Towards Novel Paradigms for Cancer Therapy

V Pavet1, MM Portal1, JC Moulin1,2, R Herbrecht2 and H Gronemeyer1

1Department of Cancer Biology, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (IGBMC), Illkirch, Alsace, France and 2Department of Oncology and Hematology, Hoˆpitaux Universitaires de Strasbourg, Strasbourg, France

Cancer is a complex progressive multistep disorder that normal cell toward a malignant derivative. This process results from the accumulation of genetic and epigenetic shapes each tumor in such a dynamic and unique way abnormalities, which lead to the transformation of normal that it is extremely difficult to determine the alterations cells into malignant derivatives. Despite enormous progress that cause, maintain and spread the disease (Greenman in the understanding of cancer biology including the et al., 2007; Wood et al., 2007). decryption of multiple regulatory networks governing cell Historically, solid tumors have been treated by growth and death, and despite the possibility of analyzing surgery for the past 4000 years (http://www.cancer.org/ (epi)genetic deregulation at the genome-wide scale, cancer- docroot/CRI/content/CRI_2_6x_the_history_of_cancer_ targeted therapy is still the exception. In fact, to date there 72.asp?sitearea ¼ ). It was only after the discovery of are still far too few examples of therapies leading to cure; X-rays at the end of the nineteenth century that treatment-derived toxicity is a major issue, and cancer radiotherapy emerged as a novel therapeutic approach. remains to be one of the largest causes of death worldwide. Whereas localized tumors could be treated by focal The purpose of this review is to discuss the state of the art of therapy, extensive or metastatic tumors and hematolo- cancer therapy with respect to the key issue of any treatment, gical malignancies required the development of systemic namely its target selectivity. Therefore, we recapitulate and anticancer therapies. Initial efforts in anticancer drug discuss current concepts and therapies targeting tumor- discovery started in the mid-twentieth century based on specific features, including oncofusion proteins, aberrant the observation that cytotoxic agents could be used to kinase activities and epigenetic tumor makeup. We analyze kill cells displaying high proliferation rate. To date, strategies designed to induce tumor-selective death such as surgery, radiotherapy and standard systemic chemother- the use of oncolytic virus, tumoricidal proteins (NS1, Eorf4, apy still comprise the standard treatment in a majority apoptin, HAMLET (human a-lactalbumin made lethal to of proliferative diseases. Indeed, novel chemotherapeu- tumor cells)) and activation of signaling pathways involved in tic (genotoxic) compounds are continuously being tumor surveillance. We emphasize the potential of the tumor developed, despite the induction of serious side effects necrosis factor-related apoptosis-inducing ligand (TRAIL) arising from the damage caused to normal tissue. pathway, an essential component of the evolutionary Although initially the development of anticancer thera- developed defense systems that eradicate malignant cells. pies relied on empirical observations, the current Finally, we discuss the necessity of targeting tumor-initiating challenge is to develop novel therapeutic paradigms cells (TICs) to avoid relapse and increase the chances of exploiting the knowledge derived from molecular, complete remission, and describe emerging concepts that cellular and systems biology studies of tumor formation might provide novel avenues for cancer therapy. and progression (Shipley and Butera, 2009; Zhenchuk Oncogene (2011) 30, 1–20; doi:10.1038/onc.2010.460; et al., 2009). Despite the pleiotropic nature of tumors, published online 11 October 2010 several characteristics are shared by almost all malignancies namely: self-sufficiency in growth signals, Keywords: apoptosis; TRAIL/Apo2L/TNFSF10; epigenetic evasion from apoptosis/immunosurveillance, insensitiv- drug; tumor-initiating cell; dependence receptor; non- ity to growth inhibitory signals, limitless replicative coding RNA potential, sustained angiogenesis and tissue invasion/ metastasis; as well as metabolic, mitotic, oxidative and DNA damage stress (Hanahan and Weinberg, 2000; Introduction Luo et al., 2009). Consequently, a plethora of therapeutic approaches targeting the corresponding path- Cancer results from the accumulation of genetic and ways/key players that support or are essential for tumor epigenetic alterations that drive the transformation of a development are being developed and explored (Ferrara et al., 2004; Bianco et al., 2007; Berdis, 2008; Lane Correspondence: Dr V Pavet or Dr H Gronemeyer, Department of and Chabner, 2009; Desgrosellier and Cheresh, 2010; Cancer Biology, Institut de Geńe´tique et de Biologie Molećulaire et Tennant et al., 2010) (Figure 1). However, irrespective Cellulaire, 1 Rue Laurent Fries, BP10142, Illkirch-Strasbourg, of the accumulating knowledge on tumor-specific Alsace 67404, France. E-mails: [email protected] or [email protected] features, at present the corresponding targeted therapies Received 6 May 2010; revised 10 August 2010; accepted 1 September have only in rare cases led to cure. Indeed, the first 2010; published online 11 October 2010 curative cancer therapy that could be understood from Novel paradigms for cancer therapy V Pavet et al 2

Figure 1 Tumor-speciﬁc features targeted for cancer therapy. General features of tumor development are depicted as red spheroids. Blue spheroids exemplify factors/processes that are integral components of the corresponding feature and which are currently being targeted for cancer therapy; numbers refer to the following publications: (1) Tennant et al., 2010; (2) Desgrosellier and Cheresh, 2010; (3) Lessene et al., 2008 (4) Lane and Chabner, 2009; (5) Bianco et al., 2007; (6) Granchi et al., 2010; (7) Ferrara et al., 2004 and (8) Berdis, 2008.

the underlying biology concerns cancers with very multifactorial events. However, tumors display certain specific somatic gene aberrations, such as defined common characteristics, some of which have been and chromosomal translocations in a subtype of acute are at the basis of anticancer drug development leukemia (Melnick and Licht, 1999). The therapeutic (Figure 1). Although it is not on the focus of this review success was due to the removal of the cause of the to enter in detail along these lines of therapy, this section malignancy, as was shown for acute promyelocytic briefly summarizes current approaches that target three leukemia (APL), which is caused by the formation of features of tumor growth, namely self-sufficiency in leukemogenic fusion proteins in hematopoietic progeni- growth signals, sustained angiogenesis (neovasculariza- tors (Melnick and Licht, 1999). Treatment of APL tion) and resistance to apoptotic stimuli. patients with compounds that eradicate the leukemo- Self-sufficiency in growth signals: A key feature of genic fusion protein (all trans-retinoic acid (ATRA), normal cell proliferation is the fact that normal cells arsenic trioxide; see below) cures most of the patients require mitogenic signals in order to switch from a (Kogan, 2009; Nasr et al., 2009). However, as a vast quiescent to a proliferative state. Under normal condi- majority of cancers arise as a result of the accumulation tions, soluble mitogenic factors are not cell auto- of multiple (epi)genetic mutations, only in very parti- nomously produced but are rather secreted by a cular cases the identification and subsequent targeting of different cell type. In contrast, a majority of tumor cells a specific aberration may be feasible. Nevertheless, the are capable of producing their own growth signals, thus example of APL demonstrates that a targetable ‘achilles promoting uncontrolled proliferation. Moreover, it has heel’ of a given tumor can, in principle, be found. been shown that cancer cells can overexpress cell surface The conceptual basis of this review is the argument receptors involved in the transduction of growth signals that the major challenges in cancer therapy are: (1) the as well as display ligand-independent signaling. These selective eradication of cancer cells, (2) the discovery of two characteristics have been reported for the epidermal robust biomarkers for predicting therapeutic response to growth factor receptors, which are transmembrane a given therapy and (3) the identification and targeting tyrosine kinase receptors involved in cell proliferation of tumor-initiating cells (TICs), which might constitute a and survival (Yarden and Ullrich, 1988). Epidermal highly dangerous compartment of a tumor that needs to growth factor receptor was the first molecular target for be eliminated for complete remission. In the following which monoclonal antibody (cetuximab) was developed sections we analyze the conceptual basis of current for cancer therapy. The interaction with cetuximab cancer therapies with respect to cancer selectivity and promotes receptor internalization and degradation, thus discuss known compounds, targets and pathways that blocking downstream signaling pathways involved in can be exploited to achieve tumor-selective killing. cell proliferation/differentiation, invasion, angiogenesis and apoptosis (Masui et al., 1986; Martinelli et al., 2009). Cetuximab has been already approved for Current therapeutic paradigms to treat cancer treatment of advanced head and neck squamous cell carcinoma and metastatic colorectal carcinoma. A Attacking hallmarks of cancer second-generation monoclonal antibody (panitumu- Targeting factors indispensable for tumor development. mab) binds with high affinity to epidermal growth Cancer is mainly the result of pleiotropic and usually factor receptor, preventing receptor dimerization,

