Drug Resistance Updates
Volume 9, Issue 1 , Pages 40-50, February 2006

Tumor-specific nuclear targeting: Promises for anti-cancer therapy?

  • Gualtiero Alvisi

      Affiliations

    • Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Australia
    • ,
  • Ivan K.H. Poon

      Affiliations

    • Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Australia
    • ,
  • David A. Jans

      Affiliations

    • Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Australia
    • ARC Centre of Excellence for Biotechnology and Development, Australia
    • Corresponding Author InformationCorresponding author at: Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton Vic. 3168, Australia.

Received 13 January 2006; received in revised form 20 February 2006; accepted 20 February 2006. published online 17 April 2006.

Article Outline

Abstract 

Recent developments in anti-cancer gene therapy suggest that the idea of a magic bullet for cancer may not be a pipe dream. Viral-based anti-cancer vectors for gene therapy have been used preferentially in this regard, but recent results from clinical trials have raised serious concerns as to their safety. For this reason, the development of non-viral vectors able to deliver drugs or suicide genes specifically to cancer cells is of paramount importance. In this context, great interest has been raised by recent reports that several proteins, including viral protein 3 (VP3 or Apoptin) from Chicken Anemia Virus, are capable of selectively killing tumor cells. Intriguingly, VP3's anti-cancer activity is strongly linked to its ability to localize more efficiently in the nucleus of cancer and transformed cells than that of normal, non-transformed cells with a tumor cell-specific nuclear targeting signal (tNTS) located at the C-terminus of the protein. Clearly, the VP3 tNTS is an exciting prospect to enhance non-viral-mediated cancer cell killing. This review will discuss recent advances in the understanding of the mechanism responsible for VP3 tumor-specific nuclear localization, including its specific phosphorylation, and the implications for the enhancement of anti-cancer therapy. It also proposes alternative strategies to develop tNTSs for anti-cancer therapies.

Keywords: Nuclear targeting, Apoptin, Cancer, Nuclear targeting signal, Apoptosis, Vehicle, Modular transporters, Therapy, Gene therapy, Drug delivery, VP3, Anti-cancer, Photosensitizers

 

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1. Introduction 

Cancer can be regarded as a heterogeneous group of proliferative diseases, resulting from the accumulation of genetic lesions. Despite considerable advances in our understanding of the molecular mechanisms of carcinogenesis and cancer progression, cancer remains the second major cause of death due to medical causes in the United States of America as well as in much of the developed world (Weir et al., 2003). Clearly, there is an urgent need for new, efficacious and specific anti-cancer approaches (Guillemard and Saragovi, 2004). Virus-based anti-cancer gene therapies, based on the ability of adenoviral or retroviral vectors to deliver suicide genes to cancer cells (Rainov and Ren, 2003), or to kill tumor cells directly (“virolytic therapy”; Everts and van der Poel, 2005, Mathis et al., 2005), are being tested in various clinical trials (www4.od.nih.gov/oba/rac/protocol.pdf). However, although one viral-based vector has been recently approved for the treatment of head and neck cancer, the outcome of several viral-based clinical trials has raised serious concerns regarding the efficacy and safety of such approaches, highlighting the need to develop alternative strategies (Hacein-Bey-Abina et al., 2003a, Hacein-Bey-Abina et al., 2003b, Raper et al., 2003, Williams and Baum, 2003, Glover et al., 2005, Young et al., 2006). Here we discuss the possibility of using recently identified tumor cell-specific nuclear targeting sequences (tNTSs) as tools for enhancing non-viral drug or suicide gene delivery to the nucleus of cancer cells.

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2. Nuclear delivery mechanisms 

A useful strategy in anti-cancer therapy is the specific delivery of high levels of either a suicide gene or a drug (see Box 1) to malignant cells, without affecting healthy cells and tissues. Viral-based vehicles represent the best option in terms of the efficiency of delivery, as viruses have evolved very specific mechanisms to deliver genetic material and proteins to host cells (Fig. 1).

Box 1.

