Oncogene (2012) 31, 1869–1883 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc REVIEW Molecular mechanisms of cisplatin resistance

L Galluzzi1,2,3, L Senovilla1,2,3, I Vitale1,2,3, J Michels1,2,3, I Martins1,2,3, O Kepp1,2,3, M Castedo1,2,3 and G Kroemer1,4,5,6,7

1INSERM, U848 ‘Apoptosis, Cancer and Immunity’, Villejuif, France; 2Institut Gustave Roussy, Villejuif, France; 3Universite´ Paris Sud-XI, Villejuif, France; 4Metabolomics Platform, Institut Gustave Roussy, Villejuif, France; 5Centre de Recherche des Cordeliers, Paris, France; 6Poˆle de Biologie, Hoˆpital Europe´en Georges Pompidou, AP-HP, Paris, France and 7Universite´ Paris Descartes, Sorbonne Paris Cite´, Paris, France

Platinum-based drugs, and in particular cis-diammine- as cisplatin or CDDP) is a largely employed platinum- dichloroplatinum(II) (best known as cisplatin), are based compound that exerts clinical activity against a employed for the treatment of a wide array of solid wide spectrum of solid neoplasms, including testicular, malignancies, including testicular, ovarian, head and neck, bladder, ovarian, colorectal, lung and head and neck colorectal, bladder and lung cancers. Cisplatin exerts cancers (Prestayko et al., 1979; Lebwohl and Canetta, anticancer effects via multiple mechanisms, yet its most 1998; Galanski, 2006). Cisplatin often leads to an initial prominent (and best understood) mode of action involves therapeutic success associated with partial responses or the generation of DNA lesions followed by the activation disease stabilization. Still, many patients (in particular of the DNA damage response and the induction of in the context of colorectal, lung and prostate cancers) mitochondrial apoptosis. Despite a consistent rate of are intrinsically resistant to cisplatin-based therapies. initial responses, cisplatin treatment often results in the Moreover, an important fraction of originally sensitive development of chemoresistance, leading to therapeutic tumors eventually develop chemoresistance (this is failure. An intense research has been conducted during the frequently observed in ovarian cancer patients) (Ozols, past 30 years and several mechanisms that account for the 1991; Giaccone, 2000; Koberle et al., 2010). The cyto- cisplatin-resistant phenotype of tumor cells have been toxicity of cisplatin (which is given intravenously as described. Here, we provide a systematic discussion of short-term infusion in physiological saline) also affects these mechanism by classifying them in alterations (1) that kidneys (nephrotoxicity), peripheral nerves (neurotoxi- involve steps preceding the binding of cisplatin to DNA city) and the inner ear (ototoxicity) (Cvitkovic et al., (pre-target resistance), (2) that directly relate to DNA– 1977; Kelland, 2007). Still, the main limitation to the cisplatin adducts (on-target resistance), (3) concerning the clinical usefulness of cisplatin as an anticancer drug is lethal signaling pathway(s) elicited by cisplatin-mediated the high incidence of chemoresistance. DNA damage (post-target resistance) and (4) affecting In the early 1980s, second-generation platinum molecular circuitries that do not present obvious links with compounds were developed with the specific aim of cisplatin-elicited signals (off-target resistance). As in some reducing the side effects of cisplatin while retaining its clinical settings cisplatin constitutes the major therapeutic anticancer properties. These efforts led to the discovery option, the development of chemosensitization strategies of cis-diammine (cyclobutane-1,1-dicarboxylate-O,O’) constitute a goal with important clinical implications. platinum(II) (carboplatin), which essentially does not Oncogene (2012) 31, 1869–1883; doi:10.1038/onc.2011.384; display nephro- and neurotoxicity, yet forms the same published online 5 September 2011 types of DNA adducts (see below) as cisplatin, although with a reduced potency (Harrap, 1985). Carboplatin, Keywords: ATP7B; CTR1; ERCC1; glutathione; metal- whose most prominent side effects concern the bone lothioneins; TP53 marrow, frequently leading to reversible thrombo- cytopenia, was approved by the FDA for the treatment of ovarian cancer in 1989. As the active form of carboplatin is identical to that of cisplatin (see below), Introduction it was not surprising to find out that most cisplatin- resistant tumors also fail to respond to carboplatin. First approved by FDA (Food and Drug Administra- These observations ignited another wave of drug devel- tion) in 1978 for the treatment of testicular and bladder opment that in 2002 led to the introduction of [(1R,2R)- cancer, cis-diamminedichloroplatinum(II) (best known cyclohexane-1,2-diamine](ethanedioato-O,O0)platinum(II) (oxaliplatin) into clinical practice. Oxaliplatin exhibits Correspondence: Dr G Kroemer, INSERM, U848, Institut Gustave distinct pharmacological and immunological properties Roussy, Pavillon de Recherche 1, 39 rue Camille Desmoulins, F-94805, than cisplatin and carboplatin, in line with the fact that Villejuif, France. E-mail: [email protected] it features the bidentate ligand 1,2-diaminocyclohexane Received 30 June 2011; revised 26 July 2011; accepted 27 July 2011; in place of two monodentate ammine ligands (Kidani published online 5 September 2011 et al., 1978). However, in spite of the fact that cisplatin- Chemoresistance to cisplatin L Galluzzi et al 1870 refractory cancers are generally considered to be Aquated cisplatin avidly binds DNA, with a predilec- sensitive to oxaliplatin, clinical data suggest that there tion for nucleophilic N7-sites on purine bases. This leads may be some degree of cross-resistance (Stordal et al., to the generation of protein–DNA complexes as well as 2007). Oxaliplatin is currently employed against colo- of DNA–DNA inter- and intra-strand