European Journal of Pharmacology 625 (2009) 165173

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European Journal of Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Review

Proteins selectively killing tumor cells

Mathieu H.M. Noteborn
Molecular Genetics, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

a r t i c l e

i n f o

a b s t r a c t
All human cells have a genetic program that upon activation will cause cell death, named apoptosis. Cancer cells can grow due to unbalances in proliferation, cell cycle regulation and their apoptosis machinery: genomic mutations resulting in non-functional pro-apoptosis proteins or over-expression of anti-apoptosis proteins form the basis of tumor formation. Surprisingly, lessons learned from viruses show that cancer cannot be regarded simply as the opposite of apoptosis. For instance, adenovirus can only transform cells when both its anti- and pro-apoptotic proteins are produced. Oncolytic viruses are known to replicate selectively in tumor cells resulting in cell death. Proteins derived from viruses, i.e. chicken anemia virus (CAV)-derived apoptosis-inducing protein (apoptin), adenovirus early region 4 open reading frame (E4orf4) and parvovirus-H1 derived non-structural protein 1 (NS1), the human -lactalbumin made lethal to tumor cells (HAMLET), which is present in human milk or the human cytokines melanoma differentiationassociated gene-7 (mda-7) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have all the ability to induce tumor-selective apoptosis. The tumor-selective apoptosis-inducing proteins seem to interact with transforming survival processes, which can become redirected by these proteins into cell death. Transformation-related processes have been identied, which seem to be crucial for the tumor-selectively killing activity of these proteins. For instance, the transformation-related protein phosphatase 2A (PP2A) plays a role in the induction of tumor-selective apoptosis. The proteins mda-7, TRAIL and HAMLET are already successfully tested in rst clinical trials. Proteins harboring tumor-selective apoptosis characteristics represent, therefore, a therapeutic potential and a tool for unraveling tumor-related processes. Fundamental molecular and (pre)clinical therapeutic studies of the various tumor-selective apoptosis-inducing proteins apoptin, E4orf4, HAMLET, mda-7, NS1, TRAIL and related proteins will be discussed. 2009 Elsevier B.V. All rights reserved.

Article history: Accepted 25 June 2009 Available online 17 October 2009 Keywords: Apoptin Apoptosis Cancer E4orf4 HAMLET Mda-7 NS1 Oncolytic virus TRAIL

Contents Introduction . . . . . . . . . . . Apoptosis and cancer . . . . . . Lessons to be learned from viruses 3.1. Oncogenic viruses . . . . . 3.2. Oncolytic viruses . . . . . 4. Viral proteins . . . . . . . . . . 4.1. NS1 . . . . . . . . . . . 4.2. E4orf4 . . . . . . . . . . 4.3. Apoptin . . . . . . . . . 5. Human milk-derived HAMLET . . 6. Cytokines . . . . . . . . . . . . 6.1. Mda-7 . . . . . . . . . . 6.2. TRAIL . . . . . . . . . . 7. Summary . . . . . . . . . . . . Acknowledgements . . . . . . . . . . References . . . . . . . . . . . . . . 1. 2. 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 166 166 166 167 167 167 168 168 169 170 170 170 170 171 171

Tel.: +31 71 5274544; fax: +31 71 5274340. E-mail address: m.noteborn@chem.leidenuniv.nl. 0014-2999/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.06.068