Herpes simplex virus thymidine kinase as a suicide gene

A suicide gene programs cells to kill themselves under certain circumstances, such as upon administration of an inactive prodrug which can be processed to a toxic form. One of the most widely used suicide genes is the Herpes simplex virus 1 (HSV-1) thymidine kinase (TK). HSV-1 TK differs considerably from the human cellular TK in its substrate specificity, phosphorylating a broad spectrum of pyrimidine and purine nucleoside analogues (Pilger et al., 1999), including ganciclovir (GCV). HSV-1 but not human TK phosphorylates GCV to its monophosphate form, which is subsequently converted to the triphosphorylated derivative by cellular kinases. The active triphosphorylated metabolite is then incorporated into the newly synthetized DNA chain, thereby blocking DNA replication. Thus, HSV-1 TK is essential for GCV-induced cellular toxicity, and GCV is one of the most widely used antiviral drugs for treatment of viral infections (De Clercq, 2004). Because of its unique properties, the HSV-1 TK gene has been widely used as a suicide gene in gene therapy vectors (Vassaux and Martin-Duque, 2004).

Photodynamic therapy

Photodynamic therapy (PDT) is based on the ability of photosentitizers (PSs) such as chlorin e6, to generate singlet oxygen species in response to light exposure. PSs preferentially accumulate in tumor cells but also in some normal tissues such the skin (Jori, 1996), and their action is strongly dependent on their localization inside the target cell, as their cytotoxic action does not exceed 40nm from the site of activation (Sobolev et al., 2000). To increase their specificity, PSs can be directly administrated into the tumoral mass, in combination with direct light exposure (Sharman et al., 1999). PDT has been proven to be effective for the treatment of some cancers (Wiedmann et al., 2003), with the nucleus being the most sensitive subcellular compartment (Peng et al., 1996). For this reason, development of methods to increase PS targeting to the nucleus of tumor cells is desirable (Bisland et al., 1999, Sobolev et al., 2000).

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  • Fig. 1. 

    Intracellular delivery as mediated by viruses and modular transporters. Viral particles such as that of adenovirus and modular transporters are able to interact specifically with a cell surface receptor (1), leading to receptor-mediated endocytosis (2). Certain viral and other proteins can undergo conformational modifications triggered by the acidic endosomal pH, causing disruption of the endosome (3) and the release of the viral particle or of the modular transporter into the cytoplasm so that they can subsequently be imported into the cell nucleus (4), where the genetic material is finally able to be expressed, or the drug released (5). PM, plasma membrane; NE, nuclear envelope; CT, cellular targeting, EE, endosomal escape; NT, nuclear targeting.

Viruses can interact with the host cell via specific interaction of viral proteins with cellular receptors exposed on the extracellular surface of the cellular plasma membrane (PM; see Fig. 1.1). In the case of non-enveloped DNA viruses such as members of the Adenoviridae family, the virus can be internalized by the cells through an endocytotic mechanism subsequent to receptor binding (see Fig. 1.2). Receptor-mediated endocytosis usually leads to degradation of the internalized ligand in the lysosomal pathway, but certain viral proteins undergo conformational changes under the acid conditions of the endosome to disrupt the vesicular membrane and facilitate the release of the viral particles into the cytoplasm of the host cell (see Fig. 1.3; Seth, 1994).

In the case of most DNA viruses and retroviruses, the viral genome is ultimately translocated into the nucleus of the host cell (see Fig. 1.4), in order to be expressed and replicated, or integrated into the host genome in the case of retroviruses. The eukaryotic cell nucleus is separated from the rest of the cell by a double membrane structure – the nuclear envelope (NE) – the only passage through which is represented by the c. 120MDa, multiprotein-constituted nuclear pore complexes (NPCs). Molecules with a mass of <50kDa can freely diffuse through the aqueous channel delimited by the NPC, whereas nucleocytoplasmic transport of larger molecules is an active process mediated by members of the importin (IMP) superfamily of soluble cellular transporters, which can be of α and β types (Weis, 1998). Members of the IMP family recognize specific targeting signals on cargoes, named nuclear localization signals (NLSs) or nuclear export signals (NESs) for transport in the import and export directions, respectively (Fig. 2). NLSs and NESs were first identified on viral proteins: the first NLS characterized, the highly basic 7 amino acid sequence (PKKKRKV132), was from the large tumor antigen (T-Ag) from Simian Virus SV40 (Kalderon et al., 1984), whereas a strongly hydrophobic 11 amino acid stretch (LQLPPLERLTL83), from the human immunodeficency virus 1 (HIV-1) Rev protein was the first NES described (Meyer et al., 1996). The best-characterized nuclear import pathway is that mediated by the IMPα/β heterodimer, where IMPα recognizes the NLS (such as that of T-Ag) on the cargo protein, and IMPβ mediates the docking of the complex to the NPC (see Fig. 2, left; Weis, 1998). IMPβ itself, or one of its many homologues, is also able to interact directly with specific cargoes independently of IMPα. Subsequently the IMP-cargo complex is translocated to the nuclear side of the NPC, where binding of the guanidine nucleotide binding protein Ran in activated GTP-bound form to IMPβ results in the release of the cargo into the nucleus. Certain IMPβ homologues mediate nuclear protein export, the best known being CRM-1 (Fukuda et al., 1997), whose function can be inhibited specifically by the drug leptomycin B (LMB; see Fig. 2, right; Kudo et al., 1998).