Oncogene Novel paradigms for cancer therapy V Pavet et al 3 phosphorylation and signaling, and thus inhibiting effects can be very serious and life threatening, depend- proliferation and promoting apoptosis. Panitumumab ing on tumor type/localization, the age of the patient, has been approved for the treatment of metastatic clinical settings and combinatorial therapies (Chen colorectal cancer showing preliminary activity in phase and Cleck, 2009). To date, neither biomarkers of I/II clinical trials, and phase III clinical trials are efficacy nor markers of early toxicity for anti-VEGF currently ongoing (Van Cutsem et al., 2007). An therapy exist (Jain et al., 2009). This constitutes a major interesting addition to this concept is that rexinoids challenge in the field that will require a combined effort exclusively exert their apoptotic action under conditions among basic and applied research in order to achieve in which growth factor signaling is impaired (Shankar- this important goal. anarayanan et al., 2009), suggesting that the combina- Evasion from apoptosis: Apoptosis is a mechanism tion of both anticancer treatments might be an attractive having a key role not only during mammalian develop- strategy. Although the clinical activity of cetuximab and ment but also in adult organisms. Maintaining a proper panitumumab antibodies has been consistently reported, regulation of apoptotic responses is mandatory for tumors with activated K-RAS present little to no assuring proper tissue development and homeostasis, response to therapy, and potentially mutations in B-raf while altered responses to normal apoptotic signals is might have the same effect (Lievre et al., 2006; Di one of the hallmarks of cancer, often related to defects Nicolantonio et al., 2008; Karapetis et al., 2008). Other in the apoptotic machinery in cancer cells. Triggering of potential markers of response to these compounds apoptosis can be achieved via the activation of two include polymorphisms of the cyclooxygenase (Lurje different pathways, the extrinsic and the intrinsic et al., 2008), and the FCGR2A and FCGR2B (Fc g apoptotic cascades (Figure 2). The extrinsic pathway receptors) genes (Zhang et al., 2007). Larger trials operates through the activation of membrane-bound including biomarker analysis are required to validate death receptors (DRs). The activation of the extrinsic these markers as predictors of response to therapy, thus cascade by the administration of receptor agonists, as allowing a more precise identification of patients who well as sensitization to this apoptogenic pathway, are would profit from this therapeutic approach. important strategies that are being explored for cancer Sustained angiogenesis: Solid tumors frequently therapy (discussed in the section ‘Exploiting the activate neovascularization pathways (angiogenesis) endogenous tumor-surveillance system for cancer to overcome death by nutrient/oxygen depletion. therapy’). The intrinsic pathway involves disruption of A tumor-induced switch of its microenvironment to an the mitochondrial membrane integrity and is tightly angiogenic state, in which several proangiogenic factors controlled by pro- and anti-apoptotic proteins of the are upregulated, characterizes this survival phenomen- B-cell lymphoma-2 (BCL-2) family, which comprises 5 on. Among those, vascular endothelial growth factor antiapoptotic (BCL-2, BCL-XL, BCL-W, MCL-1 and (VEGF) represents a major mediator of neovasculariza- A1) and 10 proapoptotic (BAX, BAK, BIM, BID, tion. In general terms, antiangiogenic cancer therapy PUMA, NOXA, BAD, BMF, HRK and BIK) proteins. seeks to promote a poor vascularization within the In addition, proteins known as inhibitors of apoptosis tumoral mass, resulting in insufficient perfusion and (IAPs) also have a crucial role through inhibiting the promoting cell death. Two different kinds of agents have apoptotic cascade by either blocking caspase activation been developed to target the VEGF system, including or promoting caspase degradation by the proteosome. those directly binding VEGF ligands, thus avoiding To date, eight IAPs (XIAP, IAP-2, cIAP-1, cIAP-2, their interaction with the receptors (VEGFR), and those ML-IAP, NAIP, survivin and apollon) have been directly binding VEGFR (Grothey and Galanis, 2009). described, with X-linked IAP (XIAP) displaying the The first validated antiangiogenic agent for cancer most potent antiapoptotic effect. However, endogenous treatment was a monoclonal antibody (bevacizumab) IAP inhibitors, such as Smac (second mitochondria- targeting VEGF (Ferrara et al., 2004). This antibody, derived activator of caspase)/DIABLO (diablo homolog- which clears VEGF from circulation thereby limiting the Drosophila), Omi/HtrA2 and XIAP-associated factor-1 formation of tumor vessels supporting tumor growth, (XAF1), can antagonize the antiapoptotic activity of was the first monoclonal antibody approved in combi- IAPs. Finally, it is the interplay between, and equilibrium nation with chemotherapy for treatment of patients with among, pro/anti-apoptotic proteins and IAP/IPA metastatic colorectal cancer (Bergers and Benjamin, inhibitors that dictate cell fate in response to diverse 2003). More recently, it has been approved for treatment apoptotic triggers. of metastatic renal cancer, glioblastoma, metastatic As resistance of cancer cells to apoptosis has been breast cancer and non-squamous non-small cell lung related to enhanced expression of antiapoptotic proteins cancer. Besides participating in tumor neovasculariza- from both the BCL-2 family and IAPs, strategies seeking tion, the VEGF pathway is an essential component of to antagonize the action of these molecules (BCL-2 and normal development and participates in the homeostasis IAP inhibitors) have been extensively studied for cancer of many organs in the adulthood. Therefore, a broad therapy. spectrum of side effects is associated with antiangiogenic BCL-2 inhibitors: The overexpression of the antiapop- therapies, such as hypertension, hemorrhage, arterial totic protein BCL-2 in several types of cancer promotes trombo-embolic events, proteinuria, renal dysfunction cancer cell survival without affecting cell proliferation. or gastrointestinal perforation. Although most of these This supports the hypothesis that restoring apoptosis adverse effects are manageable, in particular cases side in cancer cells by modulating the levels/function of

Oncogene Novel paradigms for cancer therapy V Pavet et al 4

Figure 2 TRAIL signaling pathway. (a) TRAIL is believed to bind as a homotrimeric molecule to its four receptors. Two decoy receptors (DcR1 and DcR2) either lack or present a truncated intracellular domain, thus not triggering intracellular signaling upon TRAIL binding. The death receptors 4 and 5 (DR4 and DR5) harbor an intracellular death domain (DD) that, upon TRAIL binding, recruits the adaptor protein FADD. This adaptor in turn recruits procaspase 8 and/or 10, forming the death-inducing signaling complex (DISC). (b) Upon DISC formation, initiator caspases 8 and/or 10 get cleaved and activated. Activated initiator caspases act directly on the effector caspases 3, 6 and/or 7, leading to cell death via the extrinsic pathway (in type I cells); alternatively, they can cleave and activate Bid, triggering the intrinsic death pathway, thereby amplifying the apoptotic signaling (type II cells). (c) Recent studies suggest that under certain conditions, nonapoptotic intracellular signaling can be triggered upon DISC formation, resulting in cell proliferation, migration and survival. The proposed model of these pathways is depicted.

antiapoptotic proteins could help to remove cancerous displayed tumoricidal activity in vitro and in vivo (Sun cells. The BCL-2 family is characterized by speciﬁc et al., 2008). Interestingly, phase I clinical trials of a homology regions named BH (BCL-2 homology) do- broad-spectrum inhibitor of the antiapoptotic Bcl-2 mains. Whereas all ﬁve antiapoptotic proteins and BAX/ family (obatolax mesylate) showed that it is well BAK proapoptotic proteins present four BH domains tolerated, has biological activity and displays modest (BH1, BH2, BH3 and BH4), the remaining eight single-agent activity in heavily pretreated chronic lym- proapoptotic proteins only present the BH3 domain phocytic leukemia patients (Tan et al., 2009). Another (thus called BH3-only proteins). The BCL-2 antiapopto- strategy involves the use of an antisense oligonucleotide tic protein contains a BH3 binding groove that accom- to target Bcl-2 mRNA (oblimersen sodium). The admin- modates the BH3 domain of proapoptotic proteins, istration of this molecule in different combinatorial therefore impeding their action and blocking apoptosis. approaches (that is, with chemotherapy among others) This structural feature provides the platform to develop showed activity for treatment of chronic lymphocytic peptides and chemical compounds that bind to the groove leukemia and malignant melanoma in phase III clinical of the antiapoptotic protein, thus reliving the apoptotic trials (Bedikian et al., 2006; O’Brien et al., 2007). block and promoting cell death (Lessene et al.,2008). IAP inhibitors: Overexpression of IAPs can confer However, initial phase I and II clinical trials administer- resistance to a broad variety of apoptotic stimuli and ing the BH3-mimetic gossypol were not encouraging. constitutes a marker of poor prognosis in hematologic More recently, gossypol derivates and analogs have been and solid cancers. Suppression of IAP activity/levels was developed. Among those, AT-101 is well tolerated and is shown to either directly trigger apoptosis or sensitize currently tested in phase I/II clinical trials for prostate, cancer cells to proapoptotic triggers currently used as lung and esophageal cancer, glioblastoma and B-cell anticancer treatment. Thus, strategies mimicking the lymphoproliferations. A semisynthetic gossypol analog action of endogenous IAP inhibitors such as Smac (apogossypolone) with improved pharmacologic activity (SMAC mimetics), blocking the interaction of XIAP