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1. Introduction Cancer is one of the most life-threatening diseases worldwide (Syrigos et al., 2008; Gonzalez-Angulo et al., 2007). In most cases, anticancer therapies fail and, therefore, novel improved therapies are urgently needed (Sanchez-Munoz et al., 2008; Damber and Aus, 2008). Cancer cells can be described as derailed cells due to a large variety of genetic mutations (Tusell et al., 2008; Olopade et al., 2008). Unbalances in cell proliferation, cell cycle regulation and/or cell death are proven to be crucial for tumor formation (Briggs et al., 2009; Mishra et al., 2009). Identication of these aberrant molecular processes of cancer and related diseases has unraveled partially the development of tumor formation and has resulted in the generation of potential novel anticancer therapies (Carracedo et al., 2008; Tanaka and Arii, 2009). In general, cancer-related proteins are regarded as the ones that play a role in tumor cell formation. The last years, however, proteins have been described that become selectively active in tumorigenic backgrounds. The rst group of proteins consists of e.g. proteins causing cell proliferation and/or inhibiting cell death (Whibley et al., 2009), whereas proteins of the latter group induce apoptosis selectively in tumor cells (Backendorf et al., 2008; Ashkenazi, 2008; Hallgren et al., 2008; Eager et al., 2008). A growing group of viral and cellular proteins redirect tumor-related survival processes for inducing cell death. These apparent paradoxes of requirement of antiapoptosis processes enabling tumor-selective cell death have attracted the attention of researchers worldwide. Their efforts have gathered intriguing fundamental and applied results (Bruno et al., 2009; Noteborn, in press). In this report, we will discuss the principles underlying tumorselective cell death and their tremendous scientic and clinical relevance. The relationship between apoptosis and cancer development with a focus on oncogenic versus oncolytic viruses is described. The origin and thus far known molecular mechanisms of the tumorselectively killing proteins apoptin, E4orf4, HAMLET, mda-7, NS1 and TRAIL and their potential for clinical applications will be extensively discussed. 2. Apoptosis and cancer The death and subsequent removal of cells in a human body is occurring during its complete life time (Iannolo et al., 2008). In early embryonic development, apoptosis is e.g. essential for shaping our organs. At later stages, tissue homeostasis is based on cell renewal of cells, which underwent apoptosis (Fan and Bergmann, 2008). Apoptosis is essential for correct functioning of our immune system: apoptotic mechanisms are needed e.g. to control the development of thymocytes or the shaping of the T-cell repertoire (Giovanetti et al., 2008). At all life stages, apoptosis and cell growth processes are strictly coordinated and in balance (Fan and Bergmann, 2008). The genetically regulated apoptosis machinery can be distinguished in two main pathways: the intrinsic or mitochondrial pathway and the extrinsic or death receptor pathway (Qiao and Wong, 2009). Caspases, a family of cysteine proteases, play in both pathways a crucial role and their activation will result in the execution of apoptosis (Salvesen and Riedl, 2008). The apoptosis machinery contains pro- and anti-apoptosis proteins counteracting each other (Davenport, 2001). The Bcl-2 family consists of both pro- and antiapoptotic members. Over-expression of e.g. the anti-apoptotic Bcl-2 proteins is known to neutralize apoptosis signals, whereas e.g. the pro-apoptotic BAX will transfer an apoptotic signal into the execution of apoptosis (Levine et al., 2008). The tumor suppressor p53 is one of the well-known pro-apoptotic proteins in mediating apoptotic signals (Riley et al., 2008). Apoptosis is also essential in eliminating derailed cells. Various events such as DNA damage or chromosomal abnormalities trigger

apoptosis in normal cells. Unfortunately, cancer cells acquired mutations within their genome that force them to survive these apoptotic signals (Soussi and Wyman, 2007). Over-expression of the anti-apoptotic Bcl-2 protein e.g. due to chromosomal translocations is known to result in e.g. breast cancer and B-cell lymphoma (Lessene et al., 2008). Non-functional or absent pro-apoptotic p53 protein has also been linked to the development of a large panel of various tumors (Vazquez et al., 2008). In addition, derailed up regulated expression of caspase inhibitors also prevents cancer cells in undergoing apoptosis (LaCasse et al., 2008). Cancer cells are characterized by many derailed processes resulting in an unbalance between cell survival and apoptosis. To understand the processes underlying tumor formation, one has to characterize all aberrant processes in both survival and apoptosis networks and their connections (Russo et al., 2006). 3. Lessons to be learned from viruses 3.1. Oncogenic viruses Since it was known that certain viruses cause cellular transformation resulting in tumor formation, many studies were carried out with these viruses and their proteins to unravel interactions with cellular processes resulting in cellular transformation (Levine, 2009). For instance, human papilloma virus (HPV) E6 protein was shown to degrade tumor suppressor p53 protein (Murray-Zmijewski et al., 2008). In cervix tumor cells, HPV E6 and E7 proteins disrupt the normal checkpoint regulating cell cycle entrance and proliferation. Therefore, E6 and E7 proteins are crucial for the development of cancer (Thomas et al., 2008; Howie et al., 2009). One might argue that prevention of apoptosis by e.g. HPV E6 results in cellular transformation and subsequently tumor formation? Transforming adenoviruses, however, harbor besides anti-apoptotic proteins also proteins with an apoptotic activity. To transform cells both adenoviral E1A and E1B proteins are required (White, 2006). E1B 55K protein interacts with the tumor suppressor p53 protein and by doing so it inhibits p53's apoptotic potential. Unlike E1B 55K whose anti-apoptotic activity is only p53-related, E1B 19K blocks apoptosis not only by p53 but by other diverse stimuli including both death receptor signaling and mitochondrial mediated apoptosis, suggesting that it acts as a general apoptosis inhibitor. Expression of E1A alone results in induction of apoptosis. E1A-induced apoptosis can be suppressed by both E1B proteins. The E1A function is essential for cellular transformation, but toxic for a cell and, therefore, as part of the transformation process requires apoptosis suppressors such as E1B 19K and E1B 55K (White, 2006). Actually, it sounds paradoxical that apoptosis can be part of cell transformation. How can an apoptotic activity such as E1A contribute to cell transformation induced by adenoviruses? E1A induces a DNA damage response, which accounts for the proteasome-mediated degradation of anti-apoptotic Bcl-2-related proteins such as Mcl-2, but also for accumulation of p53 (Grinstein and Wernet, 2007). These studies reveal that one has to consider that even functional opposite processes as apoptosis and survival can act together. Recently, Cao et al. (2007) have reported that the oncogene Ras is an integral component of the E1A pathway, which illustrates that transforming and apoptosis proteins are closely related with each other. In this respect, it is interesting to mention that the cellular oncogene BRAF harbors senescence and apoptosis potential, whereas in a metastatic melanoma setting activated BRAF is one of the proliferative forces (Dhomen and Marais, 2007). BRAF induces apoptosis and senescence via pathways mediated by insulin-like growth factor binding protein 7 (IGFBP7), which is down-regulated in tumor cells (Wajapeyee et al., 2008; Ruan et al., 2007). Recombinant IGFBP7 was proven to induce selectively apoptosis in BRAF-positive melanoma cell lines and systemically administered IGFBP7 reduced tumor