adducts (East- rectal cancer in association with 5-fluorouracil and man, 1987b). Although the signaling pathways that are folinic acid (the so-called FOLFOX protocol) (Giacchetti triggered by protein/cisplatin/DNA complexes have et al., 2000; de Gramont et al., 2000; Rothenberg et al., been largely ignored, great efforts have been spent to 2003; Goldberg et al., 2004), and may also be useful elucidate the molecular cascades that are activated by for the treatment of lung cancer (Raez et al., 2010). DNA–DNA inter- and intra-strand adducts. The Of note, other platinum derivatives that have recently latter—and notably 1,2-intrastrand ApG and CpG entered clinical trials, such as amminedichloro(2-methyl- crosslinks—have been indicated as the most prominent pyridine) platinum (picoplatin) and (OC-6-43)-bis(acetato) cisplatin-induced DNA lesions and have been suggested amminedichloro(cyclohexylamine)platinum (satraplatin), to account for most, if not all, cisplatin cytotoxicity have not yet been shown to provide significant advantages (Kelland et al., 1993). This notion, which in the past has over cisplatin, oxaliplatin and carboplatin (Choy, 2006; generated a vivid debate, nowadays appears as an Eckardt et al., 2009). Moreover, in specific clinical settings, oversimplification, especially in consideration of the fact cisplatin represents by far the most prominent, if not the that: (1) only B1% of intracellular cisplatin binds to sole, therapeutic option (Armstrong et al., 2006). nuclear DNA (Gonzalez et al., 2001) and (2) cisplatin Circumventing cisplatin resistance remains therefore a (as well as oxaliplatin) has been shown to exert critical goal for anticancer therapy and considerable significant cytotoxicity in enucleated cells (cytoplasts) efforts have been undertaken to solve this problem (Mandic et al., 2003; Berndtsson et al., 2007; Obeid throughout the past three decades. Here, we briefly et al., 2007). Irrespective of this, the best-characterized introduce the modes of action of cisplatin and then mode of action of cisplatin involves the DNA-damage systematically present the molecular mechanisms that response and mitochondrial apoptosis (Jamieson and can account for the cisplatin-resistant phenotype. Lippard, 1999; Cohen and Lippard, 2001). Finally, we suggest combination strategies that might Cisplatin-induced lesions cause distortions in DNA be exploited for reverting cisplatin resistance in tumors. that can be recognized by multiple repair pathways (Bellon et al., 1991). Among these, the nucleotide excision repair (NER) reportedly constitutes the most prominent mechanism for the removal of cisplatin Mode of action adducts (Chaney and Sancar, 1996; Furuta et al., 2002). However, proteins belonging to the mismatch The detailed description of the molecular mechanisms repair (MMR) system also participate in the recognition that underlie the anticancer potential of cisplatin goes and resolution of cisplatin lesions (Kunkel and Erie, largely beyond the scope of this review, and can be 2005). When the extent of damage is limited, cisplatin found elsewhere (Kelman and Peresie, 1979; Sanderson adducts induce an arrest in the S and G2 phases of the et al., 1996; Siddik, 2003). Here, we will provide key cell cycle, a phenomenon that exerts cytoprotective facts that explain the molecular basis of cisplatin effects by (1) allowing repair mechanisms to re-establish resistance. DNA integrity and (2) preventing potentially abortive Cisplatin exerts anticancer effects via an intertwined or abnormal mitoses (Vitale et al., 2011). Conversely, if signaling pathway that might be separated into one DNA damage is beyond repair, cells become committed nuclear and one cytoplasmic module. As such, cisplatin is to (most often apoptotic) death. inert and must be intracellularly activated by a series of The major signaling cascade that bridges cisplatin- aquation reactions that consist in the substitution of one induced DNA lesions to apoptosis involves the or both cis-chloro groups with water molecules (el- sequential activation of the ataxia telangiectasia Khateeb et al., 1999; Kelland, 2000). This reaction occurs mutated (ATM)- and RAD3-related protein (ATR, a spontaneously in the cytoplasm, owing to the relatively sensor of DNA damage) and checkpoint kinase 1 low concentration of chloride ions (B2–10 mM when (CHEK1, the most prominent substrate and down- compared with B100 mM in the extracellular milieu), and stream effector of ATR), which in turn phosphorylates leads to the generation of highly reactive mono- and bi- the tumor suppression protein TP53 on serine 20, aquated cisplatin forms (Eastman, 1987a; Michalke, allowing for its stabilization (Shieh et al., 2000; Appella 2010). These molecules are prone to interact with a wide and Anderson, 2001; Damia et al., 2001; Zhao and number of cytoplasmic substrates, and in particular with Piwnica-Worms, 2001). Activated TP53 exerts lethal endogenous nucleophiles such as reduced glutathione functions via nuclear and cytoplasmic mechanisms that (GSH), methionine, metallothioneins and proteins (via eventually lead to mitochondrial outer membrane their cysteines) (Timerbaev et al., 2006). Thus, cytoplas- permeabilization or increased signaling via death recep- mic cisplatin has the potential to deplete reduced tors followed by cell death (Kroemer et al., 2007; Galluzzi equivalents and to tilt the redox balance toward oxidative et al., 2011) In response to cisplatin, CHEK1 has also stress (which facilitates DNA damage; see below), but is been shown to activate various branches of the mitogen- also susceptible to inactivation by a number of cytopro- activated protein kinase (MAPK) system, including those tective antioxidant systems (Koberle et al.,2010). mediated by extracellular signal-regulated kinases, c-JUN