Oncogene Novel paradigms for cancer therapy V Pavet et al 5 with caspases (XIAP-BIR2 inhibitors) or modulating the (‘differentiation therapy’). This differentiation is be- expression levels of XIAP or survivin (XIAP/survivin lieved to occur as a result of the activation of RARa- antisense oligonucleotides) have been developed to target genes by ATRA binding to PML–RARa, which treat cancer. Whereas several phase I/II clinical trials would dissociate abnormally bound corepressor-histone administering either XIAP (AEG35156) or survivin deacetylase (HDAC) complexes from the fusion protein. (LY2813008, YM155 and EM1421) antisense oligonu- However, it was subsequently observed that liposomal cleotides as single agents or in combination with ATRA as single agent induced the degradation of the chemotherapy are currently ongoing, the development leukemogenic protein itself, achieving complete remis- of XIAP-BIR2 inhibitors is still in preclinical phase sion in human APL patients (Tsimberidou et al., 2006). (Fulda, 2009). Additional insight into the critical role of the fusion The interaction of the endogenous Smac protein with protein was obtained from a mouse model of APL, in IAPs is mediated by its N-terminal tetrapeptide AVPI which leukemia cells were serially transplanted from one (Ala1-Val2-Pro3-Ile4). Based on structural information, animal to another, resulting in transmission of the several small molecules mimicking AVPI (and thus Smac disease. In this model, a sub-population of the leukemic inhibitory function) were designed in order to derive cells (referred to as leukemia-initiating cells (LICs)) stable compounds that could be administered as ther- persisted after ATRA treatment, although ATRA apeutics. These include monovalent Smac-derived pep- induced differentiation in the bulk of cells. However, tides as well as mono- and bi-valent Smac-derived combining ATRA with arsenic trioxide (an agent peptidomimetics. To date, four Smac mimetic compounds approved for second-line APL therapy) completely (GDC-0152, LCL161, AEG40826/HGS1029 and AT- wiped out the fusion protein and eradicated both LICs 406) have been approved for clinical trials (Flygare and and bulk APL cells, and led to complete remission (Nasr Fairbrother, 2010). Whether mono- or bi-valent mole- et al., 2008). cules would present the higher activity/less toxic effects in The discovery that PML–RARA is the cause of APL humans remains to be established. coincided with the identification of RARA as a gene coding for the RAR-a, and provided a rationale for the use of ATRA as an agent that specifically attacks the Destroying the cause of cancer and inducing differentia- fusion protein. To date, ATRA-differentiative capacity tion: oncofusion proteins as targets. Somatic chromo- and ATRA/arsenic-induced degradation of PML– somal rearrangements can cause the formation of RARa fusion protein is believed to account for the abnormal fusion proteins, leading to cancer as a result therapeutic response. From a conceptual point of view, of the deregulation of key factors involved in the control these results argue that identifying and targeting cancer- of cell growth, survival or death. One of the best causing aberrant proteins would ultimately lead to cure. understood fusion proteins is the prototypic PML– Unfortunately, APL is a rather specific case in which the RARa (promyelocytic leukemia–retinoic acid receptor) fusion protein generates a pre-leukemic state, and as that causes APL and originates from the translocation little as one additional ‘hit’ is believed to suffice for t(15;17)(q22;q21). This chromosomal rearrangement progression to the fully leukemic state. Moreover, the juxtaposes a portion of the PML and RARA genes presence of the leukemogenic fusion protein remains (other X-RARa oncofusions are similarly leukemogenic; essential for the disease to develop. In contrast, a vast Melnick and Licht, 1999). The aberrant PML–RARa majority of solid cancers display multiple (epi)genetic fusion protein displays a plethora of molecular features alterations and somatic mutations that increase during distinct from RARa, including altered homo- and progression (Shah et al., 2009), involving not only hetero-oligomerization, different DNA-binding site re- coding sequences but also non-coding RNAs. Further- pertoires, changes in the interaction with key factors like more, o10% of (epi)mutations found in tumors are p53 or abnormal recruitment of epigenetically active indeed ‘driver’ mutations causally involved in tumori- complexes to RARa targets (Grignani et al., 1998; He genesis, whereas the rest are ‘passenger’ mutations that et al., 1998; Lin et al., 1998, 2004; Di Croce et al., 2002; neither contribute to the onset nor to the development Bernardi et al., 2004; Insinga et al., 2004; Kamashev of the disease (Greenman et al., 2007; Wood et al., 2007). et al., 2004; de Stanchina et al., 2004; Zeisig et al., 2007). It is conceivable that tumor cells become independent of However, which of these abnormal characteristics, if not the initial (epi)mutation(s) as a result of the inherent all, are responsible for the initiation of leukemogenesis genetic instability. As patients are usually diagnosed has remained elusive. Another open question that was with a developed tumor, it is unlikely that the search raised in view of the long latency for induction of a full and targeting of cancer-causing aberrations might be a leukemic phenotype observed in murine models, is general applicable therapeutic rationale (Huang et al., whether or not a secondary mutation is required to 1988; Warrell et al., 1993; Soignet et al., 1998). develop the disease (‘second hit’ hypothesis) (Yuan et al., 2001; Schessl et al., 2005; Yamashita et al., 2005). Importantly, the presence of the PML–RARa onco- Inhibiting tumor-activated kinases that regulate cancer fusion protein remains essential for maintenance of cell proliferation and survival. Deregulation of kinase the leukemic phenotype. activity is a major mechanism by which cancer cells Initially, it was thought that ATRA treatment would evade physiological constraints controlling cell growth cure patients because of the differentiation of blasts and survival. The drugability of these molecules and the

Oncogene Novel paradigms for cancer therapy V Pavet et al 6 dependency of tumor cells on kinase activity for survival proven clinical value of these drugs in the therapy of make them important targets for cancer therapy. CML and gastrointestinal stromal tumors, key ques- Aberrant kinase activity can result from the generation tions such as whether (an aberrant) a kinase or a set of of fusion proteins formed by chromosomal transloca- kinase(s) can be identified as driver(s) of the disease, and tions or by aberrant expression/activity of the protein whether the identified kinase is also required for the itself. Chronic myeloid leukemia (CML) represents a maintenance/survival of the corresponding TICs, re- well-characterized example for the expression of an main to be answered. Moreover, as the mutational aberrant kinase. In CML, the chromosomal transloca- status of the tumor cell and the tumor microenviron- tion t(9;22)(q34;q11) generates the ‘Philadelphia (Ph) ment may modulate tyrosine kinase activity, their effect chromosome’ harboring a fusion between the ABL1 on the action of the inhibitor needs to be clarified (for tyrosine kinase gene on chromosome 9 and the ‘break- extensive reviews about the cancer kinome and kinome point cluster region’ (BCR) on chromosome 22, result- (poly)pharmacology, see Zhang et al., 2009; Knight ing in the expression of a BCR–ABL fusion protein. et al., 2010). Like PML–RARa in the case of APL, the BCR–ABL protein is not only the initial cause but also the singular driver of CML throughout its course, thereby identify- Attacking the tumor epigenome. The term ‘epigenetics’ ing it as an ideal drug target. BCR–ABL displays describes mitotically inheritable changes of gene expres- constitutive kinase activity, which results in the deregu- sion that do not involve changes of the DNA sequence. lated activation of signaling cascades that promote cell This is achieved by DNA methylation of CpG dinucleo- proliferation and survival (Shtivelman et al., 1985; tides and, in recent years, this term is also applied for the Kantarjian et al., 2006). The first compound that gene-regulatory potential of post-translational modifi- successfully inhibited the aberrant BCR–ABL kinase cations of histones (Klose and Bird, 2006). It is now well activity and displayed clinical efficacy was imatinib established that the epigenetic status of a cell is severely mesylate (Druker, 2001), which has considerably in- altered upon malignant transformation. Cancer cells creased the survival rate of CML patients (88% for 6 concomitantly display global genomic DNA hypo- years) and decreased the need of hematopoietic stem cell methylation and gene-specific promoter hypermethyla- transplantation (Hochhaus et al., 2009). At present, two tion. Whereas DNA hypomethylation may contribute to other inhibitors have been approved and can be used to the tumorigenic process by inducing chromosomal treat patients refractory to imatinib (Kantarjian and instability and reactivation of transposons and onco- Cortes, 2006; Cortes et al., 2007; le Coutre et al., 2008). genes, hypermethylation of CpG islands may result in Notably, although imatinib was originally designed to the silencing of tumor-suppressor genes, a hallmark specifically target BCR–ABL, this compound also during cancer onset and progression (Feinberg and inhibits the c-Abl, DDR1 (discoidin domain receptor Vogelstein, 1983; Gama-Sosa et al., 1983; Fraga and family, member 1), platelet-derived growth factor Esteller, 2005; Weber et al., 2005). More recently, receptor and c-kit kinases, and has shown therapeutic changes in the methylation pattern of DNA regions activity for gastrointestinal stromal tumors, which often coding for micro-RNAs (miRs) that modulate the overexpress c-kit (Demetri et al., 2002). expression of oncogenes or tumor suppressors have The clinical success of imatinib has inspired the been reported (Datta et al., 2008; Huang et al., 2009; mapping of the full range of abnormal protein kinases Pallasch et al., 2009). Inversely, it has been observed present in human cancers (tumor kinome), and a large that oncofusion proteins can epigenetically silence miRs repertoire of kinase inhibitors is continuously being that are critically involved in lineage specification (Fazi expanded (Futreal et al., 2004; Janne et al., 2009). et al., 2007). Altogether, these studies argue that locally Ongoing activities try to match cancer kinome aberra- altered methyl-CpG densities can result in the deregula- tions with the target profiles and selectivity of these tion of multiple regulatory pathways, which are relevant inhibitors. for tumorigenesis. Furthermore, abnormal function/ Several conceptual issues have to be considered when recruitment of histone-epigenetic modulators is also using kinases for drug targeting. The validation that the recognized to have a role in tumorigenesis (Feinberg and kinase to be targeted (like in CML) is the singular driver Tycko, 2004; Esteller, 2008). Consequently, epigenetic and/or indispensable for persistence of the disease enzymes have been identified as valuable drug targets, as should be provided. As de novo or acquired resistance aberrant DNA methylation and histone modifications to these kinds of inhibitors has been reported, strategies can be reverted by the administration of epigenetic to minimize this risk have to be developed. In this drugs (‘epi-drugs’). respect, the identification of molecular markers of Inhibitors of DNA methylation (cytosine analogs; sensitivity/resistance to specific compounds is a chal- 5-azacytidine and 5-aza-20-deoxycytidine) are currently lenge. Finally, at present it is not possible to predict ‘if’ used for the treatment of myelodysplastic syndrome and and ‘how many’ matches between increased kinase the first two HDAC inhibitors, vorinostat and romi- activity in cancers and kinase inhibitors can be found; depsin, have been approved by the FDA for the technological advances like fragment-based drug dis- treatment of cutaneous T-cell lymphoma (http://clin- covery (Blundell et al., 2002) and crystallization of icaltrials.gov) (Drummond et al., 2005; Grant et al., kinase-inhibitor complexes should accelerate and im- 2007; Xu et al., 2007). With the identification of large prove drug development. In summary, despite the families of histone methyltransferases and demethylases