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growth of BRAF-positive tumors in xenografted mice (Wajapeyee et al., 2008). In addition, reactivation of IGFBP7 by DNA methylation resulted in inhibition of cancer cell growth under in vitro conditions (Lin et al., 2008). These features suggest that IGFBP7 represents a novel approach for melanoma treatment for it induces selectively apoptosis in melanoma cells, but leaves normal cells unharmed (Chien and Lowe, 2008). 3.2. Oncolytic viruses Various naturally occurring or genetically engineered oncolytic viruses are known to kill preferentially tumor cells (Vh-Koskela et al., 2007; Kirn and Thorne, 2009). Cancer cells have been changed genetically and have gained chromosomal instability, which enables cancer cells to grow out of control. These growth advantages might result in failures in the cellular viral defense system. Upon infection of cancer cells with oncolytic viruses they cannot control virus replication and will undergo cell death (Cattaneo et al., 2008). These features show that cancer cells can still undergo apoptosis. The right death signal, however, has to be provided, which can interact with one or more of the derailed cancer processes for normal cells are not sensitive to death signals of oncolytic viruses. It is intriguing to notice that oncolytic viruses can nd the Achilles heel of a cancer cell resulting in killing them. The fact that oncolytic viruses harbor a tropism for tumor cells has made oncolytic virus-based anticancer therapy to become very popular (Russell and Peng, 2008; Haseley et al., 2009). Several oncolytic virus therapies alone or in combination with chemo or radiation therapy have entered clinical trials (Kumar et al., 2008; Prestwich et al., 2008). One of the well-studied oncolytic viruses is the autonomous parvovirus-H1 (Raykov et al., 2007). Various animal studies showed that treatment with parvovirus-H1 resulted in a signicant tumor regression without detectable side effects (Cornelis et al., 2004). Recently, Angelova et al. (2009) reported that parvovirus-H1 treatment improved gemcitabine-based therapy, which illustrates that co-treatment with an oncolytic virus results in an effective chemotherapy at a lower dose of chemo compounds reducing the side effects. Besides the promising clinical applications of oncolytic viruses, they also represent tools for identifying tumor-related survival processes, which can be switched into an apoptotic one. Autonomous parvovirus such as parvovirus-H1 interferes with intracellular signaling. Parvovirus-H1 strongly requires phosphorylation of its nonstructural protein NS1 by members of the protein kinase C family (Lachmann et al., 2008). Proteins of the ezrinradixinmoesin family have been reported to be involved in the progression of the parvovirus-H1 life cycle (Nuesch et al., 2009). In the following Section 4, more detailed studies on the parvovirus-H1-derived protein NS1 in relation to its tumor-selective apoptosis induction will be described. On one hand, viruses can transform human cells into tumor cells, whereas on the other hand viruses preferentially can use tumorrelated processes to spread selectively in tumor cells and kill them. The ability of tumor cells to block apoptosis induction, which normally occurs in response to deregulated oncogenic signaling, is an essential hallmark of cancer and is known to be induced by oncogenic DNA viruses. However, alternative cell death programs still seem to appear to be functional in cancer cells and can be triggered by naturally occurring or genetically engineered oncolytic viruses (Noteborn, in press; Bruno et al., 2009). Lessons to be learned from both oncogenic and oncolytic viruses is that tumor-selective apoptosis can be regarded as part of derailed cancer processes, which have paved the path of our understanding of the intriguing appearance of tumorselective apoptosis. In the following sections, a panel of the viral proteins apoptin, E4orf4 and NS1 as well as the cellular proteins HAMLET, TRAIL and

