 kb of the promoter, which led to the identification of a region, whose deletion provokes a marked increase in transcriptional activity upon cytokine stimulation. Furthermore, this region is shown to repress a viral-driven luciferase construct, mainly at basal levels. An AP-1-like sequence present in this region that is specifically recognized by nuclear proteins is shown to be involved in the repressive effect. This element is capable of repressing a viral promoter, and its deletion augments cytokine-stimulated transcription. These findings are confirmed in various cell lines and suggest a general mechanism for the control of basal levels of NOSII expression, to avoid unnecessary toxicity under normal conditions.

International audience; 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.

Expression of the human inducible nitric oxide synthase (hiNOS) is generally undetectable in resting cells, but stimulation by a variety of signals including cytokines induces transcription in most cell types. The tight transcriptional regulation of the enzyme is a complex mechanism many aspects of which remain unknown. Here, we describe an octamer (Oct) element in hiNOS proximal promoter, located close to the TATA box. This site constitutively binds Oct-1 and its deletion abrogates cytokine-induced transcription, showing that it is indispensable though not sufficient for transcription. Increasing the distance between Oct and the TATA box by inserting inert DNA sequence inhibits transcription, and footprinting of this region shows no other protein binding in resting cells, suggesting an interaction between the two complexes. Chromatin immunoprecipitation assays detect the presence of Oct-1, RNA polymerase II and trimethyl K4 histone H3 on the proximal promoter in resting cells, confirming that the gene is primed for transcription before stimulation. RT-PCR of various fragments along the hiNOS gene shows that transcription is initiated in resting cells and this is inhibited by interference with Oct-1 binding to the proximal site of the promoter. We propose that, through interaction with the initiation complex, Oct-1 regulates hiNOS transcription by priming the gene for the rapid response required in an immune response.

Even cancer is far from being considered a channelopathy; the field of ion and protein channel research in cancer is highly important as an emerging and proven point of intervention in disease. Like membrane receptors, ion channels are directly connected with and sensitive to the extracellular environment. During the last decade, the number of ionchannel types expressed in various cancers, including melanoma, was rapidly increased. Moreover several ion channels are selectively expressed in aggressive cancers and seem to be implicated in metastasis development. The growing number of patents relative to cancer therapy targeting channel proteins testifies to the interest of such novel therapeutic approaches. The physiological significance of ion channels and transporters, as illustrated by the award of four Nobel Prizes in Physiology or Medicine (1963; 1991) and Chemistry (1997, 2003), is now accepted and established. Unlike transporters and exchangers, channel proteins form a pore through membranes allowing the selective passage of one or more ions (e.g. K+, Na+, Cl-), molecules (water) or charged atoms, through the lipid bilayer that is impermeable to these compounds. The modalities of channel opening or activation are diverse and varied: this can be performed by an external molecular stimulus (e.g. ligand), by a mechanical stimulus (e.g. cell volume, membrane tension or stretch), by electric stimuli (e.g. changes in membrane potential), by an intracellular second messenger (e.g. calcium, cAMP). Thus the classification of the channels (IUPHAR classification) is based on the channel activation mode and on the selective permeability of molecular species specific to each channel. Channel proteins are involved in the control of numerous and various physiological functions. Basically, these channels are responsible for a universal property for cellular membranes: the existence of resting membrane potential. Ion channels, mainly studied in excitable cells like muscle and neurons, are responsible for the transmission of the electric signals triggering physiological and biological phenomena such as nerve conduction or the cellular phenomenon of excitation contraction coupling. Ion transporter (Na+/K+-ATPase or simply known as sodium pump) is the membrane pump that generates the Na+ and K+ gradients across the plasma membrane, driving many physiological processes. Another

International audience; Edelfosine is an inhibitor of SK3 channel-mediated cell migration. However, this compound bears adverse in vivo side effects. Using cell SK3 dependent cell-migration assay, patch-clamp, 125I-apamin binding, and in vivo experiments we tested the ability of 15 lipid derivatives with chemical structures inspired from edelfosine to inhibit SK3 channels. Using a structure-activity relationship approach we identified an edelfosine analog named Ohmline (1-O-hexadecyl-2-O-methyl-sn-glycero-3-lactose) with potent inhibitory effects on the SK3 channel. Its potency was greater for SK3 channels than for SK1 channels; it did not affect IKCa channels and only slightly but not significantly affected SK2 channels. This is the first SKCa channel blocker that can be used to discriminate between SK2 and SK1/SK3 channels and represents a useful tool to investigate the functional role of SK3 channels in peripheral tissues (that do not express SK1 channels). This compound, which acts with an IC50 of 300 nM, did not displace apamine from SKCa channels and had no effect on non-specific edelfosine targets such as protein kinase C (PKC), receptors for platelet activating factor (PAF) and lysophosphatidic acid (LPA), as well as non-cancerous cells. This is promising because the pitfalls associated with the use of edelfosine-like compounds have been that their effective and high concentrations are often cytotoxic due to their detergent-like character causing normal cell lysis. Finally, Ohmline reduced metastasis development in a mice model of tumor indicating that this compound could become a lead compound for the first class of lipid-antimetastatic agent.

