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VRACs swallow platinum drugs

Thomas Voets, Bernd Nilius & Rudi Vennekens

Platinum-based drugs such as cisplatin react covalently with the N7 nitrogen in essential structural component of VRACs and carboplatin are on the WHO model list guanines (Galluzzi et al, 2012). This causes (Qiu et al, 2014; Voss et al, 2014). LRRC8 of essential medicines, as highly effective distortions in the DNA, which can provoke proteins form a small family of integral chemotherapeutic drugs for the treatment cell cycle arrest and ultimately programmed membrane proteins with four transmem- of various solid tumors. These drugs react cell death (Kelland, 2007; Galluzzi et al, brane helices and include five homologues with purine residues in DNA, thereby caus- 2012). However, before cisplatin and related (LRRC8A-E) in mammals and most other ing DNA damage, inhibition of cell division, drugs can exert their antiproliferative effects, chordates (Abascal & Zardoya, 2012). and eventually cell death. However, the they first need to be taken up by the targeted Current evidence indicates that functional mechanisms whereby platinum-based cells (Galluzzi et al, 2012). Cisplatin and VRAC channels are heteromultimers that drugs enter cancer cells remained poorly carboplatin are relatively polar, compared to contain the obligatory LRRC8A subunit understood. In this issue, Planells-Cases other classes of small-molecule (anticancer) supplemented with at least one of the other et al (2015) provide evidence that cells drugs. Consequently, their passive diffusion four isoforms (Voss et al, 2014). Based on take up cisplatin and carboplatin via through the plasma membrane is slow, and limited homology with pannexins, a hexam- volume-regulated anion channels (VRACs), other transport pathways, including trans- eric architecture has been proposed, but this more specifically VRACs composed of porters and channels, have been postulated. stoichiometry still demands experimental LRRC8A and LRRC8D subunits. In particular, the copper transporter CTR1 verification (Abascal & Zardoya, 2012). has been proposed to mediate cisplatin In a genomewide screen for genes that See also: R Planells-Cases et al uptake (Ishida et al, 2002), although this mediate cisplatin and carboplatin resistance, has been disputed (Ivy & Kaplan, 2013). Planells-Cases et al (2015) discovered that ifty years ago, while testing the effect Planells-Cases et al (2015) now propose that gene-inactivating insertions in the LRRC8A of electrical fields on the growth of VRACs act as entry channels for cisplatin and LRRC8D genes resulted in significantly F bacteria, researchers discovered that and carboplatin. VRACs are ubiquitous increased survival of cancer cells when metal complexes of platinum released from channels that typically open in response to grown in the presence of the two platinum- the electrodes had a strong inhibitory effect cell swelling or any other maneuver that based drugs. Moreover, cells deficient in on bacterial cell division (Rosenberg et al, dilutes the cellular ionic content (Nilius either LRRC8A or LRRC8D exhibited 1965). This serendipitous discovery fueled et al, 1997; Pedersen et al, 2015). VRACs strongly reduced cellular platinum levels, further successful research into the anti- are best known for their key role in the suggesting that resistance to cisplatin and proliferative effects of different platinum- homeostasis of cell volume, but they have carboplatin is due to impaired cellular containing molecules in mammalian cancer also been implicated in cellular processes uptake of these drugs rather than to effects cells. By the end of the 1970s, a first including regulation of the membrane poten- on downstream processes. Interestingly, platinum-based drug, cisplatin (Fig 1A), was tial, release of transmitter and cell prolifera- hypotonic cell swelling caused a striking approved for the treatment of testicular and tion (Nilius et al, 1997). increase in cisplatin/carboplatin uptake. ovarian cancers. Currently, cisplatin and Earlier work had already suggested a Overall, the results support the idea that the second- and third-generation platinum-based functional role for VRACs in cisplatin- platinum-based drugs can permeate VRACs drugs such as carboplatin and oxaliplatin induced cell death. However, since the composed of LRRC8A and LRRC8D subunits. are amongst the most widely used and effec- molecular nature of the functionally well- It may seem dubious to assume that the tive anticancer drugs (Kelland, 2007). characterized VRACs had remained elusive pore of an anion-selective channel could In the last decades, the biochemical for decades (Pedersen et al, 2015), the exact accommodate a large, non-charged, plat- mechanisms whereby cisplatin and related role of these channels in platinum drug- inum complex. Since cisplatin and oxali- compounds inhibit cell proliferation have induced effects remained murky. Recently, platin do not carry charge, it is technically been largely unraveled. Briefly, once inside the chloride channel field breathed a sigh of impossible to directly measure their flux cells, platinum-based drugs undergo aqua- relief when two groups independently iden- through VRACs using electrophysiological tion (i.e. the incorporation of water mole- tified leucine-rich repeat-containing protein techniques. Nevertheless, earlier work has cules) and enter the nucleus where they 8A (LRRC8A; also dubbed SWELL1) as an provided ample indications that VRACs may

Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium. E-mail: [email protected] DOI 10.15252/embj.201593357

ª 2015 The Authors The EMBO Journal 1 The EMBO Journal Platinum-based drugs enter via VRACs Thomas Voets et al

LRRC8D-containing VRACs, irrespective of Cl – cell volume changes, which could then be Cisplatin Cisplatin used to increase the effectiveness of plat- Cl NH Carboplatin inum-based drugs. Some caution is required Pt 3 NH VRACs here, as several earlier studies have linked Cl 3 VRAC activity to cell cycle progression, and Taurine pharmacological agents that inhibit VRACs O Cell were generally found to act as potent inhi- membrane S bitors of cell proliferation (Voets et al, HO NH2 O 1995; Shen et al, 2000; Pedersen et al, Cytosol 2015). Therefore, pharmacological upregu- Carboplatin Cl – Cl – lation of VRAC function in vivo may have O NH unwanted stimulatory effects on cell O 3 Pt growth. It is also known that various CELL DEATH O NH3 widely used drugs, including for instance O the anti-estrogen tamoxifen and the antide- 12–14Å pressant fluoxetine (Prozac), potently inhibit VRACs (Pedersen et al, 2015) and VRAC pore may thus potentially influence the uptake LRRC8B,C,E LRRC8A LRRC8D of platinum drugs by cancer cells in patients. Finally, knowledge about the entry mechanisms of platinum-based drugs might also be exploited to prevent their entry into Figure 1. Permeability of VRACs for platinum-based drugs. non-cancer cells. For instance, one of the Left: structure and dimension of platinum-based drugs in comparison with chloride ions and the VRAC- most common and burdensome side effects permeant osmolyte taurine. Right: VRACs are composed of the obligatory LRRC8A subunit complemented with of platinum-based drugs is the development additional LRRC8B-E subunits. VRACs containing LRRC8D exhibit high permeability for the platinum-based anticancer drugs cisplatin and carboplatin, allowing drug entry into cells to cause cell death. The hexameric of neuropathic pain, which is due to an stoichiometry of VRACs is an educated guess based on limited homology with pannexin/connexin channels. effect of the drugs on peripheral nerves and the surround tissue (Avan et al, 2015). Peripheral inhibition of LRRC8D-containing be permeable to larger (and not necessarily LRRC8D is about 40 amino acids longer than VRACs might be a strategy to prevent these negatively charged) molecules, including in the other isoforms (Abascal & Zardoya, adverse effects. amino acids, sugars, ATP, and the sulfonic 2012). It is therefore tempting to speculate acid taurine, and estimates of the diameter that this first extracellular domain of LRRC8 References of the VRAC pore are in the range of proteins forms a reentrant pore loop in Abascal F, Zardoya R (2012) LRRC8 proteins 12–14 A˚ (Pedersen et al, 2015). As such, the VRACs and that the extended loop in share a common ancestor with pannexins, molecular dimensions of cisplatin and carbo- LRRC8D results in an enlarged pore that and may form hexameric channels involved platin would be at least compatible with facilitates permeation of platinum-based in cell-cell communication. BioEssays 34: pore permeation (Fig 1). Planells-Cases et al drugs and organic osmolytes. 551 – 560 (2015) provide evidence that permeability to In addition to providing important Avan A, Postma TJ, Ceresa C, Cavaletti G, such larger molecules is dependent on the insights into the VRAC pore, the new Giovannetti E, Peters GJ (2015) Platinum- subunit composition of VRACs, in that chan- results may have significant clinical impact. induced neurotoxicity and preventive nels containing the LRRC8D subunit conduct Reduced cellular uptake is one key mecha- strategies: past, present, and future. Oncologist more cisplatin and taurine (Fig 1). This nism, whereby cancer cells can become 20: 411 – 432 suggests that LRRC8D differs from the other resistant to platinum-based drugs (Galluzzi Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, LRRC8 isoforms in regions that contribute to et al, 2012). In this respect, Planells-Cases Kepp O, Castedo M, Kroemer G (2012) the VRAC pore. At present, the localization et al (2015) provide a retrospective analysis Molecular mechanisms of cisplatin resistance. and structure of the VRAC pore is unknown. of two small cohorts of ovarian cancer Oncogene 31: 1869 – 1883 In fact, it cannot yet be excluded that other patients that received treatment with plat- Ishida S, Lee J, Thiele DJ, Herskowitz I (2002) non-LRRC8 proteins contribute to the VRAC inum-based drugs, which reveals that lower Uptake of the anticancer drug cisplatin pore. Mutating a threonine residue at the top expression levels of LRRC8D correlate with mediated by the copper transporter Ctr1 in of the first transmembrane domain (TM1) of lower survival rates. If these results are yeast and mammals. Proc Natl Acad Sci USA 99: LRRC8A caused mild changes in anion selec- further confirmed, assessment of LRRC8D 14298 – 14302 tivity, suggesting that this residue may be in expression may be tested in cancer patients Ivy KD, Kaplan JH (2013) A re-evaluation of the the vicinity of the permeation pathway (Qiu to predict the efficacy of platinum-based role of hCTR1, the human high-affinity et al, 2014). Sequence alignments of all drugs and to adjust the treatment strategy copper transporter, in platinum-drug entry LRRC8 proteins indicate that the first extra- accordingly. It might even be considered to into human cells. Mol Pharmacol 83: cellular loop connecting TM1 and TM2 in develop agonists that selectively activate 1237 – 1246