Oncogene Novel paradigms for cancer therapy V Pavet et al 7 and the recognition of their impact on carcinogenesis 15 years. These include viral mutants, in which genes (Shi and Whetstine, 2007; Spannhoff et al., 2009), both critical for viral growth in normal cells but not in tumor pharmaceutical companies and academic institutions cells were deleted, pseudotyped viruses, in which normal have initiated programs to specify the (patho)physiolo- viral tropism was ablated, and engineered viruses gical role of the various family members, to develop binding to speciﬁc surface receptors that are expressed inhibitors and to validate their therapeutic use (Spannh- exclusively/preferentially on tumor cells (Alemany et al., off et al., 2009). 2000; Khuri et al., 2000; Kirn, 2000; Fukuhara et al., The use of epi-drugs for therapy has both advantages 2005, 2009; Kasuya et al., 2005; Liu and Kirn, 2008; and drawbacks. On the positive side is the fact that epi- Dorer and Nettelbeck, 2009). Interestingly, oncolytic enzymes are drugable, non-genotoxic and act transi- viruses can kill apoptosis-resistant tumor cells, and do ently. The observation that a block of total HDAC not show cross-resistance with existing therapies. activity is tolerable to cutaneous T-cell lymphoma Although some promising results were observed in patients reveals a high plasticity of epigenomes, parti- clinical trials, the predicted replication selectivity has cularly those of normal cells. Ideally, inhibiting the not (yet) been reached. Reasons for this comprise the action of aberrant epi-enzymes would allow the reacti- incomplete knowledge of the complex virus–cell inter- vation of the epigenetically silenced endogenous defense actions, the leakiness of cellular promoters in the viral systems. On the negative side are the problems to genome and the interplay of the virus with the immune generate selective epi-drugs in the context of large system (Liu et al., 2007). families of epi-enzymes, and the presently incomplete A second virus-based approach involves the exogen- knowledge about their (patho)physiological functions. ous administration of viral-derived proteins to induce Moreover, the transient action of epi-drugs may also be tumor-selective cell death (Noteborn, 2009). Such a disadvantage, as it could foster development of activities have been demonstrated for the adenovirus- resistance. In this respect, the combination of several derived E4orf4, the parvovirus H1 protein NS1 and epigenetic activities is an option that is currently being chicken anemia virus-derived apoptin (Guelen et al., explored. An entirely open question is the epigenetic 2004; Maddika et al., 2005; Maddika et al., 2006; Rohn makeup of TICs that may well differ from the bulk of and Noteborn, 2004) (Table 1). During natural infec- tumor cells and represent particularly attractive targets. tion, these proteins orchestrate the cytolytic events that are requisite for successful spreading of the virus. Strategies to selectively kill tumor cells Interestingly, it has been shown that overexpression of Administration of virus and virus-derived proteins for these viral-derived proteins is cytotoxic for tumor but cancer therapy. Several viruses, including species dis- not for normal cells. playing an inherent tumor selectivity (that is, parvo- The postulated mechanisms of action of these proteins viruses, vesicular stomatitis viruses, Newcastle disease are not fully elucidated and appear to be diverse. NS1 virus), and engineered viruses (adenoviruses, herpex was shown to interact with casein kinase II of the host simple virus, measles virus) can replicate selectively cell, thus inducing casein kinase II-dependent cytoske- in cancer cells, thereby leading to cancer cell-selective letal changes that lead to apoptosis (Nuesch and lysis (oncolysis). Oncolytic viruses displaying various Rommelaere, 2006, 2007). E4orf4 is an adenovirus mechanisms of action have been developed over the past protein that does not require the classical caspase

Table 1 Proteins inducing tumor-selective cell death Protein Dependency Mechanism Therapeutic approach Therapeutic status References

p53 Bcl-2

NS1 ? No Autophagy Vaccinia delivery Preclinical Nuesch et al., 2006, 2007, 2008 Adenovirus delivery Parvovirus-H1 infection E4orf4 No No Apoptosis like Adenovirus delivery Preclinical Mitrus et al., 2005; Landry et al., Mitotic catastrophe Electroporated DNA 2006; Li et al., 2009a, b Apoptin No No Apoptosis Recombinant protein Preclinical Guelen et al., 2004; Backendorf Intrinsic pathway Viral delivery et al., 2008; Sun et al., 2009 Plasmid DNA HAMLET No No Apoptosis In vitro reconstituted Phase I and II Mossberg et al., 2007; Autophagy HAMLET clinical trials Hallgren et al., 2008 MDA-7 No Yes Apoptosis Adenovirus delivery Phase I and II Lebedeva et al., 2007; Eager et al., Intrinsic/extrinsic Plasmid DNA clinical trials 2008; Emdad et al., 2009 pathway TRAIL No Yes Apoptosis DR4/DR5 agonistic Phase I and II Holoch and Grifﬁth, 2009; Extrinsic/intrinsic antibodies clinical trials Newsom-Davis et al., 2009 pathway Recombinant protein Viral delivery

Abbreviations: BCL-2, B-cell lymphoma-2; DR, death receptor; HAMLET, human a-lactalbumin made lethal to tumor cells; MDA-7, melanoma differentiation-associated gene-7; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. ‘?’ Stands for unknown.