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1871 N-terminal kinases and stress-activated protein kinases (Persons et al., 2000; Wang et al., 2000; Dent and Grant, 2001; Yeh et al., 2002). The relative contribution of these signaling modules to the cytotoxic effects of cisplatin remain to be deciphered, as contrasting reports can be found in literature (Dent and Grant, 2001). Intriguingly, although ATM (another important sensor of DNA damage) appears to participate in cisplatin- induced cell cycle arrest but not cell death (Sancar et al., 2004; Wang et al., 2006), its major downstream target, CHEK2, has been shown to convey lethal signals in response to cisplatin in an ATM-independent fashion (Damia et al., 2001; Pabla et al., 2008). Thus, there appear to be multiple mechanisms that underlie the cytotoxic and antiproliferative potential of cisplatin (Figure 1). The cisplatin-resistant phenotype of cancer cells can derive from alterations in any of these molecular circuitries as well as from changes that affect the intracellular uptake of cisplatin or the execution of the apoptotic program.

Mechanisms of pre-target resistance

There are at least two mechanisms by which cancer cells elude the cytotoxic potential of cisplatin before it binds to cytoplasmic targets and DNA: (1) a reduced intra- cellular accumulation of cisplatin and (2) an increased sequestration of cisplatin by GSH, metallothioneins and other cytoplasmic ‘scavengers’ with nucleophilic properties (Table 1). A wide array of (mostly natural) anticancer agents is associated with the so-called multidrug resistance, a phenomenon whereby drugs are subjected to increased efflux via relatively nonselective members of the ATP- binding cassette (ABC) family of ATPases like the P-glycoprotein (Molnar et al., 2010). This is not the case of cisplatin, whose limited intracellular accumulation most often (although not always; see below) derives Figure 1 Modes of action of cisplatin. Because of the relatively from reduced uptake (Smith et al., 1993; Wada et al., low (compared with the extracellular microenvironment) concen- 1999; Baekelandt et al., 2000). Irrespective of the under- tration of chloride ions, intracellular cisplatin quickly becomes aquated and hence highly reactive. Aquated cisplatin can indeed lying mechanisms, several cisplatin-resistant cancer cells bind a plethora of nucleophilic species, including cysteine and exhibit consistent reductions in the accumulation of methionine residues on proteins and DNA bases. In the nucleus, cisplatin (Loh et al., 1992; Mellish et al., 1993). this leads to the generation of inter- and intra-strand adducts that For a long time, cisplatin was believed to enter cells are recognized by the DNA damage-sensing machinery. If the prominently by passive diffusion across the plasma extent of damage is beyond repair, cisplatin adducts trigger the activation of a DNA damage response (DDR) that frequently membrane, mainly because the uptake of cisplatin, involves the ATR kinase, CHEK1 and CHEK2 and the tumor- which is highly polar, is relatively slow when compared suppressor protein TP53. In turn, TP53 transactivates several genes with that of chemically similar anticancer agents that whose products facilitate mitochondrial outer membrane permea- are actively transported (Yoshida et al., 1994; Kelland, bilization (MOMP), thereby triggering intrinsic apoptosis, as well as genes that encode for components of the extrinsic apoptotic 2000). More recently, however, the copper transporter 1 pathway. MOMP (alone or with the contribution of death (CTR1), a transmembrane protein involved in copper receptor-ignited, BID-transduced signals) sets off the caspase homeostasis, turned out to play an important role in cascade as well as multiple caspase-independent mechanisms that the uptake of cisplatin. Ctr1À/À mouse embryonic eventually seal the cell fate. Several other signaling pathways link fibroblasts accumulate much less cisplatin than their cisplatin-induced DNA damage to MOMP and cell death (not shown, see the main text for further details). In the cytoplasm, wild-type counterparts, and are indeed two- to three- the interaction between cisplatin and GSH, metallothioneins fold more resistant to its cytotoxic effects (Ishida or mitochondrial proteins like the VDAC results in the depletion et al., 2002; Katano et al., 2002; Holzer et al., 2006). of reducing equivalents and/or directly sustains the generation of Accordingly, cells pre-treated with copper (the main reactive oxygen species (ROS). ROS can directly trigger MOMP or exacerbate cisplatin-induced DNA damage, thereby playing a dual CTR1 substrate) are protected from cisplatin cytotoxicity role in cisplatin cytotoxicity. (More et al., 2010), whereas copper chelators result in

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1872 Table 1 Mechanisms of pre-target resistance Factor Mode of action Relevance Reference

Reduced uptake CTR1 Plasma membrane copper Downregulated in CDDP-resistant cancer Ishida et al., 2002; Katano et al., transporter. cell lines. 2002; Holzer et al., 2006; CTR1 depletion increases CDDP resistance. Ishida et al., 2010 Copper chelators enhance the uptake and efficacy of CDDP in vitro and in vivo.

Increased efflux ATP7A/ATP7B Copper-extruding P-type ATPases Upregulated in CDDP-resistant cancer Nakayama et al., 2002; Nakayama involved in the regulation of ion cell lines. et al., 2004;Safaei et al., 2004; homeostasis. ATP7B expression levels may predict the Aida et al., 2005 efficacy of CDDP chemotherapy in patients with ovarian cancer. MRP2 Member of the ABC family of Overexpressed in CDDP-resistant cancer Koike et al., 1997; Cui et al., 1999; plasma membrane transporters. cell lines. Liedert et al., 2003; Korita et al., Mediates the ATP-dependent Modulation by antisense cDNA enhances 2010; Yamasaki et al., 2011 cellular efflux of CDDP. CDDP sensitivity. Expression levels affect the efficacy of CDDP regimens in ESCC and HCC patients.

Increased inactivation GSH/g-GCS/GST GSH scavenges electrophiles and CDDP-resistant cells often exhibit elevated Lewis et al., 1988; ROS. g-GCS catalyzes GSH synthesis. levels of GSH, g-GCS and GST. Chen and Kuo, 2010 GST conjugates GSH to CDDP, No conclusive clinical evidence. thus facilitating its extrusion. Metallothioneins Intracellular thiol-containing proteins May bind and inactivate CDDP. Kelley et al., 1988; involved in the detoxification of No conclusive clinical evidence. Kasahara et al., 1991 metal ions.

Abbreviations: ABC, ATP-binding cassette; CDDP, cisplatin; cDNA, complementary DNA; CTR1, copper transporter 1; ESCC, esophageal squamous cell carcinoma; g-GCS, g-glutamylcysteine synthetase; GSH, reduced glutathione; GST, glutathione S-transferase; HCC, hepatocellular carcinoma; MRP2, multidrug resistance protein 2; ROS, reactive oxygen species.

increased cisplatin accumulation and exacerbate cyto- expression levels might predict the sensitivity of ovarian toxicity (Ishida et al., 2010). Of note, clinically relevant and endometrial cancers to cisplatin chemotherapy concentrations of cisplatin reportedly downregulate (Nakayama et al., 2002, 2004; Aida et al., 2005). Still, CTR1, owing to internalization followed by protea- in line with the multifactorial nature of cisplatin efflux some-mediated degradation (Holzer and Howell, 2006). (and resistance; see below), small molecules that inhibit This mechanism may account (at least in part) for specific ABC transporters (for example, the P-glycopro- multiple instances of acquired cisplatin resistance. tein-specific inhibitor 5-bromotetrandrine) appear to be Early reports suggested that ABC ATPases like unable per se to restore cisplatin accumulation and multidrug resistance protein (MRP)1, MRP2, MRP3 sensitivity (Jin et al., 2005). and MRP5 would also mediate some extent of cisplatin Aquated cisplatin avidly binds to cytoplasmic nucleo- resistance by increasing cisplatin export (Borst et al., philic species, including GSH, methionine, metallothio- 2000). In particular, results from genetic manipulations neins and other cysteine-rich proteins. On one hand, this (that is, overexpression, RNA interference) pointed to may underlie at least part of the cytoplasmic effects of MRP2 as the major ATPase responsible for an increased cisplatin, resulting in the depletion of antioxidant efflux of cisplatin in resistant cells (Koike et al., 1997; reserves and in the establishment of oxidative stress Cui et al., 1999; Liedert et al., 2003). Recent reports (Slater et al., 1995). On the other hand, nucleophilic reinforced the notion that MRP2 expression levels might species act as cytoplasmic scavengers, thereby limiting predict the responsiveness of tumors to platinum-based the amount of reactive cisplatin (Kasahara et al., 1991; therapies (Korita et al., 2010; Yamasaki et al., 2011). Sakamoto et al., 2001). Thus, elevated levels of GSH, Following the discovery of the role of CTR1 in cisplatin of the enzyme that catalyzes GSH synthesis (that is, uptake, however, attention was attracted by two copper- g-glutamylcysteine synthetase), or of the enzyme that extruding P-type ATPases, ATP7A and ATP7B. These mediates the conjugation between cisplatin and GSH proteins are upregulated in cisplatin-resistant cancer cell (that is, glutathione S-transferase) have been observed lines (Safaei et al., 2004), and their transfection-enforced in the context of cisplatin resistance, both in vitro and overexpression has been shown to drive the acquisition ex vivo (in cancer cell lines that were derived from one of the cisplatin-resistant phenotype (Samimi et al., ovarian carcinoma patient before and after the devel- 2004). Importantly, clinical studies indicate that ATP7B opment of resistance) (Lewis et al., 1988). Of note,