International audience; Edelfosine is an inhibitor of SK3 channel – mediated cell migration. However, this compound bears adverse in vivo side effects. Using cell SK3 dependent cell-migration assay, patch-clamp, 125I-apamin binding, and in vivo experiments we tested the ability of 15 lipid derivatives with chemical structures inspired from edelfosine to inhibit SK3 channels. Using a structure-activity relationship approach we identified an edelfosine analog named Ohmline (1-O-hexadecyl- 2-O-methyl-sn-glycero-3-lactose) with potent inhibitory effects on the SK3 channel. Its potency was greater for SK3 channels than for SK1 channels; it did not affect IKCa channels and only slightly but not significantly affected SK2 channels. This is the first SKCa channel blocker that can be used to discriminate between SK2 and SK1/SK3 channels and represents a useful tool to investigate the functional role of SK3 channels in peripheral tissues (that do not express SK1 channels). This compound, which acts with an IC50 of 300 nM, did not displace apamin from SKCa channels and had no effect on non-specific edelfosine targets such as protein kinase C (PKC), receptors for platelet activating factor (PAF) and lysophosphatidic acid (LPA), as well as non-cancerous cells. This is promising because the pitfalls associated with the use of edelfosine-like compounds have been that their effective and high concentrations are often cytotoxic due to their detergent-like character causing normal cell lysis. Finally, Ohmline reduced metastasis development in a mice model of tumor indicating that this compound could become a lead compound for the first class of lipid-antimetastatic agent.

This paper deals with the problem of counting nuclei in low-resolution fluorescence images. The low resolution is a consequence of the usage of a large field of view, this to reduce the number of experiments, implying time saving and money saving. But the small size of nuclei increases the risk of error in counting. In this work we used some image processing basic operators in order to extract potential shapes. These shape are then characterized (size, shape, edges) to decide if they are noise or not. Two approaches are presented for this: the first one correspond to a translation of some basic knowledge used by practitioners; the second one uses a supervised classification technique where it is the computer who discovers the knowledge by the analysis of a database of cases. This leads to a fast technique which gives very good results, validated by cell biology experts.

International audience; Lost of adenomatous polyposis coli gene (Apc) disturbs the migration of intestinal epithelial cells but the mechanisms have not been fully characterized. Since we have demonstrated that SK3/KCa2.3 channel promotes cancer cell migration, we hypothesized that Apc mutation may affect SK3/KCa2.3 channel-mediated colon epithelial cell motility. We report evidence that SK3/KCa2.3 channel promotes colon epithelial cells motility. Following Apc mutation SK3/KCa2.3 expression is largely reduced leading to a suppression of the SK3/KCa2.3 channel mediated-cell migration. Our findings reveal a previously unknown function of the SK3/KCa2.3 channel in epithelial colonic cells, and suggest that Apc is a powerful regulator SK3/KCa2.3 channel.

International audience; This paper deals with the problem of counting nuclei in low-resolution fluorescence images. The low resolution is a consequence of the usage of a large field of view, this to reduce the number of experiments, implying time saving and money saving. But the small size of nuclei increases the risk of error in counting. In this work we used some image processing basic operators in order to extract potential shapes. These shape are then characterized (size, shape, edges) to decide if they are noise or not. Two approaches are presented for this: the first one correspond to a translation of some basic knowledge used by practitioners; the second one uses a supervised classification technique where it is the computer who discovers the knowledge by the analysis of a database of cases. This leads to a fast technique which gives very good results, validated by cell biology experts.

Oct-1 (POU2f1) and Oct-2 (POU2f2) are members of the POU family of transcription factors. They recognize the same DNA sequence but fulfil distinct functions: Oct-1 is ubiquitous and regulates a variety of genes while Oct-2 is restricted to B-cells and neurones. Here we examine the interplay and regulatory mechanisms of these factors to control the inducible nitric oxide synthase (iNOS, NOS2). Using two breast cancer cell lines as a comparative model, we found that MCF-7 express iNOS upon cytokine stimulation while MDA-MB-231 do not. Oct-1 is present in both cell lines but MDA-MB-231 also express high levels of Oct-2. Manipulation of Oct-2 expression in these cell lines demonstrates that it is directly responsible for the repression of iNOS in MDA-MB-231. In MCF-7 cells Oct-1 binds the iNOS promoter, recruits RNA PolII and triggers initiation of transcription. In MDA-MB-231 cells, both Oct-1 and Oct-2 bind the iNOS promoter, forming a higher-order complex which fails to recruit RNA PolII, and as a consequence iNOS transcription does not proceed. Unravelling the mechanisms of transcription factor activity is paramount to the understanding of gene expression patterns that determine cell behaviour.