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  • Fig. 2. 

    Schematic representation of nucleocytoplasmic transport. (A) Nuclear import occurs when IMPβ1, either alone or through IMPα, recognizes an NLS on the cargo protein in the cytoplasm (1) and mediates the docking of the complex to the NPC (2) followed by translocation into the cell nucleus. Binding of RanGTP to IMPβ1 (3) results in the cargo being released into the nucleus (4). (B) Nuclear export occurs when cellular export receptors, such as the IMPβ homologue CRM-1, recognize a NES on the cargo protein in the presence of RanGTP (1) and mediate the docking of the complex to the NPC and its translocation to the cytoplasm (2). Upon hydrolysis by Ran of GTP to GDP (3), facilitated by Ran GTPase-activating protein (not shown), the cargo is released into the cytoplasm (4).

The three functions described above – cell targeting (CT), endosomal escape (EE), and nuclear targeting (NT; see Fig. 1) – are desirable elements of an efficient cancer-specific targeting agent (Glover et al., 2005). Modular recombinant transporters, assemblies of independently acting domains able to fulfill some or all of these functions (see Table 1 and Fig. 1), have been shown to be capable of targeting reporter genes or drugs specifically to cancer cells in culture (see below; Rosenkranz et al., 2003). That modular transporters of this type may be suitable for targeted delivery in the clinic in the future is indicated by the observation that liver-targeted luciferase expression has been achieved in mice after intravenous injection of a modular transporter complexed with plasmid DNA (Nishikawa et al., 2000).

Table 1.
CTEENTPS agentReference
A. Modular proteins used to deliver photosensitizing (PS) agents to mammalian cellsa
Insulin––Chlorin e6Akhlynina et al. (1995)
Insulin–opT-NLS(1); T-ag NLSChlorin e6Akhlynina et al. (1997)
InsulinAdenoviral particlesopT-NLSChlorin e6Akhlynina et al. (1999)
α-MSHDT TDopT-NLSChlorin e6Rosenkranz et al. (2003)
Bacterio chlorin P6
EGF––Aluminium/CobaltLutsenko et al. (1999)
Disulfonated phthalocyanines
EGF––Tin (IV) chlorin e6Gijsens et al. (2000)
Polylysine–A124SV40LTaChlorin e6Bisland et al. (1999)
Maleic acid––Chlorin e6Liu and Hamblin (2005)
CTEENTDNA bindingReference
B. Modular proteins used to deliver DNA to mammalian cellsb
––NLSV404 peptide(2)NLSV404 peptideRitter et al. (2003)
Integrin binding domain of Yersinia pseudotuberculosis invasin protein––Non specific DNA binding domain from Histone H1 (SPKR)4Fortunati et al. (2000)
H fragment of the tetanus toxinDT TDGAL4 BDcGAL4 BDBox et al. (2003)
scFv(FRP5)DT TDGAL4 BDGAL4 BDUherek et al. (1998)
scFv(FRP5)PE TDGAL4 BDGAL4 BDFominaya and Wels (1996)
TGF-αPE TDGAL4 BDGAL4 BDFominaya et al. (1998)
Invasin–GAL4 BDGAL4 BDPaul et al. (1997)
Anti-HIV gp 120 Fab––ProtamineChen et al. (1995)
Ad5 penton proteinAd5 penton protein–Polylysine and protamineMedina-Kauwe et al. (2001a)
EGF-like domain of the heregulin-α(1) isoformAd5 penton protein–PolylysineMedina-Kauwe et al. (2001b)
Letal factor domain from anthrax toxinLetal factor domain from anthrax toxinGAL4 BDGAL4 BDGaur et al. (2002)
––T-ag NLSHuman histone H1Gaur et al. (2002)
FMDV RGD sequence–β-GaldPolylysineAris et al. (2000)
FMDV RGD sequence–T-ag NLSPolylysineAris and Villaverde (2003)
––OpT-NLSPolylysineChan et al. (2000)
Transferrin–OpT-NLSPolylysineChan and Jans, 1999a, Chan and Jans, 1999b)
α-MSH–OpT-NLSGAL4BDChan and Jans (2001)
GalactoseFusogenic peptide (mHA2)–Poly-L-OrnithineNishikawa et al. (2000).