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1873 glutathione S-conjugates are readily extruded by cells NER and ICR activity in patient material are missing), via MRP1 or MRP2, possibly explaining why the latter although ERCC1 constitutes one of the rate-limiting has been more robustly associated with cisplatin factors for NER (Niedernhofer et al., 2001, 2004; resistance than other ABC ATPases (Ishikawa, 1992). Ahmad et al., 2008). Moreover, although the absence Genetic manipulations of human and murine cells have of ERCC1 consistently correlates with cisplatin respon- also linked increased levels of metallothioneins, a class siveness, both in vitro and in vivo (in patients), the same of low-molecular-weight thiol-containing proteins that does not hold true for ERCC1 overexpression, which in are involved in the binding and detoxification of heavy some instances resulted in increased, rather than metal ions, to the cisplatin-resistant phenotype (Kelley decreased, sensitivity (Bramson and Panasci, 1993). et al., 1988; Kasahara et al., 1991). However, conclusive This might be because of disequilibria in the components clinical data on this correlation are missing. of complex DNA repair pathways such as NER (Coquerelle et al., 1995). However, it remains formally possible that ERCC1 levels affect cisplatin resistance via Mechanisms of on-target resistance hitherto uncharacterized NER- and/or ICR-indepen- dent pathways. Irrespective of this issue, ERCC1 The recognition of inter- and intra-strand DNA expression constitutes a very promising biomarker for adducts and the consequent generation of an apoptotic the prediction of cisplatin responsiveness in patients signal is often impaired in cisplatin-resistant cancer cells (Olaussen, 2009), and has already begun to be exploited because of a variety of defects. Alternatively, cisplatin- in this sense in clinical settings. resistant cells acquire the ability to repair adducts at an Cisplatin-induced DNA lesions can be detected (but increased pace, or become able to tolerate unrepaired not repaired) by the MMR system, which normally DNA lesions, thanks to a particular class of DNA handles erroneous insertions, deletions and mis-incor- polymerases that mediate the so-called translesion porations of bases that can arise during DNA replica- synthesis (Table 2). tion and recombination (Vaisman et al., 1998; Kunkel The majority of cisplatin lesions are removed from and Erie, 2005). MMR-related proteins that participate DNA by the NER system (Wood et al., 2000; Shuck in the recognition of GpG interstrand adducts include et al., 2008). In this setting, damaged nucleotides are MSH2 and MLH1 (Mello et al., 1996; Vaisman et al., excised from DNA upon incision on both sides of the 1998). According to accepted viewpoints, MMR pro- lesion, followed by DNA synthesis to reconstitute teins would attempt to repair cisplatin adducts, fail, and genetic integrity (Gillet and Scharer, 2006). At least 20 hence transmit a proapoptotic signal (Vaisman et al., proteins participate in NER, including excision repair 1998). In line with this model, MSH2 and MLH1 are cross-complementing rodent repair deficiency, comple- often mutated or underexpressed in the context of mentation group 1 (ERCC1), a single-strand DNA acquired cisplatin resistance (Aebi et al., 1996; Drum- endonuclease that forms a tight heterodimer with mond et al., 1996; Brown et al., 1997; Fink et al., 1998), ERCC4 (also known as xeroderma pigmentosum although NSCLC patients with high MSH2 expression complementation group F (XPF)) and incises DNA on who do not undergo cisplatin treatment upon tumor the 50 side of bulky lesions such as cisplatin adducts resection have a better prognosis than patients with low (Biggerstaff and Wood, 1992; Sijbers et al., 1996; MSH2 levels (Kamal et al., 2010). This apparent Ahmad et al., 2008). Early reports pointed to a discrepancy simply reflects the intrinsic difference correlation between NER proficiency and cisplatin between naive, previously untreated tumors (for which resistance in multiple preclinical models (Li et al., high MSH2 levels constitute a good prognostic indica- 1998, 2000; Metzger et al., 1998), and subsequent tor) and cancers that have acquired resistance upon studies supported this notion at the clinical level. Thus, cisplatin exposure (which are often associated with ERCC1 expression (be it measured at the mRNA or reduced MSH2 expression). Thus, at least in some protein level) has been negatively correlated with clinical settings, a high DNA repair capacity appears to survival and/or responsiveness to cisplatin-based regi- protect against tumor relapse (Kamal et al., 2010) but mens in several human neoplasms including bladder may prevent patients to benefit from DNA-damaging (Bellmunt et al., 2007), colorectal (Shirota et al., 2001), agents. Of note, defects in MLH1 and MSH6 (another gastric (Metzger et al., 1998), esophageal (Kim et al., component of the MMR system) are associated with 2008), head and neck (Handra-Luca et al., 2007; Jun increased level of translesion synthesis, the phenomenon et al., 2008) and ovarian cancers (Dabholkar et al., whereby DNA synthesis is not blocked but proceeds 1992), as well as non-small cell lung cancer (NSCLC) beyond cisplatin adducts (Bassett et al., 2002). Transle- (Olaussen et al., 2006). ERCC1 also participates in sion synthesis, which is also known as replicative bypass, interstand crosslink repair (ICR), and ICR proficiency is mediated by the concerted activity of a specific group appears to be reduced and augmented in cisplatin- of DNA polymerases including POLH, POLI, POLK, sensitive and cisplatin-resistant tumor cells, respectively REV1, REV3 and REV7 (Shachar et al., 2009). POLH (Zhen et al., 1992; Usanova et al., 2010). and the REV3–REV7 heterodimer appear to be It should be noted that increased levels of ERCC1 involved in the replicative bypass of GpG adducts (Alt does not necessarily (and have never been formally et al., 2007; Shachar et al., 2009). Defects in POLH and shown to) correspond to increased NER and ICR REV3 have been linked to increased sensitivity to proficiency in patients (as methods that reliably measure cisplatin in multiple tumor cell lines, in vitro (Wittschie-

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1874 Table 2 Mechanisms of on-target resistance Factor Mode of action Relevance Reference