mda-7 harboring tumor-selective apoptosis will be described. Besides their benet for therapeutic applications, the processes underlying their tumor-selective apoptosis induction will be extensively discussed. Table 1 shows the various tumor-selective apoptosis proteins and their origins. 4. Viral proteins 4.1. NS1 In glioma cells, parvovirus-H1 NS1 protein is able to induce death in cells resistant to TRAIL, cisplatin, or both, even when Bcl-2 is overexpressed (Di Piazza et al., 2007). Tumor-selective apoptosis-inducing viral proteins such as the parvovirus-H1 non-structural protein NS1 require interference with events that are characteristic for permissive tumor cells to induce their tumor-selective apoptosis. Recently, Nuesch and Rommelaere (2006) have analyzed which proteins interact with NS1 and might be responsible for its tumor-selective apoptosis activity. They have e.g. determined that NS1 interacts with the catalytic subunit of casein kinase II (CKII). The cellular protein CKII specically interacts with the helicase domain within NS1. This NS1CKII interaction points to interference by NS1 with intracellular signaling processes. The interaction of NS1 with CKII results in CKII-dependent cytoskeletal changes followed by apoptosis (Nuesch and Rommelaere, 2007). NS1 acts as an adaptor molecule, linking the cellular protein kinase CKII to tropomyosin and thus modulating the substrate specicity of the kinase. This action results in an altered tropomyosin phosphorylation pattern. Inhibition of endogenous CKII protein protects permissive tumor cells from parvovirus-H1induced cell death (Nuesch and Rommelaere, 2007). Parvovirus-H1 infections induce characteristic changes within the cytoskeleton laments of tumor cells, which results nally in the degradation of actin bers and the appearance of so-called actin patches. Br et al. (2008) has proven that gelsolin, a multifunctional protein cleaving actin laments, is crucial in these actin-related processes for gelsolin co-localizes with the characteristic actin patches. Although the role of these gelsolin-containing actin patches remains to be solved, it is suggestive to assume that they fulll a signaling function. Actin structures are known to act as scaffolds for signaling processes. Their aberrant performance induced by NS1, therefore, affects cellular signaling pathways (Br et al., 2008). The authors suggest that their observations imply the involvement of a lipid-dependent signaling cascade. Besides interaction of NS1 with CKII, it has also other cellular interaction partners involved in the NS1-induced cell death (Nuesch and Rommelaere, 2006). In addition, parvovirus-H1 infections were found to result in activation of both phosphoinositoldependent kinase signaling, i.e. PDK1 and PK, as evidenced by changes in their subcellular distribution and overall (auto) phosphorylation (Lachmann et al., 2008). The thus far known apoptosis characteristics of the parvovirus-H1 protein NS1 are shown in Table 2. The here described studies on the interaction of NS1 with cellular processes have revealed indications explaining its tumor-selective apoptosis. Future studies have to be performed to unravel whether the determined NS1-interacting factors represent successful drug targets for the development of novel anticancer therapies.

Table 1 Origins of proteins selectively killing tumor cells. Proteins Apoptin E4orf4 HAMLET Mda-7 NS1 TRAIL Origin Chicken anemia virus Adenovirus Human breast milk Human cells Parvovirus-H1 Human cells References Backendorf et al. (2008) Landry et al. (2006) Hallgren et al. (2008) Eager et al. (2008) Nuesch and Rommelaere (2007) Ashkenazi (2008)

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Table 2 Main characteristics of tumor-selective virus proteins. Characteristics p53-independent apoptosis Blocked by Bcl-2 Acts via caspase pathways Cytochrome c release Interference with APC/C Interference with CKII Nuclear localization Phosphorylated to be active Degradation of cytoskeleton Interaction with PP2A Ceramide elevation Mitotic catastrophe Apoptin Yes No Yes Yes Yes Yes Yes Yes E4orf4 Yes No No No Yes Yes Yes Yes Yes Yes NS1 Yes No Yes Yes Yes Yes Yes Yes References (Backendorf et al., 2008; Lavoie et al., 2000; Di Piazza et al., 2007) (Backendorf et al., 2008; Landry et al., 2006; Di Piazza et al., 2007) (Danen-van Oorschot et al., 2000; Landry et al., 2006; Di Piazza et al., 2007) (Danen-van Oorschot et al., 2000; Li et al., 2009; Di Piazza et al., 2007) (Heilman et al., 2005, 2006) (Nuesch and Rommelaere, 2007) (Backendorf et al., 2008) (Rohn et al., 2002; Gingras et al., 2002; Lachmann et al., 2008) (Li et al., 2009; Br et al., 2008) (Shtrichman et al., 1999) (Liu et al., 2006b) (Li et al., 2009)