aopT-NLS, Simian Virus 40 large tumor antigen (T-Ag) derived sequence CGPGSDDEAAADAQHAAPPKKKRKVGY; T-Ag NLS, T-Ag derived sequence PKKKRKVEDPYC; α-MSH, Melanocyte stimulating hormone; DT TD, Diphteria toxin translocation domain; EGF, epidermal growth factor; A124SV40LTa, T-Ag derived sequence APPKKKRKVEDP.

bNLSV404 peptide, Simian virus SV40 large tumor antigen (T-ag) derived sequence PKKKRKVGPKKKRKVGPKKKRKVGPKKKRKVGC; GAL4 BD, Saccharomyces cerevisiae GAL4 DNA binding domain; scFv(FRP5), antibody fragment specific for the tumor-associated ErbB2 antigen; DT TD, Diptheria toxin translocation domain; PE TD, Pseudomonas exotoxin translocation domain; TGF-α, Transforming growth factor alpha; FMDV, Foot-and-mouth disease virus; α-MSH, Melanocyte stimulating hormone; T-Ag NLS, T-Ag derived sequence PKKKRKVEDPYC; OpT-NLS, T-Ag derived sequence CGPGSDDEAAADAQHAAPPKKKRKVGY.

cAn NLS has been described within the GAL4 BD (Silver et al., 1984, Chan et al., 1998; Chan and Jans, 1999a, Chan and Jans, 1999b).

dA cryptic NLS has been described within the β-Gal (McInnis et al., 1995).

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3. Importance of nuclear delivery of modular transporters 

Several studies have shown that the efficiency of foreign gene expression depends on the ability of the modular transporter to deliver the coding sequences to the nucleus of the target cells and that addition of NT modules can improve gene expression (Chan et al., 2000, Chan and Jans, 1999a, Chan and Jans, 1999b, Chan and Jans, 2001, Chan and Jans, 2002). For example, Villaverde and coworkers described a modular protein (249AL) comprising the β-galactosidase (β-Gal) protein as a carrier, a N-terminal polylysine stretch as a DNA compacting and protecting module, and the foot-and-mouth disease virus (FMDV) RGD sequence as a CT module able to interact with cell surface integrins. 249AL is able to associate with a firefly luciferase expressing plasmid without undergoing any appreciable diminution of β-Gal enzymatic activity and can facilitate expression of the luciferase reporter in integrin-expressing CaCO2 cells with an efficiency comparable to that of a non-targeted liposome-based commercial transfection system (Aris et al., 2000). Subsequent inclusion of an NT module by adding the T-Ag NLS, generated the NLSCt protein which transfected CaCO2 cells with 30-fold higher efficiency than that of 249AL. The implication is that the efficiency of transgene expression can be facilitated by increasing the nuclear delivery of the transgene (Aris and Villaverde, 2003).