Increased NER proficiency ERCC1 Single-strand endonuclease that— ERCC1 expression negatively correlates Dabholkar et al., 1992; Metzger et al., in association with ERCC4/XPF— with CDDP clinical responses in multiple 1998; Shirota et al., 2001; Olaussen incises DNA on the 50 side of bulky human cancers. et al., 2006; Handra-Luca et al., 2007; lesions (such as CDDP adducts). Proposed predictor of CDDP-based Bellmunt et al., 2007; Kim et al., 2008; Also implicated in the ICR. chemotherapy sensitivity in multiple Jun et al., 2008; Olaussen, 2009 clinical settings. MMR deficiency MLH1 Component of a multiprotein MLH1 deficiency is sometimes Aebi et al., 1996; Drummond et al., complex that excides and repairs associated with CDDP resistance 1996; Brown et al., 1997; Fink et al., DNA mismatches. (and increased TLS). 1998; Gifford et al., 2004 Implicated in DNA damage MLH1 promoter methylation predicts signaling and apoptosis. poor survival in relapsing ovarian cancer patients. MSH2 Forms MSH2–MSH6 and MSH2– Mutated or underexpressed in some Aebi et al., 1996; Brown et al., 1997; MSH3 heterodimers that detect tumors with acquired CDDP resistance. Fink et al., 1998; Kamal et al., 2010 DNA lesions including base–base Low MSH2 levels predict CDDP benefits mismatches. in patients with resected lung cancer. When repair cannot be accomplished, High MSH2 levels are a positive signals for the activation of cell death. prognostic factor for untreated lung cancer patients.

Increased TLS POLH DNA polymerase that substitutes POLH upregulation correlates with Alt et al., 2007; Ceppi et al., 2009; stalled replicative polymerases and shorter survival in CDDP-treated NSCLC Shachar et al., 2009 includes nucleotides opposite to the patients. DNA lesion. Implicated in the bypass of CDDP adducts. REV3/REV7 Catalytic (REV3) and structural REV3 defects correlate with increased Wittschieben et al., 2006; Shachar (REV7) subunits of the TLS DNA CDDP sensitivity in cancer cell lines. et al., 2009; Roos et al., 2009 polymerase z. REV overexpression is associated with Implicated in the bypass of CDDP CDDP resistance, in vitro. adducts. Conclusive clinical data are missing.

Increased HR proficiency BRCA1/BRCA2 Critical components of the BRCA1/2-deficient tumors respond better Narod and Foulkes, 2004; Edwards HR DNA repair system. to CDDP. et al., 2008; Sakai et al., 2008 Also involved in the regulation Secondary mutations that restore BRCA of transcription and cell cycle function favor acquired chemoresistance. progression.

CDDP-binding proteins VDAC Protein of the OM that mediates Aquated CDDP binds VDAC. Yang et al., 2006; Kroemer et al., 2007; vital functions but also participates Depletion/or inhibition of VDAC Tajeddine et al., 2008 into the PTPC. increases CDDP resistance. Might also be involved in post-target resistance.

Abbreviations: BRCA1, breast cancer 1, early onset; BRCA2, breast cancer 2, early onset; CDDP, cisplatin; ERCC1, excision repair cross-complementing rodent repair deficiency, complementation group 1; HR, homologous recombination; ICR, interstrand crosslink repair; MMR, mismatch repair; NER, nucleotide excision repair; NSCLC, non-small cell lung cancer; OM, mitochondrial outer membrane; PTPC, permeability transition pore complex; TLS, translesion synthesis; VDAC, voltage-dependent anion channel; XPF, xeroderma pigmentosum complementation group F.

ben et al., 2006; Roos et al., 2009), whereas REV3 together, these observations suggest that the expression overexpression reportedly increases cisplatin resistance levels of components of the MMR and translesion (Wang et al., 2009). Moreover, POLH expression levels synthesis systems may constitute useful predictors of correlate with overall survival in lung cancer patients cisplatin responsiveness in clinical settings, although (Ceppi et al., 2009), REV3 was found to be upregulated compelling data on this issue have not yet been reported. in glioma samples, correlating with tumor grade (Wang Cisplatin-induced inter-strand adducts can lead to the et al., 2009), and the methylation-dependent silencing of so-called double-strand breaks, DNA lesions that are MLH1 has been shown to predict poor survival in normally repaired in the S phase of the cell cycle (or ovarian cancer patients (Gifford et al., 2004). Taken shortly after) by the machinery for homologous