Oncogene Novel paradigms for cancer therapy V Pavet et al 8 pathways in order to trigger cell death (Landry et al., icidal activity in humans (Gustafsson et al., 2004; 2006), and it leads to changes in cell morphology Mossberg et al., 2007; Noteborn, 2009) (Table 1). That (including multiple nuclei and increase in cell volume) HAMLET has been proven active in vivo, and that no that suggest a tumor cell-selective G2/M arrest, leading major toxic effects have been documented, is encoura- to mitotic catastrophe (Li et al., 2009a, b). Finally, ging. However, as local administration is necessary, a apoptin induces apoptosis in a wide variety of human very restrictive spectrum of cancer types can be targeted. cancer cell lines via classical apoptotic pathways (Rohn Efforts to determine the molecular mechanisms under- and Noteborn, 2004; Maddika et al., 2005, 2006; lying HAMLET-induced cell death and its cancer Backendorf et al., 2008). Several independent in vivo selective action are required in order to provide the analyses using human xenografted tumors have proven molecular rationale(s) of its mode of action, hence that administration of apoptin, as well as its recombi- expanding the spectrum of potential therapeutic targets nant version protein transduction domain 4 (PTD4)- to exploit the HAMLET-induced tumor-selective cell apoptin, results in a significant reduction of tumor death pathway(s). growth without major side effects (Peng et al., 2007; Sun et al., 2009). An important feature shared by these three Exploiting the endogenous tumor-surveillance system for viral-derived anticancer proteins is the fact that they do cancer therapy. induce cell death in a p53-independent manner and that Melanoma differentiation-associated gene-7 (mda-7): the tumoricidal activity is not affected by the over- The expression of this human endogenous cytokine expression of Bcl-2 (Table 1). Hence, NS1, Eorf4 and (also known as interleukin-24) was first described in apoptin circumvent blocks in the apoptosis machinery normal melanocytes and keratinocytes, and later re- often observed in tumor cells and could induce ported in tissues and cells of the immune system alternative types of cell death (that is, mitotic cata- (Jiang et al., 1995; Wang and Liang, 2005). The strophe). Although these molecules could represent observation that the expression of this interleukin serious candidates for the development of novel antic- decreases during pathologic progression of melanocytes ancer paradigms, further insights into the molecular into melanomas suggested a potential role as tumor mechanisms by which these factors achieve tumor suppressor (Huang et al., 2001; Ellerhorst et al., 2002). selectivity are essential for their validation and devel- Although the exact biological functions of mda-7 are opment as cancer therapeutics. not fully elucidated, it is clear that it presents a dual role as a classical cytokine via its receptors and as a cytotoxic agent acting intracellularly in a receptor-independent HAMLET: a cellular-derived complex displaying tumori- manner (Sieger et al., 2004; Ekmekcioglu et al., 2008). It cidal activity. The tumoricidal activity of a substance has been demonstrated that whereas normal cells are not purified from human milk, a-lactalbumin bound to oleic affected by mda-7 expression, it induces antiproliferative acid (also referred to as ‘human a-lactalbumin made effects as well as downregulation of survival signals lethal to tumor cells’ (HAMLET)), was discovered by leading to apoptosis in several cancer cell lines by serendipity when the role of milk fractions on bacterial modulating different pathways (p38-mitogen-activated attachment to lung carcinoma cells was studied protein kinase (MAPK), protein kinase RNA-regulated (Hakansson et al., 1995). Apart from inhibiting bacterial (PKR), c-Jun-N-terminal kinase (JNK), b-catenin and attachment, this fraction also displayed tumoricidal phosphatidylinositol-3-kinase (PI3K)/Akt signaling activity and suggested a potential use of HAMLET as pathways). This interleukin also induces Fas/FasL an anticancer agent (Table 1). This complex has been signaling, thus supporting apoptosis via the extrinsic reported to induce cell death in a plethora of cancer cell apoptotic cascade (Gopalan et al., 2005; Shanker et al., lines, whereas normal cells are resistant (Svanborg et al., 2007). 2003; Hallgren et al., 2008). It has been postulated that Besides its apoptotic potential in tumor cells, mda-7 HAMLET might interact and perturb the functioning of also inhibits cancer cell migration and invasion, and the mitochondria (Kohler et al., 1999), proteosome displays a substantial antiangiogenic activity within (Gustafsson et al., 2009) and histones (Duringer et al., tumors (Lebedeva et al., 2007). Interestingly, phase I 2003), inducing features of multimembrane autophago- clinical trials using mda-7 as single agent for the somes and suggesting that macroautophagy might treatment of malignant melanoma demonstrated sig- contribute to tumor cell death (Aits et al., 2009). nificant activity and good tolerability (Lebedeva et al., Although it has been demonstrated that normal and 2007; Eager et al., 2008; Emdad et al., 2009) (Table 1). tumor cells display different features for the subcellular However, it has been observed that subsets of tumor localization and internalization of HAMLET (suggest- cells are either inheritably resistant to mda-7 or can ing the existence of active shuttling mechanisms), neither acquire resistance upon exposure to the cytokine. the receptors involved nor the mechanisms orchestrating Although some molecules/pathways participating in the protein uptake have been identified. the acquisition of the resistant phenotype have Despite limited knowledge on the molecular mechan- been identified (that is, Bcl-2; activation of the isms orchestrating HAMLET action, two clinical pilot kRas signaling pathway among others), neither the experiments using local treatment of skin papillomas molecular basis underlying resistance in different cancer and intravesical instillation of bladder cancer with cells nor the molecular basis of tumor selectivity have HAMLET revealed that this complex displays tumor- been unraveled (Sarkar et al., 2008).

Oncogene Novel paradigms for cancer therapy V Pavet et al 9 Tumor necrosis factor-related apoptosis-inducing been developed, including the administration of recom- ligand (TRAIL): TRAIL, a member of the extrinsic binant human TRAIL (Apo2L/AMG951); the use of death pathway, is a type II trans-membrane protein that activating humanized antibodies directed against the binds as a homotrimer to the extracellular part of its death receptors DR4 (mapatumumab/HGS-ETR1) or four membrane-bound TRAIL-specific receptors DR5 (lexatumumab/HGS-ETR2/AMG655); and the (Figure 2a) with similar affinity. Among the four adenoviral delivery of the TRAIL coding sequence into receptors, two DRs (death receptors DR4/TRAIL-R1 tumor cells (Ad5-TRAIL) (Holoch and Griffith, 2009). and DR5/TRAIL-R2) present an intracellular death Initial reports from phase I and II trials indicate that, domain that, upon TRAIL binding, recruits adaptor contrary to what was expected based on in vitro data, proteins (FADD (Fas-associated death domain)) and hepatic or renal toxicity were not clinically significant initiator caspases (procaspases-8/10), thereby transdu- and, when observed, they were modest and usually in cing the apoptotic signal by activating the caspase patients presenting hepatic problems as baseline. In- cascade via the extrinsic pathway (Figure 2b). In some deed, the most common adverse effects included nausea, cells, TRAIL will also operate by activating the intrinsic fatigue, constipation and leucopenia (in B10–20% of pathway, crosstalk mediated by the action of Bid the patients) and did not lead to the discontinuation of (Figure 2b). The other two TRAIL receptors (decoy the treatment. Furthermore, no immunogenicity against receptors (DcR1/TRAIL-R3 and DcR2/TRAIL-R4)) mapatumumab or lexatumumab has been observed. either lack or have a truncated death domain, thus Clinical responses ranging from stable disease, partial binding the ligand without inducing apoptosis responses and even a complete response have been (Figure 2a). Several reports indicate that the TRAIL reported after administering either untagged versions of pathway is a natural component of the endogenous recombinant human TRAIL or antibodies targeting tumor-surveillance system in mammals (Smyth et al., death receptors as monotherapy (Plummer et al., 2007; 2001; Takeda et al., 2001, 2002, 2004; Cretney et al., Tolcher et al., 2007; Greco et al., 2008; Hotte et al., 2002; Zerafa et al., 2005). Moreover, TRAIL exerts a 2008; Leong et al., 2009; Mom et al., 2009; Trarbach potent tumoricidal activity in cancer cells in vitro and et al., 2010; Wakelee et al., 2010). Taken together, safety in vivo, causing negligible effects on normal cells when results and clinical responses encourage further disease- exogenously administered, an important feature of this directed assays of these agents. cascade regarding its therapeutic potential (Walczak These clinical trials have also indicated that several et al., 1999; Mitsiades et al., 2001; Naka et al., 2002; human tumors might be resistant to TRAIL mono- Georgakis et al., 2005; Motoki et al., 2005; Pukac et al., therapy. Moreover, proliferative effects in response to 2005). Early in vitro studies raised concerns regarding TRAIL have been reported in vitro using glioma and the potential toxicity of TRAIL toward normal hepa- small cell lung cancer cell lines (Belyanskaya et al., 2008; tocytes, prostate, endothelial and brain cells (Jo et al., Vilimanovich and Bumbasirevic, 2008) (Figure 2c). In 2000; Leverkus et al., 2000; Nitsch et al., 2000; Ozoren other case, mice in which Bcl-xl-overexpressing Colo357 et al., 2000; Nesterov et al., 2002; Li et al., 2003). pancreatic ductal carcinomas were orthotopic trans- However, later reports indicated that this toxic effect, planted formed more metastases upon TRAIL treat- observed under in vitro culturing conditions, might ment (Trauzold et al., 2006), and finally two studies with result from the use of tagged-TRAIL (histidine, FLAG blasts from leukemia patients treated ex vivo report or leucine-tagged TRAIL), whereas the recombinant effects of TRAIL exposure ranging from triggering of untagged version of the cytokine was nontoxic on cell death to induction of cell proliferation (Ehrhardt normal cells (Lawrence et al., 2001; Qin et al., 2001). et al., 2003; Hasegawa et al., 2005). TRAIL-induced Furthermore, recent studies have shown that whereas proliferative effect has also been reported in synovio- the tagged versions of TRAIL might result in toxicity on cytes from human rheumatoid patients who exhibit a primary human hepatocytes in vitro, the same com- poorly understood biphasic response to the cytokine, as pounds displayed very modest toxic effect on hepatic it triggers death in an initial phase but support explants obtained from healthy donors, indicating that proliferation for the persisting/resistant cell fraction the use of primary human hepatocytes as model for (Morel et al., 2005). These results suggest that in the case analyzing toxicity effects might not be the most suitable of TRAIL resistance in cancer cells or specific inflam- (Volkmann et al., 2007). The same report shows that matory conditions, cells may switch the TRAIL hepatic explants obtained from patients suffering from response from apoptotic to proliferative. Although there hepatic diseases (hepatitis C viral infections or liver is evidence that the nuclear factor-kB, p38, JNK and steatosis) were susceptible to the toxic action of the extracellular signal-regulated kinase pathways modulate cytokine, suggesting that the clinical use of TRAIL the proliferative responses, it will be important to might be cautiously considered in patients presenting precisely define which factors and complexes (for hepatic inflammatory diseases. example, death-inducing signaling complex members) Despite our limited understanding of the molecular are modulating two different responses to the same basis of the cancer-selective action of TRAIL, multiple trigger (Harper et al., 2001; Ehrhardt et al., 2003; clinical trials have been/are being conducted in order to Varfolomeev et al., 2005; Belyanskaya et al., 2008; define its therapeutic potential as a single agent or in Vilimanovich and Bumbasirevic, 2008) (Figure 2c). combinatorial settings (Table 2; http://clinicaltrials.gov). Moreover, even if an extensive number of molecules To date, three principal pharmacological strategies have and mechanisms have been shown to participate in

