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Sig1R silencing inhibits CRC HCT-116 cell motility

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K+ channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition. Cancer Res; 76(3); 607–18. ©2015 AACR.

Sig1R is associated to β1 integrin and promotes β1 integrin / hERG association without altering β1 integrin membrane expression

Control experiments showing specificity of PLA assay between hERG and β1 integrin

Sig1R promotes hERG- and AKT-dependent FDM-induced cell spreading, VEGF secretion and endothelial transmigration

FDM induces a stable increase in hERG current in a Sig1R-dependent manner but does not alter channel maturation

Simple Summary Medulloblastoma is the most frequent pediatric brain cancer. Despite great improvements in the treatment of this disease over the last decades, survivors are subject to debilitating adverse effects that strongly impair their quality of life. There is an urgent need to find efficient anticancer therapies with fewer toxic effects. In this study, we suggest that an FDA- and EMA-approved antiangiogenic compound named axitinib may display effective antitumoral effects and low toxicity towards children as compared to a reference treatment currently used in clinical protocols. We also show that this compound can enter the brain compartment and exert antitumoral effects in vivo. Our study paves the way towards a clinical trial of repurposing axitinib to a pediatric brain cancer indication. Abstract Background: Despite the improvement of medulloblastoma (MB) treatments, survivors face severe long-term adverse effects and associated morbidity following multimodal treatments. Moreover, relapses are fatal within a few months. Therefore, chemotherapies inducing fewer adverse effects and/or improving survival at relapse are key for MB patients. Our purpose was to evaluate the last-generation antiangiogenic drugs for their relevance in the therapeutic arsenal of MB. Methods: We screened three EMA- and FDA-approved antiangiogenic compounds (axitinib, cabozantinib and sunitinib) for their ability to reduce cell viability of five MB cell lines and their low toxicity towards two normal cell lines in vitro. Based on this screening, single-agent and combination therapies were designed for in vivo validation. Results: Axitinib, cabozantinib and sunitinib decreased viability of all the tested tumor cells. Although sunitinib was the most efficient in tumor cells, it also impacted normal cells. Therefore, axitinib showed the highest selectivity index for MB cells as compared to normal cells. The compound did not lead to acute toxicity in juvenile rats and crossed the blood–brain barrier. Moreover, axitinib efficiently reduced the growth rate of experimental brain tumors. Analysis of public databases showed that high expression of axitinib targets correlates with poor prognosis. Conclusion: Our results suggest that axitinib is a compelling candidate for MB treatment.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-020-01502-2.

Medulloblastoma (MB), the most common brain pediatric tumor, is a pathology composed of four molecular subgroups. Despite a multimodal treatment, 30% of the patients eventually relapse, with the fatal appearance of metastases within 5 years. The major actors of metastatic dissemination are the lymphatic vessel growth factor, VEGFC, and its receptors/co-receptors. Here, we show that VEGFC is inversely correlated to cell aggressiveness. Indeed, VEGFC decreases MB cell proliferation and migration, and their ability to form pseudo-vessel in vitro. Irradiation resistant-cells, which present high levels of VEGFC, lose the ability to migrate and to form vessel-like structures. Thus, irradiation reduces MB cell aggressiveness via a VEGFC-dependent process. Cells intrinsically or ectopically overexpressing VEGFC and irradiation-resistant cells form smaller experimental tumors in nude mice. Opposite to the common dogma, our results give strong arguments in favor of VEGFC as a negative regulator of MB growth., Manon Penco-Campillo, Yannick Comoglio et al. show that VEGFC decreases the proliferation and migration of medulloblastoma cells, as well as their ability to form pseudo vessels. Cells expressing high levels of VEGFC also form smaller tumors when subcutaneously injected into the flank of nude mice, thus highlighting a negative regulatory role for VEGFC on tumor growth.

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K+ channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition. Cancer Res; 76(3); 607–18. ©2015 AACR.

International audience; Angiogenesis is a finely tuned process, which is the result of the equilibrium between pro- and antiangiogenic factors. In solid tumor angiogenesis, the balance is highly in favor of the production of new, but poorly functional blood vessels, initially intended to provide growing tumors with nutrients and oxygen. Among the numerous proteins involved in tumor development, several types of ion channels are overexpressed in tumor cells, as well as in stromaland endothelial cells. Ion channels thus actively participate in the different hallmarks of cancer, especially in tumor angiogenesis and metastasis. Indeed, from their strategic localization in the plasma membrane, ion channels are key operators of cell signaling, as they sense and respond to environmental changes. This review aims to decipher how ion channels of different families are intricately involved in the fundamental angiogenesis and metastasis hallmarks, which lead from a nascent tumor to systemic dissemination. An overview of the possible use of ion channelsas therapeutic targets will also be given, showing that ion channel inhibitors orspecific antibodies may provide effective tools, in the near future, in the treatmentof carcinomas.