Intranuclear delivery is also important for cellular transporters designed to deliver photosentitizing (PS) agents to cancer cells (see Box 1). Using bovine serum albumin (BSA) as a carrier linked to insulin as a CT module, Sobolev and coworkers (Akhlynina et al., 1993, Akhlynina et al., 1995) generated a modular targeting protein able to specifically deliver conjugated clorin e6 to the human hepatoma cell line PLC/PRF/5, resulting in reduction of the EC50 by about 100-fold when compared to that of free clorin e6 after light exposure. Interestingly, addition of a functional “optimized” T-Ag NLS (opT-NLS) as an NT module significantly decreased the EC50 about 7-fold, compared to a functionally impaired NLS mutant derivative (Akhlynina et al., 1997). Finally, addition of an EE moiety in the form of adenovirus particles mixed with the modular transporter further enhanced the efficiency (Akhlynina et al., 1999).

These and other studies show that the addition of an NT module to a modular transporter significantly enhances the efficacy of both gene transfer (Chan et al., 1998, Chan et al., 2000, Chan and Jans, 1999a, Chan and Jans, 1999b, Chan and Jans, 2001, Singh et al., 1999, Aris and Villaverde, 2003, Ritter et al., 2003) and sensitivity of target cells to nuclear acting toxic agents such as PSs (Akhlynina et al., 1997, Akhlynina et al., 1999, Bisland et al., 1999, Brokx et al., 2002; see also Table 1).

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4. Tumor cell-specific nuclear delivery 

Several proteins, including TRAIL, Adenovirus E4orf4 and the Chicken Anemia Virus protein 3 (VP3, also known as Apoptin) have been shown to kill tumor cells selectively (Danen-van Oorschot et al., 1997, Lavoie et al., 1998, Walczak et al., 1999), raising great hopes for their utilization in anti-cancer therapy (Branton and Roopchand, 2001, MacFarlane, 2003, Rohn and Noteborn, 2004, Tavassoli et al., 2005). While nuclear localization is important, but not essential, for E4orf4 mediated induction of apoptosis (Miron et al., 2004), VP3's ability to kill tumor cells selectively is correlated with its nuclear localization specifically in tumor and not “normal” cells (Danen-van Oorschot et al., 2003). VP3 is a 121-amino acid protein rich in Ser and Thr residues, which induces programmed cell death (PCD) in chicken thymocytes (Noteborn et al., 1994), and in several human tumor but not normal cells, through a p53-independent, Bcl-2-resistant process (Zhuang et al., 1995). However, its ability to induce PCD in primary human fibroblasts has been reported recently (Guelen et al., 2004), suggesting that further studies are required to verify VP3's selectivity in terms of tumor cell killing.

Although not completely understood, the molecular basis of VP3's tumor-specific nuclear localization has been intensively investigated (Oro and Jans, 2004, Wang et al., 2004, Poon et al., 2005a, Poon et al., 2005b, Rohn et al., 2005, Wagstaff and Jans, 2006). Regardless of its proapoptotic activity, understanding of the mechanisms and sequences responsible for VP3's tumor-cells specific nuclear localization would allow use of the latter as an NT module in modular transporters designed to specifically localize specifically in the nucleus of tumor but not normal cells. The determinants of VP3 intracellular localization thus far characterized (see Fig. 3, Fig. 4) are:

1.A bipartite basic NLS, located at the C-terminus of the protein (aa 80-121), necessary for VP3 nuclear accumulation as revealed by analysis of deletion and point mutant derivatives (Danen-van Oorschot et al., 1997, Danen-van Oorschot et al., 2003, Wang et al., 2004, Poon et al., 2005a, Poon et al., 2005b). This motif is able to confer nuclear localization on heterologous proteins such as green fluorescent protein (GFP; Danen-van Oorschot et al., 2003, Wadia et al., 2004, Poon et al., 2005a, Poon et al., 2005b) and maltose-binding protein (MBP; Rohn et al., 2005). Intriguingly, VP3(74–121) binds to IMPβ1 with three times higher affinity compared to VP3(1–121), suggesting that the NLS of VP3 activity may be regulated by intramolecular masking through the VP3 N-terminus (Wagstaff and Jans, 2006).

2.A CRM-1 recognized NES (residues 97–105) inactive in tumor cells, which contributes to determine tumor cell-specificity of VP3 localization (Poon et al., 2005a). The NES is located between the arms of the bipartite NLS, and thus aa 74–121, encompassing both the VP3 NLS and NES, represent a tNTS.

