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-␣). 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-B by degrading its main inhibitor, I-B␣. HSP27 overexpression increases NF-B nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ⌻NF-␣, and interleukin 1␤. HSP27 does not affect I-B␣ phosphorylation but enhances the degradation of phosphorylated I-B␣ by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-B␣. A protein complex that includes HSP27, phosphorylated I-B␣, 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-B␣. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-B activity.

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 2þ entry and cancer cell migration through an interaction with the Ca 2þ 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-offunction, 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 2þ 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. Ó2013 AACR.

ATP-gated P2X 7 receptors (P2X 7 R) are unusual plasma membrane ion channels that have been extensively studied in immune cells. More recently, P2X 7 R have been described as potential cancer cell biomarkers. However, mechanistic links between P2X 7 R and cancer cell processes are unknown. Here, we show, in the highly aggressive human breast cancer cell line MDA-MB-435s, that P2X 7 receptor is highly expressed and fully functional. Its activation is responsible for the extension of neurite-like cellular prolongations, of the increase in cell migration by 35% and in cell invasion through extracellular matrix by 150%. The change in cancer cell morphology and the increased migration appeared to be due to the activation of Ca 2 þ -activated SK3 potassium channels. The enhanced invasion through the extracellular matrix was related to the increase of mature forms of cysteine cathepsins in the extracellular medium, which was independent of SK3 channel activity and not associated with cell death. Pharmacological targeting of P2X 7 R in vivo was crucial for cell invasiveness in a zebrafish model of metastases. Our results demonstrate a novel mechanistic link between P2X 7 R functionality in cancer cells and invasiveness, a key parameter in tumour growth and in the development of metastases. They also suggest a potential therapeutic role for the newly developed P2X 7 R antagonists.

BackgroundBarely 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.ResultsIn 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.ConclusionsThis 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.

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 Ca-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 Ca 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 Ca homeostasis in epithelial cancers.

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.

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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, (125)I-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 IC(50) 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.