Oncogene Novel paradigms for cancer therapy V Pavet et al 10 Table 2 Completed and ongoing clinical trials exploiting the TRAIL signaling pathway Receptor engaged Approach Combination Tumor Clinical trail

Phase Status ID

Death receptors TRAIL-R1/DR4 Agonist Mapatumumab Cisplatin/ Cervical cancer Ib/II R NCT01088347 antibody (HGS-ETR1) radiotherapy (monoclonal- Sorafenib HCC I/II R NCT00712855 anti-DR4) Bortezomib Multiple myelo- II ANR NCT00315757 ma Carboplatin NSCLC II ANR NCT00583830 Paclitaxel — NSCLC II C NCT00092924 — Lymphoma II C NCT00094848 Non-Hodgkin TRAIL-R2/DR5 Lexatumumab INF-g Kidney cancer I ANR NCT00428272 (HGS-ETR2) Neuroblastoma (monoclonal- Sarcoma anti-DR5) Lymphoma

AMG-655 Doxorubicin Sarcoma Ib/II ANR NCT00626704 (monoclonal- Gemcitabine Pancreas Ib/II ANR NCT00630552 anti-DR5) FOLFOX6 CRC Ib/II ANR NCT00625651 Bevacizumab Carboplatin NSCLC Ib/II ANR NCT00534027 Paclitaxel Panitumumab CRC Ib/II ANR NCT00630786 AMG479 Solid Ib/II R NCT00819169 Bortezomib Lymphoma Ib R NCT00791011 Vorinostat AMG479 CRC II R NCT00813605 FOLFIRI

CS-1008 — Solid/Lymphoma I C NCT00320827 (monoclonal- Gemcitabine Pancreas II ANR NCT00521404 anti-DR5) Irinotecan CRC II R NCT00969033 Paclitaxel NSCLC II R NCT00991796 Carboplatin Paclitaxel Ovarian cancer II R NCT00945191 Carboplatin FOLFIRI CRC I R NCT01124630 Sorafenib Hepatic cancer II R NCT01033240

Apomab Cetuximab CRC Ib ANR NCT00497497 (PR095780) Irinotecan (monoclonal- Bevacizumab anti-DR5) FOLFIRI Rituximab NHL II C NCT00517049 Carboplatin NSCLC II ANR NCT00480831 Paclitaxel Bevacizumab — Chondrosarcoma II C NCT00543712T Bevacizumab CRC Ib C NCT00851136 FOLFLOX

Death and decoy receptors TRAIL-R1/DR4, Recombinant Apo2L/TRAIL Rituximab Low-grade NHL Ib/II ANR NCT00400764 TRAIL-R2/DR5, human TRAIL Cetuximab CRC I R NCT00671372 TRAIL-R3/ Irinotecan DcR1, AMG-951 FOLFLOX CRC I R NCT00873756 TRAIL-R4/DcR2 Bevacizumab Carboplatin NSCLC II ANR NCT00508625 Paclitaxel Bevacizumab

Abbreviations: ANR, active not recruiting; C, completed; CRC, colorectal cancer; DR, death receptor; HCC, hepatocellular carcinoma; IFN-g; interferon-g; NHL, non-Hodgkin’s lymphoma; NSCLC, non-small cell lung carcinoma; R, recruiting; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Oncogene Novel paradigms for cancer therapy V Pavet et al 11 TRAIL-induced apoptosis and/or resistance in cancer cells (Zhang and Fang, 2005; Thorburn et al., 2008), it has not been possible to determine a single ‘common factor’ regulating TRAIL sensitivity in different types of tumors. Thus, these data suggest that a ‘master regulator’ triggering the switch from TRAIL resistance in normal cells to TRAIL sensitivity in tumors may not exist, but that it is rather a balance between different molecules and/or signaling pathways that will dictate the final growth or apoptosis-stimulatory activity of the cytokine. Taken together, data from basic research and clinical trials not only support the promise of cancer-selective apoptosis through activation of the TRAIL pathway, but also point out the need for extensive and carefully monitored studies in which the regulatory networks controlling the apoptotic and proliferative activities of this signaling are deciphered. Therefore, an urgent need is to identify biomarkers and sensitivity assays that permit to recognize patients who will profit from the therapeutic potential of this pathway and would not suffer from potential non-desired proliferative effects. It is important to emphasize that resistance to TRAIL- Figure 3 A plethora of compounds modulate the sensitivity to induced apoptosis in cancer does not represent a TRAIL-induced apoptosis by regulating the expression/activity of roadblock for the use of the TRAIL signaling pathway different molecular players along the pathway. Known molecular players involved in TRAIL-induced apoptosis are depicted in the as a therapeutic target. Indeed, it is well established that pink circle (TRAIL, DISC, pro-/anti-apoptotic proteins, mito- resistance can be relieved by a poorly understood chondria, XIAPs and IAPs). Compounds displayed in concentric phenomenon generally referred to as ‘sensitization’. A partial circles (retinoids, chemo-radiotherapy, Smac mimetics, plethora of sensitizing agents, including ionizing radia- HDAC inhibitors, proteosome inhibitors, rexinoids, cAMP and demethylating agents) synergize with the TRAIL-apoptotic path- tion, chemotherapeutic drugs, cytokines as well as way by regulating the expression/activity of the molecular players HDAC, proteosome or phosphatidylinositol-3-kinase depicted in the pink circle. As evidenced, whereas some compounds (PI3K)/Akt inhibitors can reverse the TRAIL-resistant modulate TRAIL sensitivity by targeting different components of phenotype (Figure 3) (Altucci et al., 2001, 2005; the cascade (that is, HDAC inhibitors concomitantly modulate the Clarke et al., 2004; Insinga et al., 2005; Nebbioso expression of TRAIL, DISC members as well as pro-/antiapoptotic proteins and induce mitochondrial activation), others et al., 2005; Newsom-Davis et al., 2009). Furthermore, present a more restricted action (that is, Smac mimetics regulate the administration of inhibitors of the canonical nuclear activity of IAPs). factor-kB pathway, which has been shown to regulate resistance to TRAIL-induced apoptosis, induces sensitization of cancer cells to TRAIL (Ravi et al., 2001). avenues to exploit the TRAIL pathway as anticancer Interestingly, first reports from clinical trials combining treatment (Pavet et al., 2010). mapatumumab with cisplatin and gemcitabine have In summary, the observations that TRAIL (1) is part demonstrated that this combination can be safely of an evolutionarily shaped endogenous tumor defense administered. Response numbers of this phase I trial system, (2) induces apoptosis in a tumor-selective were encouraging, as 26/37 patients showed a decrease manner, (3) resistance can be overcome by various in tumor lesions and 12 showed partial response. modes of ‘sensitization’, (4) displays activity in pre- However, it is worth highlighting that the value of these clinical models (human tumor xenografts) (5) shows numbers is limited given the non-randomized nature of promising results in clinical trials and (6) can be used in the study. at least four different pharmacological modes, identify Recombinant proteins and humanized antibodies are the TRAIL cascade as one of the most exciting and without doubt well established as therapeutics. Never- promising pathways for the development of non- theless, the inherent disadvantages of proteins (synth- genotoxic cancer-selective therapeutics. To date, 6 esis, purity, stability, production costs) provide a strong clinical trials have been completed and data have been rationale for the development of more ‘drug-like’ released, whereas 25 targeting the TRAIL pathway activators of the TRAIL cascade. In that regard, the alone or in several combinatorial approaches are recent demonstration that specific multivalent DR5- currently ongoing (Table 2). It is worth mentioning that selective synthetic peptides activate the TRAIL pathway initial trials have enrolled patients mostly bearing in a tumor-selective manner even when combined with advanced, relapsed, refractory cancers. Therefore, the sensitizing compounds is a promising development. tumoricidal efficiency of inducers of TRAIL pathway in Moreover, these novel TRAILDR5/mim mimics displayed less-advanced stages of the malignancy is still unknown. anticancer activity in vivo as single agents in human Moreover, it has been very recently demonstrated that tumor xenograft models (Figure 4), opening new TRAIL in combination with all-trans-retinyl acetate can