3.VP3 T108, adjacent to the NES, is specifically phosphorylated by an unidentified kinase in tumor but not normal cells (Rohn et al., 2002). Phosphorylation at this site appears to be responsible for tumor cell-specificity of VP3 nuclear localization, by inhibiting NES-mediated nuclear export in tumor cells (Poon et al., 2005a), and possibly also by enhancing its nuclear import (Rohn et al., 2002, Rohn et al., 2005). The phosphomimetic E108 VP3 mutant derivative shows enhanced nuclear localization in normal cells, reaching levels of nuclear accumulation comparable to that achieved in tumor cells (Poon et al., 2005a), but is not responsive to LMB treatment, consistent with the idea that phosphorylation at T108 primarily inhibits NES activity.

4.A leucine-rich sequence (LRS), at residues 33–46, which appears to mediate VP3'S interaction with the promyelocytic leukemia (PML) protein (Poon et al., 2005a), the organizer of the PML nuclear bodies (PML NBs). PML NBs are structured protein complexes associated with the nuclear matrix, associated with several functions such as apoptosis, DNA replication and repair, transcription regulation and RNA transport (Takahashi et al., 2004). The LRS lies within the VP3 multimerization domain (residues 26–69; Leliveld et al., 2003a), and although proposed to be a putative NES (Danen-van Oorschot et al., 1997), this has been disproved (Wang et al., 2004, Poon et al., 2005a). Intriguingly, mutation of the LRS sequence not only ablates VP3 co-localization with PML, but also reduces VP3 nuclear accumulation in tumor and normal cells, and LMB responsiveness in normal cells. Thus, association with PML and thereby PML NBs may be a prerequisite for VP3 nuclear export (Poon et al., 2005a). VP3 has also been reported to bind to other nuclear components such as the kinase HIPK2 (Poon et al., 2005a), the human death effector domain-associated factor (Danen-van Oorschot et al., 2004), DNA (Leliveld et al., 2003b) and APC-1, a subunit of the anaphase-promoting complex/cyclosome involved in the assembly and regulation of the cyclosome complex (Teodoro et al., 2004). VP3 has also been reported to interact with cytoplasmic protein Hippi, the protein interactor of the Huntingtin interacting protein 1 (Hip-1; Cheng et al., 2003). Thus, it seems possible that VP3 subcellular localization stems from several, competing processes within the cell such as nuclear and cytoplasmic retention as well as facilitated nuclear import and export.

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  • Fig. 3. 

    Summary of the targeting sequences of VP3 and domains involved in protein–protein interaction. The single letter code is used for amino acid; NLS, nuclear localization signal; NES, nuclear export signal; PML, promyelocytic leukemia protein; Hippi, protein interactor of the Huntingtin interacting protein 1; APC1, subunit of the anaphase-promoting complex/cyclosome.

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  • Fig. 4. 

    Model for VP3 tumor-specific nuclear localization. VP3 is recognized by IMPβ1 and transported into the nucleus in both normal and tumor cells (1). Once inside the nucleus, VP3 is released from IMPβ1 upon binding of RanGTP and localizes to the PML NBs (2). In tumor but not in normal cells, VP3 phosphorylation on T108 prevents recognition by CRM1 (3) and export to the cytoplasm (4). Thus, VP3 localizes more strongly in the nucleus in tumor rather than in normal cells, as illustrated in the confocal microscopic images of 1BR3 (normal; left) and 1BR3/N (transformed; right) cells expressing GFP-VP3 (lower panels); note that VP3 is, in fact, able to accumulate in the nuclei of non-tumor cells, but to a significantly reduced extent compared to in transformed cells (see Poon et al., 2005a, Poon et al., 2005b).

As mentioned above, modular transporters ideally combine CT, EE and NT functions in order achieve efficient delivery of drugs or DNA encoding toxic genes to the nuclei of target cancer cells. Importantly, VP3(74–121) not only exhibits tumor cell-specific nuclear localization (Poon et al., 2005a, Poon et al., 2005b), but has been reported to induce apoptosis in cancer cells (Danen-van Oorschot et al., 2003), as well as bind DNA (Leliveld et al., 2004), raising the possibility that this portion of VP3 could possess DNA binding, tumor cell-specific nuclear targeting and proapoptotic abilities. Further analysis is required to demonstrate if this is the case.