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1875 recombination (HR) (Smith et al., 2010). Two critical proficient and -deficient tumor cell lines (O’Connor components of the HR system are encoded by BRCA1 et al., 1997; Branch et al., 2000), and also in vivo, in the and BRCA2, two genes that are frequently mutated in clinical setting (Hengstler et al., 2001). Thus, ovarian familial breast and ovarian cancers (Venkitaraman, cancer patients harboring wild-type TP53 reportedly 2002; Narod and Foulkes, 2004). Notably, HR-deficient have a higher probability to benefit from cisplatin-based cancers have a different phenotype and are often more chemotherapy than patients with TP53 mutations sensitive to crosslinking agents including cisplatin than (Gadducci et al., 2002; Feldman et al., 2008). Moreover, their HR-proficient counterparts (Bryant et al., 2005; testicular germ cell tumors, which are particularly Farmer et al., 2005; Ratnam and Low, 2007). For sensitive to cisplatin, are one of the few cancers in instance, BRCA1/2-deficient ovarian cancers metasta- which TP53 is rarely, if ever, inactivated (Peng et al., size to viscera more frequently than sporadic epithelial 1993). Other members of the TP53 protein family, ovarian cancers (which most often remain confined to notably the transactivation-incompetent TP63 variant the peritoneum), yet are generally more responsive to DNp63a, have recently been shown to transduce platinum compounds and associated with better prog- prosurvival signals in response to cisplatin (Yuan nosis (Ben David et al., 2002; Chetrit et al., 2008; et al., 2010), but the putative clinical implications of Gourley et al., 2010). Moreover, it has been shown that these observations remain to be established. cisplatin resistance can develop in initially cisplatin- Intriguingly enough, tetraploid cancer cells have been sensitive tumors because of secondary mutations that shown to endure DNA-damaging agents (including compensate for BRCA1/2 deficiency and re-establish cisplatin and oxaliplatin) far better (410-fold) than HR (Edwards et al., 2008; Sakai et al., 2008). their diploid counterparts (Castedo et al., 2006; Vitale Altogether, these observations suggest that the HR et al., 2007). This phenomenon can be reverted by the status, at least in specific clinical settings, has an depletion/inhibition of TP53, its target ribonucleotide important prognostic and predictive value. reductase M2 B (RRM2B) or CHEK1 (Castedo et al., As the catalog of cisplatin interactors that may be 2006; Vitale et al., 2007), implying that the cisplatin- implicated in its cytotoxicity has not yet been entirely resistant phenotype of tetraploid cancer cells relies on elucidated, cytoplasmic proteins may also be responsible complex mechanisms that go beyond a merely stoichio- for (at least part of) the cisplatin-resistant phenotype. With metric (on-target) process whereby the double amount regardtothis,cisplatinhasbeenshowntobind of DNA entirely accounts for resistance. Altogether, mitochondrial DNA as well as the voltage-dependent these observations suggest that multiple factors, includ- anion channel (VDAC) (Yang et al., 2006), a mitochon- ing ploidy, are likely to affect the molecular mechanisms drial protein with vital and lethal functions (Kroemer et al., that underlie cisplatin resistance. 2007). Notably, VDAC-depleted cancer cells are highly Preclinical studies suggest that other proapoptotic signal resistant to CDDP treatment (Tajeddine et al., 2008), yet it transducers such as MAPK family members might is not clear whether this constitutes an example of on- contribute to the cisplatin-resistant phenotype (Mansouri target resistance or whether in this context VDAC simply et al., 2003; Brozovic and Osmak, 2007). In particular, it transduces upstream proapoptotic signals (and hence has been proposed that cisplatin-resistant cells would fail would be involved in a post-target resistance mechanism). to activate MAPK1 (also known as p38MAPK) and c-JUN N-terminal kinase in a sustained fashion in response to cisplatin (Mansouri et al., 2003; Brozovic et al., 2004). This would limit signaling through the FAS/FASL system (an Mechanism of post-target resistance inducer of extrinsic apoptosis) and hence confer cytopro- tection (Spierings et al., 2003). Contrarily to the case of Post-target resistance to cisplatin can result from a TP53, so far no correlation has been found between the plethora of alterations including defects in the signal levels of MAPKs or MAPK-related proteins and cisplatin transduction pathways that normally elicit apoptosis in sensitivity in patients. response to DNA damage as well as problems with the Alterations in any of the factors that regulate and cell death executioner machinery itself. Nonrepairable execute apoptosis, be it triggered by DNA damage or cisplatin-induced DNA damage leads to the activation oxidative stress via the mitochondrial pathway or be it of a multibranched signaling cascade with proapoptotic mediated by the extrinsic route, have the potential to outcomes (see above). Genetic and epigenetic alterations influence cisplatin sensitivity. Several dozens of proteins in the components of this complex signaling network (including death receptors, cytoplasmic adaptors, pro- and have been associated with variable levels of resistance to antiapoptotic members of the BCL-2 protein family, cisplatin, presumably reflecting the relative relevance of caspases, calpains, mitochondrial intermembrane proteins specific proteins (and the underlying pathways) in and many others) participate in these lethal cascades and different cellular and experimental settings (Table 3). most of them have been shown to modulate the response One of the most predominant mechanisms of post- to cisplatin (as well as to a plethora of other chemother- target resistance involves the inactivation of TP53 apeutic agents, drugs, toxins and stressors) in vitro (de La (Vousden and Lane, 2007), which occurs in approxi- Motte Rouge et al., 2007; Sakai et al., 2008; Tajeddine mately half of all human neoplasms (Kirsch and Kastan, et al., 2008; Wang et al., 2010; Janson et al., 2011). 1998). This has been documented in vitro, by comparing However, only some of these proteins predict cisplatin the sensitivity to cisplatin of a wide panel of TP53- responsiveness in the clinical setting.

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1876 Table 3 Mechanisms of post-target resistance Factor Mode of action Relevance Reference

BAX-like Proapoptotic members of the BCL-2 BAX/BAK-deficiency confers resistance Castedo et al., 2006; Kroemer et al., 2007; proteins protein family. to CDDP and to several other stressors, Tajeddine et al., 2008 in vitro. Conclusive clinical data are missing. BCL-2-like Antiapoptotic members of the BCL-2 Overexpression of BCL-2, BCL-XL and Han et al., 2003; Williams et al., 2005; proteins protein family. MCL-1 confers resistance to several Erovic et al., 2005; Michaud et al., stressors, in vitro. 2009; Jain and Meyer-Hermann, 2011 Clinical data link the expression levels of (http://clinicaltrials.gov) antiapoptotic BCL-2 proteins with CDDP resistance and recurrent disease. Chemical inhibitors of BCL-2-like pro- teins are being clinically tested to over- come resistance. BIRC5 Caspase inhibitor of the IAP family that is BIRC5 overexpression is associated with Kato et al., 2001; Nakamura et al., 2004; (Survivin) often upregulated in response to PI3K chemoresistance and poor prognosis in Karczmarek-Borowska et al., 2005; signaling. multiple types of cancer. Altieri, 2008; Ryan et al., 2009 Component of CPC, a complex involved BIRC5 inhibitors are currently being (http://clinicaltrials.gov) in the regulation of chromosome evaluated in clinical trials. segregation. Calpains Non-caspase proteases that participate In vitro, galectin-3 inhibition exacerbates Wang et al., 2010 in the execution of multiple cell death CDDP responses by enhancing calpain subroutines. activation. Caspases Mediate the initiator (caspase-9 and -8) In vitro, acquired resistance to CDDP is Janson et al., 2011 and executioner (caspase-3, -6 and -7) link to modifications in the caspase phase of apoptosis. activation cascade. MAPKs Members of the JNK, ERK and SAPK JNK, ERK and SAPK inhibition has been Persons et al., 2000; Wang et al., 2000; family transmit pro- and/or anti-apoptotic associated with both increased and de- Dent and Grant, 2001; Yeh et al., 2002; signals in response to CDDP, with a creased sensitivity to CDDP, depending Mansouri et al., 2003; Brozovic et al., high degree of variability in different on the experimental setting. 2004 experimental settings. Conclusive data are missing. DNp63a TP53 protein family member. In vitro, transduces prosurvival signals in Yuan et al., 2010 response to CDDP. TP53 Tumor-suppressive protein that controls CDDP-resistant tetraploid cells exhibit an Peng et al., 1993; Gadducci et al., 2002; DNA repair and apoptosis in response increased transcription of specific TP53 Castedo et al., 2006; Vousden and Lane, to stress. target genes. 2007; Feldman et al., 2008 Also implicated in senescence, autophagy Tumors harboring wild-type TP53 respond and genomic stability. better to CDDP-based chemotherapy. XAF1 Nuclear protein that antagonizes High levels of XAF1 correlate with Plenchette et al., 2007; Pinho et al., 2009 the activity of IAPs, thus acting improved progression-free survival in as a proapoptotic factor. advanced bladder cancer patients.

Abbreviations: BCL-2, B-cell lymphoma 2; CDDP, cisplatin; CPC, chromosome passenger complex; ERK, extracellular signal-regulated kinase; IAP, inhibitory apoptosis protein; JNK, c-JUN N-terminal kinase; MAPK, mitogen-activated protein kinase; MCL-1, myeloid cell leukemia sequence 1; PI3K, phosphoinositide-3-kinase; SAPK, stress-activated protein kinase; XAF1, X-linked IAP-associated factor 1.