To study the role of nitric oxide (NO) in lung metastasis of breast carcinoma, we isolated two cell clones (H and J) from the parental EMT-6 murine breast carcinoma cell line, based on their differential NO production. In vitro, EMT-6 J cells, but not EMT-6H cells, constitutively expressed inducible NO synthase (NOS II) and secreted high levels of NO. IL-1beta increased NO production in both clones, and TNF-alpha had a synergistic effect on IL-1beta-induced NO production, but NO production by EMT-6 J cells was always higher than by EMT-6H cells. Proliferation, survival and adhesion to lung-derived endothelial cells of both clones were similar and were not affected by NO. In vivo, both clones similarly located in the lungs of syngeneic mice 48 h after injection. However, EMT-6H cells were significantly more tumorigenic than EMT-6 J cells as assessed at later time points. Injection of EMT-6 J cells and simultaneous treatment of mice with aminoguanidine (AG), a NOS II inhibitor, significantly increased tumour formation. Injection of EMT-6H and EMT-6 J cells into NOS II-deficient mice resulted in a significant survival increase as compared with wild-type animals. Simultaneous administration of AG increased the death rate of NOS II-deficient mice injected with EMT-6 J cells. These results demonstrate that: (i) NO does not influence the early stages of tumour metastasis to the lungs and (ii) NOS II expression in tumour cells reduces, while NOS II expression in host cells enhances, tumour nodule development. In conclusion, the cellular origin and the local NO production are critical in the metastatic process.

NO is a molecule produced in different amounts by two types of enzymes, constitutive NO-synthases and inducible NO-synthase, the last one produce large amount of NO. In tumor outcome, its role may be either beneficial or detrimental due to its actions in the different steps of tumor growth and metastasis. This review deals with mammary tumors and the mechanisms lying behind NO effects. In human patients, increased amounts of NO were evidenced in blood, linked with the presence and activity of NO-synthase in breast tumors. Non-unequivocal correlations were established with tumor grade, invasiveness and metastatic potential. Studies in animal models have given hints to explain these discrepancies by the type of the involved NO-synthase, the amount of NO it produces, and its belonging to tumoral or stromal cells. Indeed, it was recently shown that NO produced by tumor cells inhibits, while NO produced by stromal cells facilitates tumor growth, at least in the model which was studied. On the one hand, NO toxicity against tumor cells is a well known phenomenon, but on the other hand, NO may promote tumor invasiveness due to its effect on extracellular matrix, and to its angiogenetic properties. So the role of NO in mammary tumor outcome is not clear-cut, and, at the present time, it cannot be ascribed a pronostic value in breast tumor. However, researches aimed at managing tumor cells to produce NO sufficient to induce their death may be fruitful since, be tumor targeting successful, no general toxicity would be encountered.

Nitric oxide (NO) produced by inducible nitric-oxide synthase (NOSII) is mainly regulated at the transcriptional level by the nuclear factor-kappaB (NF-kappaB). In the present study, we further analyzed the role of NF-kappaB in the in vivo transcriptional regulation of NOSII gene by comparing two clones isolated from the EMT-6 mouse mammary cancer cell line. In response to interleukin (IL)-1beta or lipopolysaccharide (LPS), EMT-6 clone J (EMT-6J) cells produce 3-fold more NO than EMT-6 clone H (EMT-6H) cells, an effect correlated with enhanced activation of NF-kappaB in EMT-6J cells. In response to IL-1beta, the kinetics of degradation of NF-kappaB inhibitors IkappaB-alpha and IkappaB-beta, the nucleo-cytoplasmic shuttling of the transcription factor and its binding to a specific DNA sequence were similar in both clones. In contrast, an IL-1beta-induced phosphorylation of serine residues in NF-kappaB p65 subunit was observed in EMT-6J, but not in EMT-6H, cells. This IL-1beta-induced phosphorylation of p65 was specifically prevented by pretreatment of EMT-6J cells with the casein kinase II inhibitor DRB. Small interfering RNA-mediated depletion of casein kinase II-alpha subunit also decreased NF-kappaB transcriptional activity and NOSII gene transcription in IL-1beta and LPS-stimulated EMT-6J cells to the levels observed in EMT-6H cells treated in the same conditions. Altogether, these data indicate that casein kinase II-mediated phosphorylation of p65 subunit can enhance the transcriptional activity of NF-kappaB in vivo. This post-translational modification of the transcription factor can be responsible for increased NOSII gene transcription and NO production in tumor cells exposed to either IL-1beta or LPS.

HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-alpha). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-kappaB by degrading its main inhibitor, I-kappaBalpha. HSP27 overexpression increases NF-kappaB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, TNF-alpha, and interleukin 1beta. HSP27 does not affect I-kappaBalpha phosphorylation but enhances the degradation of phosphorylated I-kappaBalpha by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-kappaBalpha. A protein complex that includes HSP27, phosphorylated I-kappaBalpha, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-kappaBalpha. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-kappaB activity.