4.2. E4orf4 Loss of p53 functioning is related to both tumor formation and as important also to resistance of various anticancer therapies (Gonzalez-Angulo et al., 2007, Fuster et al., 2007). The adenovirus-derived protein E4orf4 kills selectively tumor cells independent of p53 (Lavoie et al., 2000), illustrating that E4orf4 might kill tumor cells when conventional therapies will fail (Fuster et al., 2007). Preclinical studies revealed that treatment of tumors via electroporation with DNA encoding E4orf4 resulted in tumor growth inhibition (Mitrus et al., 2005). Src-family kinases modulate E4orf4 phosphorylation (Gingras et al., 2002). Shtrichman et al. (1999) have shown that E4orf4 interacts with the protein phosphatase 2A (PP2A). PP2A is known to play a relevant role in tumor cell formation (Schaffhausen and Roberts, 2009; Virshup and Shenolikar, 2009). Recently, it was shown that interaction of E4orf4 with the PP2A subunit B55 results in the down-regulation of the oncogene c-myc (Ben-Israel et al., 2008). Heilman et al. (2005) reported that E4orf4 induces tumor-selectively G2/M arrest by interacting and inhibiting the anaphase-promoting complex/cyclosome (APC/C). Like parvovirus-H1-derived protein NS1, E4orf4 expression also results in deregulation of the cytoskeleton (Smadja-Lamere et al., 2006). E4orf4 seems to use alternative cell death pathways for it does not require the classical caspase pathways and circumvents Bcl-2 blockage of apoptosis (Landry et al., 2006). Furthermore, Li et al. (2009) have proven that E4orf4 induced cell death also does not require release of mitochondrial cytochrome c. In contrast, E4orf4 expression induced changes in cell morphology, multiple nuclei in many cells and increased cell volume. These characteristics are not really related to apoptosis, but are implying that E4orf4 induces cell death via growth arrest and mitotic catastrophe (Li et al., 2009). One can conclude that E4orf4 circumvent the various blocks within the apoptosis machinery of human tumor cells, whereas it is able to trigger alternative cell death processes that seems to be absent in human normal cells. The characteristics of E4orf4 tumor-selective apoptosis are described in Table 2. 4.3. Apoptin In 1991, our laboratory discovered the chicken anemia virus (CAV)-derived protein apoptin as a small protein of 121-amino-acids, consisting of proline-rich regions, a basic C-terminus and overall containing a high percentage of serine and threonine residues (Noteborn, 2005). Apoptin was shown to induce apoptosis in chicken transformed cells and to be the main apoptosis-inducing factor of CAV (Noteborn, 2005). Several years later, a breakthrough was achieved: apoptin was proven to induce apoptosis in cell lines derived from a great variety of human tumors (Rohn and Noteborn, 2004; Maddika et al., 2006). Today, various research groups have reported that over 70 analyzed tumor cell lines were proven to be sensitive to apoptin-

induced apoptosis. On the other hand, apoptin did not induce apoptosis in normal, non-transformed human diploid cells inclusive primary human hepatocytes and stem cells, which are known to be very chemotherapy-sensitive (Backendorf et al., 2008). Apoptin can become activated by expression of SV40-derived transforming protein domains in normal human broblasts. A non-established tumor environment seems sufcient for apoptin's activation and one can conclude that apoptin recognize an early transformation event (Zhang et al., 2004). This is in accordance with the observation that apoptin can induce apoptosis in thus far all analyzed tumor cells (Backendorf et al., 2008). Apoptin acts as a multimeric protein complex, interacts in tumor cells with chromatin structures, e.g. with the chromatininteracting protein DEDAF (Backendorf et al., 2008). In tumor cells, apoptin becomes selectively phosphorylated (Rohn et al., 2002; Maddika et al., 2009). Apoptin expression enhances the level of the tumor suppressor ceramide in tumor cells, indicating the involvement of sphingolipids in apoptin-induced cell death (Liu et al., 2006b). Evidence has been obtained that apoptin acts mainly via interaction with the anaphase promoting complex/cyclosome (APC/C) complex, inducing G2/M cell cycle arrest resulting in p73/PUMA-mediated apoptosis (Nuesch et al., 2008; Klanrit et al., 2008). RNA-interference studies revealed that inhibition of the APC/C1 protein results in apoptosis induction in p53-minus cells, which implies that APC/C represents a potential drug target for the development of a novel anticancer therapy (Heilman et al., 2006). In contrast to E4orf4, apoptin has been proven to act via classical apoptosis pathways such as cytochrome c release and activation of the central caspase pathways (Danen-van Oorschot et al., 2000; Maddika et al., 2005). Various reports have described that apoptin harbors two tumorselective apoptosis domains. One apoptotic domain is located within its N-terminal region and the other one in its C-terminal one (DanenVan Oorschot et al., 2003). The latter one is regulated by its phosphorylation status (Rohn et al., 2005). The N-terminal region of apoptin contains a characteristic hydrophobic domain, which is known to interact with various apoptin-interacting proteins and itself (Backendorf et al., 2008). This hydrophobic domain within apoptin might represent a so-called hot-spot domain enabling binding to a large range of different proteins (Shulman-Peleg et al., 2007). The various tumor-selective apoptosis characteristics of apoptin are shown in Table 2. Thus far only one protein has been characterized with apoptin-like characteristics. An open reading frame derived from a Torque Teno virus (TTV) isolate encoding the putative TTV apoptosis-inducing protein (TAIP) showed some sequence homology with apoptin (Kooistra et al., 2004). Several recent reports have speculated that TTV is more present in tumor tissue in comparison to normal ones (De Villiers and Zur Hausen, 2009). In several analyzed human hepatocarcinoma cells, TAIP induces apoptosis to a similar level as apoptin, whereas in other human tumor cells to a lesser extent, but still to signicant levels in comparison to the control non-apoptotic agents (Kooistra et al., 2004). The presence of TAIP is limited within the