Active urea transport in the rat inner medullary collecting duct: Functional characterization and initial expression cloning. Active transport of urea has been proposed to exist inthe inner medullary collecting duct (IMCD) of low-protein fed mammals for over 30 years. We perfused IMCD subsegments from rats fed a standard (18%) or a low (8%) protein diet and tested for the presence of active urea transport. We found no active urea transport in terminal IMCDs, regardless of diet. In initial IMCDs from rats fed 18% protein or fed 8% protein for one to two weeks, we again found no active urea transport. However, in rats fed 8% protein for three to four weeks, we found significant net urea reabsorption. This active urea reabsorption was inhibited when Na + ,K + -ATPase activity was inhibited by adding 1mM ouabain or removing bath potassium, suggesting a secondary active transport process. Removing sodium from the perfusate completely inhibited net urea reabsorption, demonstrating that this active urea transport is dependent upon the presence of sodium in the tubule lumen. Unlike the facilitated urea transporter, the active urea transporter was not inhibited by phloretin nor stimulated by vasopressin, suggesting that it is a distinct transport protein. To test this hypothesis, we size-separated poly(A) + -RNA preparedfrom inner medullae of rats fed 8% protein for three weeks and injected it into Xenopus laevis oocytes. RNA froma 4.4 to 8.4kb size fraction increased urea permeability fourfold compared to water-injected oocytes or injecting RNA from other size-fractions. We conclude that feeding rats a low-protein diet for three weeks induces the expression of an unique, secondary active, sodium-dependent urea transporter whose cDNA is between 4.4 and 8.4kb in size. In addition, our results suggest that it will be possible to clone the cDNA for this sodium-urea cotransporter by expression in Xenopus laevis oocytes.

International audience; In mammals, urea is the predominant end-product of nitrogen metabolism and plays a central role in the urinary-concentrating mechanism. Urea accumulation in the renal medulla is critical to the ability of the kidney to concentrate urine to an osmolality greater than systemic plasma. Regulation of urea excretion and accumulation in the renal medulla depends on the functional state of specialized phloretin-sensitive urea transporters. To study these transporters and their regulation of expression we isolated a cDNA which encodes the rat homologue (rUT2) of rabbit UT2 (You, G., C. P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M. A. Hediger, et al. Nature (Lond.). 1993.365:844-847). Rat UT2 has 88% amino acid sequence identity to rabbit UT2 and 64% identity to the recently cloned human erythrocyte urea transporter, HUT1l (Olives, B., P. Neav, P. Bailly, M. A. Hediger, G. Rousselet, J. P. Cartron, and P. Ripoch J. Biol. Chem. 1994. 269:31649-31652). Analysis of rat kidney mRNA revealed two transcripts of size 2.9 and 4.0 kb which had spatially distinct distributions. Northern analysis and in situ hybridization showed that the 4.0-kb transcript was primarily responsive to changes in the protein content of the diet whereas the 2.9-kb transcript was responsive to changes in the hydration state of the animal. These studies reveal that the expression levels of the two rUT2 transcripts are modulated by different pathways to allow fluid and nitrogen balance to be regulated independently. Our data provide important insights into the regulation of the renal urea transporter UT2 and provide a basis on which to refine our understanding of the urinary concentrating mechanism and its regulation.

International audience; Sigma-1 receptor (Sig1R) is a 25-kDa protein structurally unrelated to other mammalian proteins. Sig1R is present in brain, liver, and heart and is overexpressed in cancer cells. Studies using exogenous sigma ligands have shown that Sig1R interact with a variety of ion channels, but its intrinsic function and mechanism of action remain unclear. The human ether-a-gogo related gene (hERG) encodes a cardiac channel which is also abnormally expressed in many primary human cancers, potentiating tumor progression through the modulation of extracellular matrix adhesive interactions. We show herein that sigma ligands inhibit hERG current density and cell adhesion to fibronectin in K562 myeloid leukemia cells. Heterologous expression in Xenopus oocytes demonstrates that Sig1R potentiates hERG current by stimulating channel subunit biosynthesis. Silencing Sig1R in leukemic K562 cells depresses hERG current density and cell adhesion to fibronectin by reducing hERG membrane expression. In K562 cells, Sig1R silencing does not modify hERG mRNA contents but reduces hERG mature forms densities. In human embryonic kidney (HEK) cells expressing hERG and Sig1R, both protein co-immunoprecipitate demonstrating a physical association. Finally, Sig1R expression enhances both channel protein maturation and stability. Altogether, these results demonstrate for the first time that Sig1R controls ion channel expression through the regulation of subunit trafficking activity.