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5. Tumor cell-specific phosphorylation 

Studies on VP3 suggest the existence of tumor-specific cell-phosphorylation activity (Rohn et al., 2002, Zhang et al., 2004). A number of kinases show elevated activity in transformed cells. Protein kinase CK2 (CK2), is activated to up to 7-fold higher levels in tumor than in normal cells (Guerra and Issinger, 1999, Tawfic et al., 2001); while cytoplasmic extracts of cells obtained from SCCHN tumor surgical specimens show a 6-fold higher CK2 activity compared to those of cells obtained from normal oropharyngeal mucosa (Faust et al., 1999). These findings are interesting in light of the fact that CK2 facilitates the nuclear accumulation of a number of proteins, mainly through regulation of the NLS activity of a conserved modular domain known as the CcN motif (Rihs et al., 1991, Ueda et al., 1995, Longshaw et al., 2000, Briggs et al., 2001, Nuthall et al., 2002, Wilson et al., 2002, Alvisi et al., 2005). The CcN (CK2, cdc, and NLS) motif is a modular domain, originally described for T-Ag, that comprises phosphorylation sites for both CK2 and cyclin-dependent kinases (cdks) upstream of the NLS (Jans et al., 1991, Rihs et al., 1991). A phosphorylation site for double-stranded DNA-dependent kinase (dsDNA-PK) upstream of the cdk site has also been identified (Xiao et al., 1997). In vitro experiments indicate that CK2-mediated phosphorylation of Ser 111/112 increases the rate of nuclear accumulation of T-Ag-CcN-containing fusion proteins (Rihs et al., 1991), by increasing the affinity of recognition by IMPα/β (Hubner et al., 1997). Phosphorylation of Thr124 by cdk34 inhibits the nuclear import process (Jans et al., 1991), while dsDNA-PK mediated phosphorylation of Ser 120 enhances the maximal level of nuclear import and promotes IMPα/β binding in synergy with the CK2 site (Xiao et al., 1997). These data enabled to the development of a 25-amino acid optimized T-Ag NLS (opT-NLS), which has been used as an NT module in recombinant modular transporters for the specific targeting of PS agents to cancer cells to increase cell killing (Akhlynina et al., 1997, Akhlynina et al., 1999, Rosenkranz et al., 2003), or in recombinant modular transporter to increase reporter gene expression (Chan and Jans, 1999a, Chan and Jans, 1999b, Chan and Jans, 2001, Chan and Jans, 2002). Since CK2 is highly active in tumor cells as indicated above (Faust et al., 1999), the opT-NLS itself effectively represents a tNTS, at least for tumors showing high CK2 activity. Clearly, further studies are required to verify this interesting possibility using the approaches described by Poon et al., 2005a, Poon et al., 2005b.

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6. Engineering the T-Ag CcN motif to generate a tNTS 

The T-Ag CcN motif has been extensively studied, with the molecular mechanisms underlying the regulation of its activity well characterized (Jans et al., 1991, Rihs et al., 1991, Hubner et al., 1997, Xiao et al., 1997, Xiao et al., 1998, Fontes et al., 2003). The introduction of point mutations substituting the CK2 consensus phosphorylatin site with consensus sites for cAMP-dependent protein kinase (PKA) or Ca2+, phospholipid-dependent protein kinase (PKC) within the CcN motif, results in enhanced nuclear accumulation of β-GAL, in response to agents elevating the intracellular cAMP or activating PKC, respectively, when tested in an in vitro transport assay or in microinjected cells (Xiao et al., 1996, Xiao et al., 1998). This demonstrates that the CK2 site within the CcN motif can be substituted by sites for other kinases and yet retain functionality in terms of nuclear localization in response to specific phosphorylation; changing the kinase site changes the cellular signal that potentially drives nuclear localization. Substitution of the CK2 site with a potentially cancer-specific phosphorylation site could generate a novel specific tNTS. Screening for sequences that are selectively phosphorylated in the cytoplasm of tumor as opposed to normal cells may identify sequences that enhance tumor specificity of modular transporters for the delivery to cancer cells of suicide genes, or PS agents or other nuclear-acting drugs.