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heterogeneous TTV population. However, it's N-terminal half containing a similar hydrophobic region like apoptin is conserved throughout a broad range of TTV genotypes (De Smit and Noteborn, 2009). Interestingly, late in CAV infection of transformed chicken cells also an N-terminal apoptin product of 58 amino acids is expressed (Kamada et al., 2006). Which of the tumor-specic processes might be disturbed by the occurrence of proteins containing apoptin-like hot-spot elements? Future experiments will tell. Besides its tumor-selective apoptosis-inducing characteristics, apoptin has several other relevant features making it suitable as a novel anticancer agent: apoptin acts independently of p53 and is in certain tumor cell lines even stimulated by Bcl-2 or insensitive to BcrAbl and Bcl-xl (Zhuang et al., 1995; Schoop et al., 2004; Liu et al., 2006b) (Table 2), which suggests that apoptin can induce apoptosis in those cases where (chemo)-therapeutics might fail. The fact that apoptin is encoded by a rather small gene makes it very suitable to be applied as a novel cancer gene therapy. Under in vitro conditions, recombinant non-replicative oncolytic parvovirus-H1 vectors expressing apoptin were generated and shown to be effective in killing selectively tumor cells in vitro. As important, apoptin expression broadened the cell killing range of the used non-replicative oncolytic parvovirus-H1 vector (Olijslagers et al., 2001), which suggests that apoptin is suitable for being integrated in replicative-competent oncolytic viruses (Nuesch et al., 2008). Various independent in vivo studies based on xenografted human lung tumors, liver tumors, prostate tumors and breast tumors have shown that intratumoral delivery of apoptin via adenoviral vectors results in a signicant decrease of tumor growth and in some cases even to complete regression of the treated tumors (Visser et al., 2007; Qu and Noteborn, in press). Peng et al. (2007) reported that systemic delivery of apoptin by means of an Asor-delivery vehicle in an in situ xenografted human liver tumor in nude mice revealed an effective reduction of tumor growth without detectable side effects, even not in adjacent normal liver cells. Guelen et al. (2004) have shown that bacterially produced TAT-apoptin protein can enter both normal and tumor cells in vitro, but only kills tumor cells. The related recombinant PTD4-apoptin protein, applied on the skin covering xenografted liver, gastric or cervix tumors, shows apoptin's anti-tumor efcacy as well as its safety aspects (Sun et al., 2009). PTD4-apoptin protein is taken up by the (epi)dermal and subcutaneous layers on top of the xenografted tumors as well as by the tumor cells themselves. Whereas, the tumor cells were shown to become apoptotic, the normal cells on top of the tumor were not affected at all. PTD4-apoptin protein could also be detected in other normal tissues such as heart and liver. PTD4apoptin treatment resulted in a signicant reduction of the tumor growth, but no side effects could be detected. The overall health characteristics of PTD4-treated mice were as good as those from the control-treated groups (Sun et al., 2009). Alternatively, one could use secretable TAT-apoptin fusion proteins produced by (normal) cells and able to kill tumor cells as reported by Flinterman et al. (2009). As important, apoptin combined with several (chemo)therapeutic agents showed a better therapeutic effect in comparison to the single treatments (Qu and Noteborn, in press). For instance, Olijslagers et al. (2007) showed that chemotherapeutical agents such as etoposide, paclitaxel and methotrexate have an additive effect on apoptin's tumor-selective apoptosis activity. Liu et al. (2006a) have observed that co-treatment of acid ceramidase inhibitor LCL204 and apoptin signicantly reduced tumor growth in DU145 xenografts. A combinatorial treatment based on apoptin and the knockdown of the caspase inhibitor survivin resulted in an enhanced killing effect of apoptin (Liu et al., 2008). Recently, Schoop (2009) showed that apoptin enhances radiation treatment in more radio-resistant squamous cell carcinoma cells. The unique apoptin characteristics and the various independent gene, protein and combinatorial therapy approaches based on it show that apoptin is a serious candidate for the development of a novel

anticancer therapy. The apoptin preclinical characteristics relevant for its therapeutic applications are summarized in Table 3.