For instance, conclusive clinical data on the associa- esophageal and ovarian cancer and NSCLC patients tion between the proapoptotic BCL-2 family members (Kato et al., 2001; Nakamura et al., 2004; Karczmarek- BAX and BAK and cisplatin sensitivity are missing, but Borowska et al., 2005). Of note, survivin inhibitors elevated levels of their antiapoptotic counterparts (for example, YM155, LY2181308) are currently being including BCL-2, BCL-XL and MCL-1 (myeloid cell evaluated as single agents or in combination with leukemia sequence 1) reportedly correlate with cisplatin cisplatin for the treatment of several malignancies (Ryan resistance and tumor recurrence in multiple clinical et al., 2009) (http://clinicaltrials.gov). Along similar scenarios, including head and neck cancer, ovarian lines, high levels of XIAP-associated factor 1 (XAF1), cancer and NSCLC (Han et al., 2003; Erovic et al., 2005; a tumor-suppressor protein that antagonizes inhibitor Williams et al., 2005; Michaud et al., 2009). Moreover, of apoptosis proteins (Plenchette et al., 2007), are ongoing clinical trials are evaluating the combination of associated with improved progression-free survival in cisplatin with small molecules that inhibit BCL-2-like advanced bladder cancer patients (Pinho et al., 2009). proteins (for example, ABT-263, ABT-737) for the treatment of several neoplasms (Jain and Meyer- Hermann, 2011). Increased levels of survivin, a cas- Mechanisms of off-target resistance pase-inhibitory protein that is frequently upregulated in response to cisplatin by phosphoinositide-3-kinase Accumulating evidence suggests that the cisplatin- (PI3K)/AKT1-dependent mechanisms (Ikeguchi and resistant phenotype can also be sustained (if not entirely Kaibara, 2001), inversely correlate with cisplatin respon- generated) by alterations in signaling pathways that are siveness and favorable clinical outcome in gastric, not directly engaged by cisplatin, yet compensate for

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1877 Table 4 Mechanisms of off-target resistance Factor Mode of action Relevance Reference

Autophagy Evolutionarily conserved response Ovarian and NSCLC cells upregulate Kroemer et al., 2010; Ren et al., 2010; to multiple stress conditions. autophagy while acquiring CDDP Yu et al., 2011 Often activated in response to resistance. chemotherapy. Autophagy-targeting agents are clinically available. DYRK1B Conserved kinase that mediates DYRK1B depletion increases sensitivity Friedman, 2007; Deng et al., 2009; Gao differentiation in multiple tissues. to CDDP in vitro by favoring ROS et al., 2009; Hu and Friedman, 2010 Overexpressed or hyperactivated in generation. several neoplasms, in which it mediates antiapoptotic effects. ERBB2 Oncogenic EGFR-like receptor that is Might contribute in a dual fashion to Mitsuuchi et al., 2000; Zhou et al., 2001; (HER-2) overactivated in multiple types of cancer. CDDP resistance. Ikeguchi and Kaibara, 2001; Citri and ERBB2 conveys pro-survival signals via ERBB2 overexpression has been Yarden, 2006; Fijolek et al., 2006 PI3K and MAPK. associated with CDDP resistance in NSCLC patients. HSPs Chaperones that exert prosurvival In vitro and in vivo, HSPs protect Yamamoto et al., 2001; Miyazaki et al., functions in response to a variety cells against CDDP toxicity by several 2005; Zhang and Shen, 2007; Ren et al., of stress conditions. mechanisms. 2008 Upregulated in multiple cancers. HSP27 expression might predict CDDP chemosensitivity in ESCC patients. TMEM205 Hypothetical transmembrane protein. TMEM205 expression might be associated Shen et al., 2010 with CDDP resistance. In vivo data are missing.

Abbreviations: CDDP, cisplatin; DYRK1B, dual-specificity Y-phosphorylation-regulated kinase 1B; EGFR, epidermal growth factor receptor; ESCC, esophageal squamous cell carcinoma; HSPs, heat-shock proteins; NSCLC, non-small cell lung cancer; PI3K, phosphoinositide-3-kinase; ROS, reactive oxygen species.

(and hence interrupt) cisplatin-induced lethal signals prosurvival functions by increasing the expression of (Table 4). antioxidant enzymes such as ferroxidase, superoxide The ERBB2 protooncogene (also known as HER-2 dismutase 2 and superoxide dismutase 3 (Deng et al., or NEU) codes for a member of the epidermal growth 2009). In NSCLC and ovarian cancer cells, DYRK1B factor receptor family of tyrosine kinases and is depletion has been shown to potentiate the effects of amplified or overexpressed in multiple types of neo- subapoptotic cisplatin concentrations by favoring the plasms, including breast and ovarian cancers (Slamon establishment of lethal oxidative stress (Gao et al., 2009; et al., 1989; Hengstler et al., 1999). ERBB2 signals are Hu and Friedman, 2010). With regard to this, it would propagated via multiple downstream pathways, includ- be interesting to precisely determine to which extent ing the SHC/GRB2/SOS and the PI3K/AKT1 cascades cisplatin-induced reactive oxygen species directly trigger (Citri and Yarden, 2006). Whereas baseline signaling via cell death (via cytoplasmic mechanisms) and to which PI3K/AKT1 upregulates the cyclin-dependent kinase extent they favor cell death by exacerbating cisplatin- inhibitor 1A (CDKN1A, also known as p21Cip1 or induced DNA damage. From this perspective, GSH p21Waf1) (Mitsuuchi et al., 2000), ERBB2 overexpression appears to mediate not only pre-target resistance (by leads to CDKN1A nuclear exclusion (although an binding cisplatin and preventing its interaction with AKT1-mediated, phosphorylation-dependent mechan- DNA and other targets) but also post-target resistance ism) (Zhou et al., 2001). This is intriguing, as both (by quenching proapoptotic reactive oxygen species mechanisms might actually contribute to cisplatin generated in response to cisplatin) and perhaps off- resistance. Initially, a CDKN1A-mediated cell cycle target resistance (by rendering cells globally less arrest (as that promoted by basal PI3K/AKT1 activity) sensitive to cell death signals). would exert antiapoptotic functions by providing cells Other general stress response pathways or poorly with time for DNA repair and homeostasis re-establish- characterized mechanisms have been linked to cisplatin ment. Later on, however, cells would need to recover resistance. The former include autophagy, an evolu- proliferation, and this might be favored by the nuclear tionary conserved catabolic pathway that involves the exclusion of CDKN1A following increased PI3K/AKT1 sequestration and lysosomal degradation of organelles activity, an alteration that often occurs upon cisplatin and portions of the cytoplasm (Kroemer et al., 2010), treatment (Ikeguchi and Kaibara, 2001). Of note, and the heat-shock response, the integrated reaction of ERBB2 overexpression has been associated with a cells to high temperatures as well as to a plethora slightly subsignificant trend toward cisplatin resistance of stressful conditions that affect protein folding in NSCLC patients (Fijolek et al., 2006). (Yamamoto et al., 2001; Macleod et al., 2005; Donnelly Dual-specificity Y-phosphorylation regulated kinase and Blagg, 2008). Both ovarian and NSCLC cells have 1B (DYRK1B, also known as MIRK) is upregulated in been shown to progressively acquire cisplatin resistance multiple solid tumors (Friedman, 2007) and exerts while upregulating components of the autophagic