Newborn rat distal cells express an apical Ca2+ channel activated by dihydropyridine drugs. Similarly, in Madin-Darby canine kidney (MDCK) cells, nifedipine increased Ca2+i in a concentration-dependent manner (IC50 = 4 µM) in fura-2-loaded cells. Response to nifedipine was abolished by EGTA, suggesting that it depends on extracellular calcium. Ca2+ channel antagonist isradipine and agonist BayK8644 increased Ca2+i indicating that this effect is related to the dihydropyridine group. Diltiazem (20 µM) and gadolinium (200 µM) decreased the nifedipine effect (62 and 43%, respectively). Lanthanum (100 µM) did not change the response. Valinomycin clamping of the membrane potential did not modify nifedipine-induced increment, indicating that it was unrelated to potassium fluxes. We performed whole cell clamp experiments in MDCK cells maintained at –50 mV with perfusion solution containing 10 mM CaCl2. Nifedipine (20 µM) induced an increase in current (1.2 ± 0.3 nA), which was partially inhibited by Gd3+. No significant current was induced by nifedipine in the presence of 0.5 mM EGTA. To determine the effects of nifedipine on the membrane potential, we performed oxonol fluorescence experiments. The addition of nifedipine or Bay K8644 induced depolarization, highly dependent on external sodium. Nifedipine (20 µM) induced depolarization of 6.9 ± 0.8 mV (n = 21). EC50 to nifedipine was in the 10 µM range. We conclude that MDCK cells exhibit a dihydropyridine-activated cationic channel.

International audience; In this study, we devised a cysteine-focused point mutation analysis of the chloride channel function of trout anion exchanger 1 (tAE1) expressed in X. laevis oocytes. Seven cysteines, belonging to the transmembrane domain of tAE1, were mutated into serines (either individually or in groups) and the effects of these mutations on the chloride conductance of injected oocytes were measured. We showed that three cysteines were essential for the functional expression of tAE1. Namely, mutations C462S, C583S and C588S reduced Cl À conductance by 68%, 52% and 83%, respectively, when compared to wild type tAE1. These residual conductances were still inhibited by 0.5 mM niflumic acid. Western blot experiments demonstrated that C462 was involved in protein expression onto the plasma membrane. A mutant devoid of this residue was unable to express onto the plasma membrane, especially if several other cysteines were missing: consequently, the cysteine-less mutant of tAE1 was not functional. C583 and C588 were involved in the channel function of tAE1 as shown by anion substitution experiments proving that selectivity of the mutated pore differs from the wild type one. On the contrary, they were not involved in the Cl À /HCO À 3 exchange function of tAE1, as demonstrated by intracellular pH measurements. These and several complementary mutations allow us to conclude that a mutant of tAE1 containing the sole C462 can drive a marginal Cl À current; however, the minimal configuration necessary to get optimal functional expression of the tAE1 chloride channel is that of a mutant containing unaffected residues C462, C583 and C588.

Conclusions Les microglies provenant de la moelle osseuse offrent un potentiel formidable en vue du traitement de la maladie d’Alzheimer, puisqu’elles sont attirees vers les proteines toxiques et sont en mesure de les eliminer. Il est interessant de noter que des proteines toxiques sont presentes dans d’autres maladies neurodegeneratives, telle que l’alpha-synucleine dans la maladie de Parkinson et la superoxyde dismutase dans la sclerose laterale amyotrophique. Les cellules souches de la moelle osseuse pourront ainsi se diriger vers les differents foyers inflammatoires provoques par ces proteines toxiques. Elles ont aussi l’avantage, contrairement aux autres types de cellules souches, d’infiltrer de facon naturelle les regions endommagees et de s’adapter aux conditions inflammatoires. De plus, le prelevement des cellules souches hematopoietiques evitera le rejet des cellules genetiquement modifiees, puisque le patient servira a la fois de donneur et de receveur. Il nous reste maintenant a trouver la facon de rendre ces cellules plus resistantes, a selectionner de meilleurs phagocytes par exemple, plus specifiques a la βA (ou a d’autres proteines toxiques), tout en evitant l’emballement de la reponse inflammatoire. Un tres beau defi scientifique et medical a relever ! ‡ Bone marrow stem cells to the rescue of Alzheimer’s disease REFERENCES