5. Human milk-derived HAMLET In human milk -lactalbumin is one of the main proteins. A structural derivative of -lactalbumin harbors tumor-selective capabilities, named human -lactalbumin made lethal to tumor cells (HAMLET) (Hallgren et al., 2008). Conversion of isolated -lactalbumin to HAMLET is achieved due to binding with oleic acid resulting in release of its Ca2+ ion and a folding change (Fast et al., 2005). It is assumed that conditions for HAMLET formation resemble those in the stomach of nursing children, which can contribute in lowering the cancer incidence in breast-fed children (Svensson et al., 2000). Since the rst description of HAMLET's tumor-selective apoptosis activities, two clinical trials have been carried out successfully. HAMLET was proven to be active against skin papillomas and bladder cancers, whereas no side effects to adjacent healthy tissue could be observed (Mossberg et al., 2007; Aits et al., in press). Almost all treated skin papillomas disappeared after HAMLET treatment. Intravesical HAMLET delivery resulted in a signicant reduction in bladder tumor in 8 out of 9 treated patients. These rst clinical results show that local HAMLET might be of huge value in the future treatment of cancers. The therapeutic aspects of HAMLET are concisely shown in Table 4. HAMLET has the capability to enter efciently tumor cells and to accumulate in their nuclei, mitochondria, endoplasmic reticulum and proteasomes (Hallgren et al., 2008). HAMLET causes release of cytochrome c from the mitochondria resulting in activation of the caspase pathways (Khler et al., 2001). In the tumor nuclei, HAMLET associates with histones resulting in an irreversible disruption of the chromatin organization (Dringer et al., 2003, Permyakov et al., 2005). Like apoptin, HAMLET interferes with chromatin structures suggesting that especially these cellular structures are crucial for its tumor-selective apoptosis induction (Backendorf et al., 2008). HAMLET activates 20S proteasomes, which also contributes to cell death. HAMLET induces tumor-selective apoptosis in a p53-independent manner. Over-expression of the anti-apoptotic Bcl-2 and caspase inhibition does not rescue HAMLET positive cells. Recently, Aits et al. (2009) have provided evidence that HAMLET induces macroautophagy (Scarlatti et al., 2009). Macroautophagy is a lysosomal catabolic pathway recycling cellular molecules. In cancer cells, the macroautophagy pathways have been changed in comparison to their normal counterparts (Hyer-Hansen and Jttel, 2008), which might explain (in part) the observed tumor-selective activity of HAMLET. Future studies have to be carried out to detect in detail the precise factors enabling HAMLET's tumor-selective apoptosis for all these tumor-

Table 3 Preclinical studies on the therapeutic potential of apoptin. Therapeutic vector Non-replicative adenovirus Fowl-pox virus Plasmid DNA Asor-DNA TAT-apoptin protein PTD4-apoptin protein Combinatorial therapies Tumor Hepatocarcinoma Breast carcinoma Prostate cancer Hepatoma Lewis lung carcinoma Hepatocarcinoma Lung tumor Cervix carcinoma Gastric cancer Hepatocarcinoma Osteosarcoma Prostate cancer Squamous cell carcinoma Preclinical model Cells/mouse Cells/mouse Cells/mouse Cells/mouse Cells/mouse Cells/mouse Cells Mouse Cells/mouse Cells/mouse Cells Cells/mouse Cells References (Visser et al. 2007; Backendorf et al., 2008)

Peng et al. (2007) Guelen et al. (2004) Sun et al. (2009)

Olijslagers et al. (2007) Liu et al. (2006a) Schoop (2009)

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Table 4 Main therapeutic and mechanistic cell death aspects of HAMLET. Chemical characteristics (Hallgren et al., 2008) Complex of oleic acid and human -lactalbumin Globular opened protein structure Clinical study on: Bladder cancer Skin papilloma Clinical outcome (Aits et al., in press) 8/9 patients showed tumor regression No adverse reactions were reported 29/35 patients with complete cure No side effects were detected

Tumor-selective cell death characteristics (Hallgren et al., 2008; Aits et al., 2009) p53-independent Cytochrome c release Activation of caspase pathways Circumvents Bcl-2 over-expression & caspase inhibition Interference with chromatin structure Located in nucleus, mitochondria, ER, & proteasomes Induction of macroautophagy