Oncogene Novel paradigms for cancer therapy V Pavet et al 12 dependence receptors have been also described as modulators of the survival of endothelial cells, therefore promoting angiogenesis (Castets et al., 2009). The concept underlying the dual role of dependence receptors is that this coexistence allows them to not only regulate normal developmental process (that is, neural tube formation), but also to act as a safeguard mechanism to prevent tumor progression. As ligand- free dependence receptors induce apoptosis, they are believed to act as tumor suppressors, as hyperprolifera- tive or metastatic cells exposed to an abnormal ligand- depleted or -deﬁcient environment would undergo apoptosis. Consistently, tumor cells would either re- duce/deplete levels of dependence receptors or secrete high levels of the ligands in order to avoid cell death. This hypothesis is supported by the observations that reduced levels of several dependence receptors have been reported in different types of cancer and high expression of netrin-1 (ligand of the DCC (deleted in colorectal cancer) dependence receptor) has been recently demonstrated (recently reviewed in Goldschneider and Mehlen, 2010). Whereas reinstating the expression of dependence receptors in cancer cells that lost the receptor does not seem a suitable therapeutic approach, blocking survival signaling by interfering with ligand binding in cancer cells is promising in view of the overexpression of netrin in several types of primary and metastatic cancers. The use of decoy molecules such as the full ectodomain of DCC, shorter polypeptides encoding the DCC domains known to interact with netrin-1, or other approaches such as the use of blocking antibodies or peptidemi- metics that inhibit netrin–DCC interaction are in preclinical evaluation (Mehlen and Guenebeaud, 2010). An Figure 4 TRAIL mimics display anticancer activity in vivo. (a) Structure of the divalent TRAILmim/DR5.(b) Effect of exposure important open issue that will have to be addressed of mice bearing HCT116 human colon carcinoma xenografts to concerning the targeting of dependence receptor path- 8 mpk TRAILmim/DR5 for the indicated period (daily intraperitoneal ways for cancer therapy is the effect of such molecules injections). Red, treated group; black, vehicle group. *Po0.05; on normal cells and tissues. **Po0.005. Modiﬁed from Pavet et al. (2010).

efficiently target premalignant tumor cells (Zhang et al., Targeting TICs: a necessity to eradicate cancer?.Despite 2010). Whether TRAIL-based therapies may be utilized the significant progress that has been made in cancer for treatment of pre-neoplastic lesions or preventive diagnosis and therapy, the prognosis of most of the major therapy following surgery is an issue that deserves malignancies remains poor. Apart from acquired resis- further consideration. tance to treatment, this has been attributed to either an incomplete elimination of tumor cells or, in cases where complete remission has been observed, a relapse has been Emerging concepts for cancer therapy interpreted as reactivation of persisting ‘dormant’ tumor Dependence receptor signaling: new option for cancer cells. A key feature underlying these phenomena may be therapy?. Dependence receptors comprise a family of related to TICs (also referred to as cancer stem cells), a more than 12 membrane-bound receptors that are subset of cells within a tumor that display enhanced self- grouped together according to common function rather renewal and tumorigenic capacity. The existence of TICs than structural similarities. These receptors support cell is still a matter of controversy that is, at least in part, due differentiation, survival and migration (‘positive signal- to the vast variation in their abundance and proliferation. ing’) when bound to their cognate ligands. Importantly, Although virtually all cells of a melanoma may have the ligand-free receptor induces an entirely distinct cancer stem cell characteristics and medulloblastoma signaling cascade, resulting in cell death (‘negative TICs may be highly proliferative (Quintana et al.,2008; signaling’). Although initially identified as implicated Yang et al., 2008), TICs in general have been described as in nervous system development, these receptors were rather quiescent asymmetrically dividing cells (Holyoake subsequently shown to be involved in tumor progression et al., 2001; Guan et al., 2003). Therefore, as most of the (Mehlen and Puisieux, 2006; Goldschneider and Meh- initial and more recently developed tumoricidal drugs len, 2010; Fombonne and Thibert, 2010). More recently, target rapidly proliferating cells, TICs would be largely

Oncogene Novel paradigms for cancer therapy V Pavet et al 13 resistant to these cancer therapies and relapse is prone to products (salinomycin, abamectin and nigericin) or occur. To date, the only clinical evidence supporting this derivatives thereof (etoposide) (Gupta et al., 2009). concept involves acute myeloid leukemia, melanoma and Interestingly, salinomycin (a potassium ionophore) was breast cancer patients. When acute myeloid leukemia shown to preferentially interfere with TIC viability within patients are re-examined after chemotherapy, the persis- breast cancer populations, resulting in the loss of tence of CD34 þ CD38À cells (proposed to be LICs) expression of TIC-associated genes that correlated with predicts a high risk of relapse (van Rhenen et al.,2005). poor-prognosis tumors. Similarly, the presence of stemness markers within the In summary, most of the classical therapies currently tumor cells of melanoma and breast cancer patients used in the clinic may suffice to eliminate bulk tumor cells, (ABCB5 þ and CD44 þ , respectively) correlates with a but might not necessarily target TICs, proposed to be the higher rate of cancer progression and poor prognosis ones having the capacity to repopulate a tumor. Based on (Shipitsin et al.,2007;Schattonet al.,2008). the proof-of-concept provided by the APL, it seems rather As highlighted above, the eradication of LICs in APL obvious that tumoricidal strategies need to target TICs to upon treatment with retinoic acid and arsenic supports eradicate the disease. However, the picture is not so clear the concept that agents targeting TICs will lead to in solid tumors, as for a vast majority of them the nature/ complete cure. However, whereas in APL persistence of existenceofTICsisstillunder debate. Nevertheless, the PML–RARa fusion protein is apparently required for whether some of the therapeutic approaches discussed in the survival/proliferation of APL-LICs, the identification this review can indeed target bulk cells as well as TICs is a of similar key factors in solid tumors that arise from the topic that deserves to be intensively studied. An emerging accumulation of several driver (epi)mutations may pose area that needs to be further developed involves the serious problems. Therefore, particular attention has thorough identification and characterization of TICs, been paid to target-deregulated self-renewal pathways analyzing their genomes and epigenomes. To describe that have a critical role in TICs of solid tumors. Among the molecular signatures that distinguish TICs from the those, Hedgehog, Wnt and Notch are currently under corresponding bulk tumor cells, as well as to identify evaluation. Recently developed drugs target these signal- shared features, will not only allowustobetterunderstand ing cascades preferentially, and some agents are being the regulation of TIC stemness, but might also unravel evaluated in clinical trials (Zhou et al.,2009).Inaddition, novel targets for drug and epi-drug development. surface antigens currently used as markers for the identification of TICs can also be targeted by immu- Non-coding RNAs: potential biomarkers and therapeutic notherapeutic agents or small peptides. These molecules targets to fight cancer?. The development of new could interfere with signaling pathways orchestrated by technologies for the characterization of the human such surface molecules and exert therapeutic action. transcriptome provided evidence that almost 90% of Indeed, agents that target specific surface antigens of TICs the human genome is transcribed into RNA. Strikingly, are already in phase I/II clinical trials (Zhou et al., 2009). whereas only 2% of the transcribed RNAs display It is important to emphasize that natural compounds protein-coding capacity, the vast majority of the genome have also been reported to target TICs selectively, with is transcribed into non-coding RNAs (ncRNAs). parthenolide (PTL) being the first such compound ncRNAs are a very heterogeneous group that has been described (Guzman et al., 2005, 2007; Steele et al., 2006; classified in three families regarding their size: (1) Hassane et al., 2008; Kawasaki et al., 2009). PTL is a molecules ranging from 18 to 25 nt in length, comprising sesquiterpene lactonefoundinfeverfew(Chrysanthemum miRs and small interfering RNAs; (2) molecules from 30 parthenium), which has been traditionally used to treat to 300 nt, involving small nucleolar RNAs, piwi RNAs, migraine and rheumatoid arthritis and exerts activity as transfer RNAs, ribosomal RNAs and small-ncRNAs; nuclear factor-kB inhibitor, p53 activator (via ubiquitina- (3) molecules 4300 nt, referred to as long ncRNAs. The tion of MDM2, mouse double minute 2-p53 binding functionality of all transcribed ncRNAs is still a matter protein homolog), DNMT1 (DNA (cytosine-5-)-methyl- of controversy. Although some believe that these transferase 1) inhibitor and promoter of HDAC1 deple- molecules are just spurious transcription produced by tion by proteosome degradation (Gopal et al., 2007). pervasive transcription, others support that these Using the global transcription response to PTL, a molecules have critical roles within the cell. Experi- collection of compounds that mimic the effect of PTL mental data strongly support that several ncRNAs have were discovered and shown to target acute myeloid indeed biological function. The participation of leukemia stem cells (Hassane et al., 2008). Two of these, ncRNAs in translation (transfer RNA, ribosomal celastrol (a terpenoid) and 4-hydroxynonenal (a non- RNA), pre-mRNA splicing (small nuclear RNAs) and terpenoid lipid peroxidation product) are also natural RNA modifications (small nucleolar RNAs) were descri- products, whereas one additional hit, 15-deoxy-D12,14- bed long time ago. Moreover, it has been demonstrated PGJ2, is a prostaglandin derivative. Besides PTL, other that ncRNAs are expressed in a developmentally TIC-targeting compounds have been recently identified in regulated manner and their role in chromatin modifica- a chemical screen for molecules that target epithelial cells, tion, transcriptional regulation and post-transcriptional which have undergone an epithelial–mesenchymal transi- stabilization/degradation of mRNAs has been recently tion and display a TIC phenotype. Within a library of discovered (for a recent review, see Rana, 2007). 16 000 synthetic and natural compounds, four showed Importantly, ncRNAs affect key cellular processes consistent selective cytotoxicity for TICs; these are natural that are altered in cancer, such as cell differentiation,