International audience; Martial, Sonia, Bernadette Olivès, Laurence Abrami, Cécile Couriaud, Pascal Bailly, Guofeng You, Matthias A. Hediger, Jean-Pierre Cartron, Pierre Ripoche, and Germain Rousselet. Functional differentiation of the human red blood cell and kidney urea transporters. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F1264-1268, 1996.-The recent cloning of two urea transporters will allow to better understand their role in the urinary concentrating mechanism. This physiological approach needs to be sustained by a knowledge of their functional characteristics. We compared the pharmacological properties of the human red blood cell and kidney urea transporters (HUT11 and HUT2) in the Xenopus oocyte expression system. Both proteins allow the rapid transfer of urea but not of water. Both are inhibited by phloretin, although with different half-maximal inhibitory concentrations (IC50 75 uM for HUT11 and 230 uM for HUT2). Whereas para-chloromercuribenzene sulfonate inhibits HUT11 with an ICsO of 150 uM, it does not inhibit HUT2, whatever the concentration used. We demonstrate that thiourea diffuses through HUT11 with a Michaelis constant (K,) of 40 mM, but not through HUT2. In contrast, it inhibits urea transport through both proteins. This identification of a substrate binding site independentfrom the transport activity is the first step in the understanding of the molecular events underlying urea transport.UT2; HUT11; phloretin; para-chloromercuribenzene sulfonate

International audience; A eDNA clone (HUT2) sharing 61.1% and 89.9% sequence identity with the human erythroid (HUTll) and the rabbit (UT2) urea transporters, respectively, was isolated by homology cloning from a human kidney library. HUT2 transcripts were restricted to the kidney and the HUT2 polypeptide was not immunoprecipitated with blood group Kidd-related antibodies (anti-Jk3) in coupled transcription-translation assays. Functional expression studies in Xenopus oocytes demonstrated that HUT2-mediated urea transport was not inhibited by p-chloromercuribenzene sulfonate (pCMBS) which, however, inhibited the urea flux mediated by HUTll. These findings demonstrate that at least two distinct urea transporters are present in human tissues. By in situ hybridiza-tion, the gene encoding HUT2 has been assigned to chromosome 18q12.l-q21-1, as found previously for the Kidd/urea transporter HUT11, suggesting that both genes evolved from duplication of a common ancestor.

Feeding rats a low-protein (8%) diet (LPD) for 2 wk induces a facilitated urea transporter in rat initial inner medullary collecting ducts (IMCDs). To determine whether this is preceded by an increase in mRNA abundance, we designed degenerate polymerase chain reaction primers to the rabbit facilitated urea transporter (UT2; G. You, C. P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M. A. Hediger. Nature Lond. 365: 844–847, 1993) and amplified a 716-bp cDNA fragment to perform Northern analysis of the base or tip of rat inner medulla. In the base, the predominant transcript was a 2.9-kb band, which increased 55% after 1 wk on an LPD; there was no change in a 4-kb band. In the tip, the 4-kb band predominated, but neither band varied with an LPD. Next, we functionally characterized the induced urea transporter using microperfused initial IMCDs from rats fed an LPD for 2 wk. First, 100 pM arginine vasopressin (AVP) stimulated urea permeability (Purea); 10 nM AVP increased Purea further. Second, raising perfusate and bath osmolality to 690 mosmol/kgH2O (NaCl added) stimulated Purea; adding AVP (10 nM) increased Purea further. Third, thiourea reversibly inhibited AVP-stimulated Purea.(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; The regulation of mRNA for aldose reductase, sorbitol dehydrogenase, and the Na+/Cl-/taurine cotransporter was studied with three in vivo models in which urinary concentration is reduced: Sprague-Dawley rats undergoing a water diuresis or fed a low-protein diet or Brattleboro rats. In Sprague-Dawley rats, 3 days of water diuresis reduced inner medullary aldose reductase mRNA abundance 6.5-fold compared with untreated rats, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. When water diuretic rats were acutely deprived of water, urine osmolality increased significantly after 4 h but aldose reductase mRNA did not increase until 12 h. Heat shock protein-70 mRNA was not increased by water deprivation. Second, in rats fed a low-protein diet for 3 wk, aldose reductase mRNA increased two-fold, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. Finally, in Brattleboro rats, urine osmolality and levels of aldose reductase and taurine cotransporter mRNA increased in response to 1 day of water deprivation, whereas sorbitol dehydrogenase mRNA was unchanged. Administering vasopressin (1 U/day) to Brattleboro rats for 8 days also increased urine osmolality and aldose reductase mRNA but did not alter sorbitol dehydrogenase or taurine cotransporter mRNA. This result is consistent with the hypothesis that changes in urine osmolality induce changes in aldose reductase mRNA abundance that are independent of vasopressin. It was concluded that, in rat inner medulla: (1) aldose reductase mRNA abundance varies with changes in water balance or dietary protein, whereas sorbitol dehydrogenase and taurine cotransporter mRNA do not; and (2) heat shock protein-70 mRNA abundance is not increased during acute osmotic stress.