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7. Conclusions and future directions 

Strategies to achieve cancer cell-specific targeting – whether at the level of the whole cell, or more specifically at the level of nuclear transport – are obviously limited by the fact that cancer cells are derived from normal cells, implying that much of the fundamental signaling and transport apparatus is shared between cancer and normal cells. Hence, despite results for VP3 and other proteins discussed here, the idea that a targeting signal or approach could be uniquely functional in terms of targeting tumor as opposed to normal cells is probably naïve.

As alluded above, however, it is clear that there are several quantitative differences between cancer and normal cells in the levels of expression and activation of different signaling molecules, including protein kinases, and even in the levels of nuclear transport components (Kau et al., 2004, Poon and Jans, 2005). The ideal strategy for pursuing cancer cell-specific targeting might therefore be to exploit as many quantitative differences between tumor and normal cells as possible, and use them in combination to achieve a cumulative effect, so that much higher concentrations of drug or suicide gene are achieved in tumor as opposed to normal cells. The VP3 tNTS is clearly one targeting component that is much more efficient in conferring nuclear accumulation in tumor than in normal cells, but this appears to be a quantitative rather than all-or-nothing effect, and hence needs to be used in combination with other “tumor cell-enriched pathways” (e.g. phosphorylation cascades, receptor expression, endocytotic pathways, etc.; Guillemard and Saragovi, 2004) to achieve the equivalent of a “tumor cell-specific” effect. This can also be combined with further levels of “enrichment” such as cell/tissue-specific promoters for gene therapeutic approaches, and localized photoactivation in the case of the use of PSs in PDT.

The VP3 tNTS and modifications thereof, as well as strategies inspired by the idea of tumor cell-specific phosphorylation (see above), are great steps towards the long-term aim of cancer cell-specific therapies that do not kill or damage normal cells/tissue. Clearly, however, the key will be to find other tumor cell-specific acting moieties to use in combination with these sequences. With concerted effort, the ultimate end may be in sight, not so much of a “magic bullet” perhaps, but of a multi-component entity comprising multiple “semi-magic” moieties with higher activity in tumor than in normal cells. In combination, these cancer cell hyperactive components should afford a cumulative effect to achieve the ultimate end of efficient and above all specific tumor cell killing.

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8. Summary 


•Modular recombinant transporters, assemblies of independently acting domains, have been shown to be capable of targeting reporter genes or drugs to cancer cells specifically through cellular targeting (CT) modules.

•The addition of a nuclear targeting (NT) module increases the ability of modular transporters to deliver suicide genes or drugs to the nucleus of the target cell.

•VP3 is a protein that shows tumor cell-specific nuclear targeting as mediated by a phosphorylation regulated tumor-specific nuclear targeting sequence (tNTS).

•The activity of the best characterized nuclear targeting signal, the nuclear localization signal (NLS) from Simian virus SV40 large tumor antigen (T-Ag), is increased by protein kinase CK2 (CK2) phosphorylation upstream of the NLS.

•CK2 activity is elevated in several cancer cells so that T-Ag NLS-based modular transporters may be promising for therapy where tumors show elevated CK2 activity.

•Substitution of the CK2 site of T-Ag with consensus sites for kinases such Ca2+, phospholipid-dependent protein kinase (PKC) or cAMP-dependent protein kinase (PKA) results in its activity being increased by PKC and PKA activators, respectively. It is possible to generate tNTSs by inserting sequences phosphorylated in the cytoplasm of tumor but not normal cells in place of the T-Ag CK2 site.

•The combination of several tumor cell-specific modules, including CT and NT would increase the specificity of delivery to tumor cells of suicide genes or nuclear-acting drugs.

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Acknowledgments 

This work was supported by the Anti-Cancer Council of Victoria and the Australian National Health and Medical Research Council (fellowship #384109).

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PII: S1368-7646(06)00020-3

doi:10.1016/j.drup.2006.02.003

Drug Resistance Updates
Volume 9, Issue 1 , Pages 40-50, February 2006

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