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1878 pathway (Ren et al.,2010;Yuet al., 2011). Accordingly, autophagy inhibition reportedly can restore some extent of cisplatin sensitivity, at least in vitro (Ren et al., 2010), although inhibitors of the mammalian target of rapamy- cin kinase like temsirolimus (which stimulates rather than inhibits autophagy) have been shown to synergize with cisplatin in the killing of human oropharyngeal carcinoma cells (Gaur et al., 2011). Presumably, this apparent discrepancy reflects the existence of multiple, sometimes cell type-specific, mechanisms that lead to cisplatin resistance. Molecular chaperones that are involved in the heat- shock and unfolded protein responses, such as several heat-shock proteins, have also been shown to promote Figure 2 Strategies for reverting cisplatin resistance. Cisplatin resistance most often has a multifactorial nature, implying that cisplatin resistance via multiple, most often indirect, targeting one mechanism of resistance at a time has very low mechanisms (Yamamoto et al., 2001; Zhang and Shen, chances to result in significant chemosensitization. Thus, combina- 2007; Ren et al., 2008). Heat-shock protein 27 expres- tion strategies for blocking cisplatin resistance at multiple levels sion levels can predict the response to cisplatin-based should be designed. With regard to this, detailed information on the patient genetic and epigenetic background might be critical therapies in esophageal squamous cell carcinoma for determining which specific mechanisms should be targeted to patients (Miyazaki et al., 2005). Finally, one recent fully circumvent chemoresistance. EGFR, epidermal growth factor report demonstrates that the hypothetical membrane receptor; HSP, heat-shock protein; PTEN, phosphatase and tensin protein TMEM205 is associated with cisplatin resistance homolog; TLS, translesion synthesis. in vitro (Shen et al., 2010); yet, the clinical relevance of this observation remains to be elucidated. that aim at simultaneously limiting pre-target resistance (for example, copper chelators or other agents that maximize intracellular accumulation) and interrupting Concluding remarks post-, on- or off-target mechanisms (for example, MMR- activating agents, PI3K/AKT1 inhibitors, blockers of Cisplatin is an important therapeutic tool in the combat autophagy) may turn out to restore cisplatin sensitivity against several solid neoplasms, including (but not limited to therapeutically useful levels. In addition, chemicals that to) head and neck, ovarian and lung cancers. Unfortu- stimulate an endoplasmic reticulum stress may be useful to nately, cancer cells either intrinsically are or relatively convert cisplatin into an inducer of immunogenic cell rapidly become resistant to cisplatin, leading to relapse and death (Martins et al., 2011). Further investigation is therapeutic failure. As discussed in this review, there are at required to see if and which one of the many possible leastfourdistinctclassesofmechanisms by which cancer strategies for overcoming cisplatin resistance will match cells become resistant to cisplatin-based chemotherapy. the expectations. Of note, despite encouraging preclinical One major problem for overcoming this clinically relevant results, coordination complexes based on metals other issue is that—frequently—more than one resistance than cisplatin (for example, palladium) that have been mechanism is activated, that is, cisplatin resistance often developed to circumvent cisplatin resistance have never exhibits a multifactorial nature. This notion is supported been tested in the clinical setting (Serrano et al., 2011). The by a large amount of literature indicating that (1) a linear elucidation of the mechanisms by which tumors become correlation between cisplatin-induced cellular alterations refractory to cisplatin will lead not only to optimal and responsiveness can be found in a very restricted chemosensitization strategies, but also to the discovery of number of settings and that (2) most often the inhibition of new prognostic and predictive biomarkers. Together with single pathways that sustain cisplatin resistance fails to the tools that are currently available to clinicians, this new restore sensitivity to normal levels (some extent of residual knowledge will allow for better patient stratification and resistance remain). In addition, whereas oxaliplatin has will surely lead to the development of more efficient and recently been shown to induce a type of cell death that less toxic anticancer therapies. is immunogenic (that is, it stimulates a tumor-specific cognate immune response) (Tesniere et al., 2010), cisplatin fails to do so (Obeid et al., 2007; Kepp et al., 2011). In view of these considerations, it is tempting to Abbreviations speculate that the most successful strategies for circum- venting resistance will have to target at least two distinct ABC, ATP-binding cassette; ATM, ataxia telangiectasia mutated; ATR, ATM- and RAD3-related; BCL-2, B-cell mechanisms (Figure 2). At present, it remains to be lymphoma 2; CDKN1A, cyclin-dependent kinase inhibitor precisely determined which of these mechanisms should 1A; CHEK, checkpoint kinase; CTR1, copper transporter 1; be preferentially modulated for optimal chemosensitiza- DYRK1B, dual-specificity Y-phosphorylation regulated ki- tion, and this is likely to depend (at least in part) on nase 1B; ERCC, excision repair cross-complementing rodent clinical variables (that is, type of cancer, intrinsic or repair deficiency; GSH, reduced glutathione; HR, homologous acquired resistance). Nevertheless, combined interventions recombination; ICR, interstrand crosslink repair; MAPK,

Oncogene Chemoresistance to cisplatin L Galluzzi et al 1879 mitogen-activated protein kinase; MMR, mismatch repair; Acknowledgements MRP, multidrug resistance protein; NER, nucleotide excision repair; NSCLC, non-small cell lung cancer; PI3K, phospho- LG and LS are supported by the European Commission (Apo- inositide-3-kinase; RRM2B, ribonucleotide reductase M2 B; Sys) and the Fondation pour la Recherche Me´dicale (FRM), VDAC, voltage-dependent anion channel; XAF1, XIAP- respectively. IV is funded by the Ligue Nationale contre associated factor 1; XPF, xeroderma pigmentosum comple- le Cancer. GK is supported by Ligue Nationale contre le mentation group F. Cancer (e´quipe labellise´e), AXA Chair for Longevity Research, Cance´ropoˆle Ile-de-France, Institut National du Cancer (INCa), Fondation Bettencourt-Schueller, Fondation Conflict of interest de France, FRM, Agence National de la Recherche, LabEx Immuno-Oncology and the European Commission (Apo-Sys, The authors declare no conflict of interest. ArtForce, ChemoRes. Death-Train).

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