2 The EMBO Journal ª 2015 The Authors Thomas Voets et al Platinum-based drugs enter via VRACs The EMBO Journal

Kelland L (2007) The resurgence of platinum-based Pt-based anti-cancer drugs. EMBO J doi: Shen MR, Droogmans G, Eggermont J, Voets T, Ellory cancer chemotherapy. Nat Rev Cancer 7: 10.15252/embj.201592409 JC, Nilius B (2000) Differential expression of 573 – 584 Pedersen SF, Klausen TK, Nilius B (2015) The volume-regulated anion channels during cell Nilius B, Eggermont J, Voets T, Buyse G, identification of a volume-regulated anion cycle progression of human cervical cancer cells. Manolopoulos V, Droogmans G (1997) channel: an amazing Odyssey. Acta Physiol 213: J Physiol 529(Pt 2): 385 – 394 Properties of volume-regulated anion channels 868 – 881 Voets T, Szucs G, Droogmans G, Nilius B (1995) in mammalian cells. Prog Biophys Mol Biol 68: Qiu Z, Dubin AE, Mathur J, Tu B, Reddy K, Miraglia Blockers of volume-activated ClÀ currents 69 – 119 LJ, Reinhardt J, Orth AP, Patapoutian A (2014) inhibit endothelial cell proliferation. Pflugers Planells-Cases R, Lutter D, Guyader C, Gerhards SWELL1, a plasma membrane protein, is an Arch 431: 132 – 134 NM, Ullrich F, Elger DA, Kucukosmanoglu A, Xu essential component of volume-regulated anion Voss FK, Ullrich F, Munch J, Lazarow K, Lutter D, G, Voss FK, Reincke SM, Stauber T, Blomen VA, channel. Cell 157: 447 – 458 Mah N, Andrade-Navarro MA, von Kries JP, Vis DJ, Wessels LF, Brummelkamp TR, Borst P, Rosenberg B, Vancamp L, Krigas T (1965) Inhibition Stauber T, Jentsch TJ (2014) Identification of Rottenberg S, Jentsch TJ (2015) Subunit of cell division in Escherichia coli by electrolysis LRRC8 heteromers as an essential component composition of VRAC channels determines products from a platinum electrode. Nature of the volume-regulated anion channel VRAC. substrate specificity and cellular resistance to 205: 698 – 699 Science 344: 634 – 638

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