The gene encoding the urea transporter of human erythrocytes (HUT11 clone) has been cloned recently (Olives, B., Neau, P., Bailly, P., Hediger, M. A., Rousselet, G., Cartron, J. P., and Ripoche, P. (1994) J. Biol. Chem. 269, 31649-31652). Now, this gene has been assigned to chromosome 18q12-q21 by in situ hybridization, as also found for the Kidd (Jk) blood group locus. In coupled transcription-translation assays, the HUT11 cDNA directed the synthesis of a 36-kDa protein which was immunoprecipitated by a human anti-Jk3 antibody produced by immunized Jk(a-b-) donors whose red cells lack Kidd antigens. The anti-Jk3 antibody also immunoprecipitated a protein material of 46-60 kDa from all red cell membranes, except those from Jk(a-b-) cells. After N-glycanase digestion the 46-60-kDa component was reduced to 36 kDa. A rabbit antibody raised against the predicted NH2-terminal amino-acids of the HUT11 protein reacted on immunoblots with a 46-60-kDa component present in all human erythrocytes except those from Jk(a-b-) individuals. Jk(a-b-) red cells lack the Kidd/urea transport protein and have a selective defect of the urea transport capacity, but a normal water permeability and aquaporin-associated Colton blood group antigens. These findings indicate that the erythrocyte urea transporter is encoded by the Kidd locus and may have implications for the biology of urea transporters and their tissue-specific regulation.

International audience; In amphibian urinary bladder epithelium, vasopressin increases passive urea permeability, concomitant with the appearance of a facilitated urea transport. Amphibian oocytes from Xenopus laevis and Rana esculenta microinjected with total or fractionated poly(A +) RNA isolated from frog urinary bladder epithellal cells. After several (3-5) days at 18°C, the urea flux was assayed by measuring the uptake and efflux of [14C] urea inwater-injected and mRNA-injected oocytes. A 2 to 3-fold increase of urea transport was detected in oocytes injected either with total mRNA or with a 6-10 kilobase mRNA fraction, when compared with water-injected oocytes. This expression of urea channels was inhibited by 0.1 mM phloretin (50% inhibition) and 0.1 mM nitrophenylthiourea (up to 70% inhibition). On the contrary, no expression was detected in brain mRNA-injected oocytes. Theseresults show the specific functional expression of the phloretin and NPTU-sensitive urea channel (or carrier), from frog urinary bladder epithelial cells, providing an approach for the expression cloning of these urea channels.

Experiments were performed to characterize the P2 purinoceptor subtype responsible for cytoplasmic calcium mobilization in cells from the initial part of rabbit distal convoluted tubule (DCT). Free calcium concentration was measured in a DCT cell line (DC1) with the probe fura 2. Both ATP and UTP increased cytosolic Ca2+concentration ([Ca2+]i; EC503 and 6 μM, respectively). The order of potency for nucleotide analogs was ATP = UTP > adenosine 5′- O-[thiotriphosphate] ≫ ADP > UDP, which is consistent with the pharmacology of the P2Y2 receptor subtype. The increased [Ca2+]iresponses to ATP and UTP were strongly inhibited by suramin. Pretreatment of cells with pertussis toxin (PTX) attenuated the action of both nucleotides. Inhibition of phospholipase C with U-73122 totally blocked the [Ca2+]iresponse to ATP. Thus ATP- and UTP-stimulated [Ca2+]imobilization in DC1 cells appears to be mediated via the activation of P2Y2 purinoceptors coupled to a G protein mechanism that is partially sensitive to PTX. Calcium flux measurements showed that lanthanum- and nifedipine-sensitive calcium channels are involved in the [Ca2+]iresponse to ATP.