control groups (Gopalan et al., 2007). Adenovirus delivered mda-7 in combination with bevacizumab, which is a humanized monoclonal antibody against vascular endothelial growth factor, results in a synergistic and complete inhibitory effect on lung tumor xenografts (Inoue et al., 2007). Recently, clinical phase I trials have been carried out and show that mda-7 is well tolerated and demonstrates evidence of signicant clinical activity (Lebedeva et al., 2007; Eager, et al., 2008; Emdad et al., 2009). However, as with most treatment modalities, subsets of tumor cells are resistant to mda-7. However, e.g. combinatorial treatment of mda-7 with Bcl-2 siRNA, radiation and chemotherapy could overcome this resistance (Sarkar et al., 2008). 6.2. TRAIL The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce p53-dependent and p53-independent apoptosis preferentially in human tumor cells (Ashkenazi, 2008). TRAIL interacts with the death receptors DR4 and DR5 via activation of the extrinsic apoptosis pathway leading to the activation of caspase 8 and subsequently e.g. the executioner caspase-3. TRAIL induced apoptosis is complemented by activation of the intrinsic pathway (Mahalingam et al., 2008). Despite its tumor-selective apoptosis activity, TRAIL cannot induce apoptosis in a rather broad range of tumor cells. TRAIL resistance might be explained by the fact that TRAIL signalling also activates NF-B, which induces the anti-apoptotic regulators Mcl-1 and cIAP2 (Hall and Cleveland, 2007). Furthermore, tumor cells over-expressing the anti-apoptosis protein FLIP or Bcl-2 are resistant to TRAIL treatment (Johnstone et al., 2008). Fortunately, combinatorial therapy of TRAIL with chemotherapeutic agents such as R-roscovitine (Festa et al., 2009), which lowers the anti-apoptotic activity in cancer cells, though, is very encouraging. Clinical phase I trials have shown that TRAIL treatment is well tolerated and clinical phase II studies as monotherapy or combined with established anticancer therapeutics are underway (Bellail et al., 2009; Newson-Davis et al., 2009). Recently, Cool et al. (in press) have improved the characteristics of a recombinant form of TRAIL by in silico design, which generates TRAIL variants with a higher selectivity for TRAIL receptors DR4 or DR5 resulting in improved apoptosis activities. These promising studies and clinical trials clearly show the potential of TRAIL, but also the complexity of inducing apoptosis in a transformation background. Table 5 summarizes the relevant mechanistic and therapeutic data of TRAIL-induced apoptosis in tumor cells. 7. Summary Unbalance in the apoptotic machinery is one of the underlying mechanisms of tumor formation. Lessons to be learned from tumorinducing viruses showed that cancer cells are formed by blocking apoptosis, but also due to expression of pro-apoptotic proteins. This paradox is even strengthened by the fact that oncolytic viruses induce selectively apoptosis in transformed cells. One can assume, therefore, that despite certain apoptotic blocks, tumor cells have obtained alternative apoptosis pathways. Here, we have described a panel of tumor-selective apoptosis proteins consisting of the viral proteins apoptin, E4orf4 and NS1 as well as the cellular proteins HAMLET, mda7 and TRAIL. The tumor-selective apoptosis-inducing proteins sense specic cellular pathways underlying tumor formation, which can become redirected by them into cell death. Unraveling the synergy of transformation and the factors inducing tumor-selective apoptosis will generate a better understanding of the processes underlying cancer resulting in novel anticancer therapy strategies. The growing amount of preclinical data has shown that the different tumorselective proteins have obtained similarities and differences in their characteristic tumor-selective induction of cell death. Several of the unraveled tumor-related processes underlying tumor-selective induction of apoptosis represent potential anticancer drug targets. First

related factors might represent potential drug targets. The apoptosis characteristics of HAMLET are shown in Table 4. 6. Cytokines 6.1. Mda-7 A member of the interleukin-10 family, the melanoma differentiation-associated gene-7 (mda-7; also known as interleukin 24) has also been shown to induce caspase-dependent apoptosis in a broad spectrum of cancer cells (Fisher et al., 2007). Table 5 shows the fundamental and therapeutic aspects of mda-7-induced apoptosis. In breast cancer cells adenoviral-induced mda-7 expression triggers anti-proliferative effects by down-regulation of survival signals such as Bcl-2 and Akt, leading to apoptosis (Bocangel et al., 2006). However, it is also known that Bcl-2 over-expression can inhibit mda-7 induced apoptosis (Su et al., 2006). Secreted mda-7 can affect distant tumor cells, which indicate a by-stander effect of mda-7 treatment. Mda-7 activates the FasL/TRAIL pathways resulting in tumor-selective apoptosis (Ekmekcioglu et al., 2008; see also below). All these features of mda-7 make it an interesting novel anticancer agent (Eager et al., 2008). In vivo, treatment of subcutaneous ovarian cancer xenografts with an adenovirus expressing mda-7 resulted in signicant tumour growth inhibition when compared with that in

Table 5 Main therapeutic and mechanistic aspects of mda-7 and TRAIL. Mechanistic aspects p53-independent Down-regulation of Bcl-2 Degradation of Akt Inhibited by Bcl-2 Inhibited by FLIP Activation of TRAIL/FasL Acts via caspase-8 Mda-7 Yes Yes Yes Yes Yes Yes TRAIL Yes/no Yes Yes Yes References (Fisher et al., 2007, Ashkenazi, 2008) Bocangel et al. (2006) Bocangel et al. (2006) (Su et al., 2006, Johnstone et al., 2008) Johnstone et al. (2008) Ekmekcioglu et al. (2008) (Fisher et al., 2007, Mahalingam et al., 2008) References (Sarkar et al., 2008, Johnstone et al., 2008) (Inoue et al., 2007, Festa et al., 2009) (Emdad et al., 2009, Newson-Davis et al., 2009)

Therapeutic aspects Resistance of subset of tumor cells Part of combinatorial therapy Clinical trials

Mda-7 Yes Yes Yes

TRAIL Yes Yes Yes

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clinical trials have provided the hope that, although the occurrence of bottlenecks, this relatively young research eld will become of importance for the development of novel improved anticancer strategies. Acknowledgements This work was supported by a grant from the Dutch Royal Society of Arts and Sciences (08CDP045). References
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