Oncogene Novel paradigms for cancer therapy V Pavet et al 14 proliferation, death and survival. The H19 RNA was the This suggests that by modulating ncRNA levels, the first cancer-related ncRNA described whose expression is epigenetic makeup of a cell could be potentially modified, deregulated in cancer. Some reports show that in certain thus opening new avenues for epigenetic therapy. None- cancer cells, H19 expression decreases tumorigenicity, thus theless, several open questions remain to be answered acting as tumor suppressor. Conversely, other studies before ncRNAs can be used as therapeutics. For example, indicate that this ncRNA is elevated in different cancer it has been demonstrated that some miRs either block or types, in which it seems to display oncogenic activity. induce the translation of a given mRNA, depending on the These results highlight the fact that the expression level of proliferative status of the cell (Vasudevan et al., 2007). a given ncRNA by itself might not be sufficient for Moreover, although bioinformatic prediction indicates predicting its final oncogenic/tumor-suppressor capacity. that a given miR could have hundreds of potential target Indeed, the final effect exerted by ncRNAs may be mRNAs, the definition of the real target(s) within different dependent on cell type, developmental stage and expres- cells and the molecular basis of this selection have sion of cell/tissue-specific isoforms of the ncRNA or remained obscure. As miR expression signatures are expression of specific ncRNA-binding proteins and target associated with tumor classification, prognosis, diagnosis mRNAs. Indeed, it was recently reported that the H19 and treatment response, several clinical trials are currently ncRNA is a primary precursor encoding at least one miR ongoing with the aim of profiling or identifying ncRNAs (miR-675). Whether this or other miRs and/or ncRNAs in several types of cancer (http://clinicaltrials.gov). The processed from H19 precursor are responsible for the anticipated insight from current clinical trials and ongoing tumorigenic properties of this transcript remains to be efforts to unravel the function and regulation of ncRNAs established (Cai and Cullen, 2007). will not only help us to better understand cellular Although the aberrant expression of several small and circuitries, but may provide additional biomarkers and a long ncRNAs has been reported in cancer, and some can novel class of therapeutic targets to fight cancer. be already used as molecular markers of early disease (for example, DD3 RNA in prostate cancer) or prognostic factors (for example, MALAT-1 RNA in lung cancer), their functional role in the onset/progression of the disease Conclusions remains largely elusive. Despite this lack of knowledge, the observation that ncRNAs can regulate the tumor response Although the existence of neoplastic transformation has to different therapeutic drugs revealed their importance been acknowledged for centuries, treatment was re- not only as biomarkers but also as potential therapeutic stricted to surgery and radiotherapy until the 1950s. targets (Persson et al., 2009). As an example, the Starting in the 1980s, our increasing understanding of overexpression of the long ncRNA PCGEM1 (prostate (cancer) cell biology and the mechanisms of tumorigen- cancer gene expression marker 1) in prostate cancer esis/cancer progression allowed the development of models confers resistance against sodium selenite and ‘cancer-related therapies’. Although such mechanistic doxorubicin, thus supporting the escape of prostate cancer studies are intensified and enter a new dimension by the cells from drug-induced apoptosis (Srikantan et al., 2000). application of genome-wide technologies through a Among all ncRNAs, miRs are the ones that have been plethora of international consortia (for example, Inter- more extensively studied to date. These molecules have national Cancer Genome Consortium (ICGC; http:// been shown to display pro- and anti-tumorigenic capacity, www.icgc.org/); International Human Epigenome Con- to be either targets or regulators of main cancer- sortium (IHEC; http://www.epigenome-noe.net/WWW/ deregulated pathways (that is, p53, c-Myc among others), ihec/index.php)), today’s efforts include the discovery of and to modulate apoptotic responses. Supporting this line molecules and regulatory pathways that allow the design of thought, very recent reports have proven that modifying of ‘cancer-targeted therapies’, thus restricting side the levels of single miRs in vivo can directly induce a effects, and improving life quality and overall survival. reduction of tumor growth as well as regulate sensitivity to That cancer remains one of the largest causes of death apoptosis in cancer cells (Esquela-Kerscher et al., 2008; worldwide with a striking 13% of all human deaths per Idogawa et al., 2009). Similarly, certain miRs can year asks for a reflection about the principle concepts of modulate the response of cancer cells to TRAIL in vitro cancer therapy. Clearly, despite improvements, the and in preclinical models (Ovcharenko et al., 2007; critical issue of targeting cancer cells with high Garofalo et al., 2009). selectivity has by far not been reached for most cancers, In summary, the presently available data support the although some very specific cases are often presented as notion that ncRNAs are regulators of cell homeostasis and breakthrough in cancer therapy, such as APL and CML have central roles in a variety of processes that are relevant (see discussion above). In this study we attempted to to cancer cell growth and survival. Considering that a critically review present paradigms for cancer-selective single ncRNA can potentially orchestrate the expression of therapy and their status of development. From this several proteins engaged in different molecular pathways, analysis we derive three conceptual remarks. First, it is modulating the expression of a single RNA could unlikely that single compound satisfying the criteria of simultaneously target different molecular cascades. More- selectivity and efficacy can be found. This supports the over, it has been recently shown that ncRNAs can direct development of combination therapies that allow for epigenetic modifications including DNA methylation and dose reduction at similar efficacy, due to synergistic post-translational modifications of histones (Costa, 2008). action. Moreover, the combination of drugs exerting

Oncogene Novel paradigms for cancer therapy V Pavet et al 15 their effect through different therapeutic targets Novel, less-advanced therapeutic concepts are at minimizes the risk of resistance development. An various steps of development or validation, including attractive option in this respect is the generation of the conceptually attractive death-inducing ‘dependence compounds with multiple desired activities, such as a receptors’. At the horizon, the world of ncRNAs emerges recently described epi-drug that concomitantly targets with all the promises that a new world can offer. three different chromatin-modifying enzymes (WO 2008/125988 A1) (HDAC, sirtuin and DNMT1 inhibitory activity). Second, should there be a general cancer Conflict of interest eradication pathway that has been developed during evolution, we need to understand the mechanistic basis The authors declare no conflict of interest. and develop tools to exploit this selectivity for therapy. The apoptotic TRAIL signaling pathway is a very promising candidate in this respect, and intensive Acknowledgements research and drug development is ongoing at both pharmaceutical and academic research laboratories. Work in our laboratory is supported by Agence Nationale de la Recherche (ANR-07-PCVI-0031-01), the European Commi- Surprisingly, the mechanisms accounting for the cancer ssion (LSHC-CT-2005-518417 ‘Epitron,’ HEALTH-F4-2007- selectivity of TRAIL have still remained largely enig- 200767 ‘Apo-Sys,’ H Gronemeyer laboratory), La Ligue Contre matic. Third, the existence of TICs needs to be validated le Cancer (H Gronemeyer, laboratoire labelliseé; MMP post- and their response to current and future therapeutic doctoral fellow) and Fondation pour la Recherche Me´dicale paradigms has to be considered. (JCM master fellow).

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