**Cytotoxic Endonucleases: New Targets for Prostate Cancer Chemotherapy**

Xiaoying Wang, Marina V. Mikhailova and Alexei G. Basnakian *University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System Little Rock, Arkansas USA* 

## **1. Introduction**

266 Prostate Cancer – From Bench to Bedside

Yang, X., B. Pursell, S. Lu, T.K. Chang, & A.M. Mercurio. 2009. Regulation of beta 4-integrin

Zhang, B., X. Pan, G.P. Cobb, & T.A. Anderson. 2007. microRNAs as oncogenes and tumor

Zhang, Z., N.E. Ramirez, T.E. Yankeelov, Z. Li, L.E. Ford, Y. Qi, A. Pozzi, & M.M. Zutter.

tumor angiogenesis in a tumor cell-specific manner. *Blood*. 111:1980-1988.

epithelial-to-mesenchymal transition. *J Cell Sci*. 122:2473-2480.

suppressors. *Dev Biol*. 302:1-12.

expression by epigenetic modifications in the mammary gland and during the

2008. alpha2beta1 integrin expression in the tumor microenvironment enhances

Prostate cancer is one of the most common malignancies in Western countries and the world (Baade et al., 2009). It is the third most common cause of death from cancer in men of all ages and the most common cause of death from cancer in men over age 75. The current standard therapies for prostate cancer include radiation, surgery, hormonal therapy and chemotherapy (Debruyne, 2002; Freytag et al., 2007; Nelius et al., 2009; Rozkova et al., 2009). Chemotherapy is almost always a salvage therapy for advanced prostate cancer, and chemoresistance is emerging problem in prostate cancer therapy. Strategies to overcome the chemoresistance of prostate cancer cells have not been developed partially because mechanisms of it are unknown and likely to be numerous. Chemoresistance has a tendency to occur both to clinically established therapeutic agents and novel targeted therapeutics implicating both intrinsic and acquired mechanisms of drug resistance (Djeu & Wei, 2009). Most likely, these are the mechanisms which are universal for cytoprotection from cell death induced by various factors.

Cell death by apoptosis is one of the most universal mechanisms of cell response to injury. It plays the major role in carcinogenesis and prostate tumor progression. Suppression of apoptosis was proposed to cause inappropriate survival of genetically aberrant cells during carcinogenesis (Vineis, 2003). Cancer cells seem to be designed to propagate and survive in a new and hostile environment by suppressing their natural mechanisms of cell death. The neoplastic transformation of prostate epithelial cells is known to be associated with decreased apoptotic cell death (Inokuchi et al., 2009; Shilkaitis et al., 2000). The progression of prostate cancer, in particular, androgen-independent prostate cancer or prostate adenocarcinomas, was also shown to be associated with decreased apoptosis (Raffo et al., 1995; Singh & Lokeshwar, 2009). The latter is the predominant form of tumor cell demise caused by chemotherapeutic agents and it plays an important role in cancer chemosensitivity and radiosensitivity (Arnold & Isaacs, 2002; Debes & Tindall, 2004). Targeting various mechanisms of apoptosis to cure prostate cancer has been suggested in many studies, naming potential molecular targets and key apoptotic regulators such as upstream and downstream caspases, p53, Phosphatase and tensin homolog (PTEN), prostate apoptosis response gene-4 (Par-4), Bcl-2 (B-cell lymphoma 2) protein, transcription factor

Cytotoxic Endonucleases: New Targets for Prostate Cancer Chemotherapy 269

essential for clean up after cell death, removal of DNA from blood plasma, and destroying "foreign" DNA from bacteria and viruses consumed by cells (Buzder et al., 2009). These roles of cytotoxic endonucleases are less relevant to prostate cancer and thus will not be

Although all cells and tissues seem to express all endonucleases, the spectrum of them differs between the tissues. The reason for such redundancy of the cytotoxic endonucleases is not known, which allows speculation about the importance of DNA destruction from

The most expressed and active endonuclease in normal prostate is DNase I, previously also known as Ca/Mg-dependent endonuclease (Kyprianou et al., 1988; Kyprianou & Isaacs, 1988). Ca/Mg-dependent endonuclease-mediated DNA fragmentation is used as a marker of apoptosis in prostate cancer. The degradation of genomic DNA into nucleosome-sized fragments is an early event in castration-induced androgen withdrawal that involves death of the androgen-dependent epithelial cells following an increase of endonuclease activity

DNase I is found in all studied species and tissues (Lacks, 1981). It is expressed mainly in tissues of the digestive system, though the specific activity of the enzyme varies between the organs (Gonzalez et al., 2001; Jacob et al., 2002; Lacks, 1981). In digestive tissues (intestine, pancreas, salivary glands), it is a secreted enzyme intended to hydrolyze DNA in the alimentary tract. In non-digestive tissues (including prostate), the role of DNase I is not known. Bovine or mouse DNase I bind specifically to G-actin and blocks its polymerization (Lacks, 1981). The enzyme from all sources endonucleolytically cleaves double- or single-stranded DNA to 3'OH/5'P-end oligonucleotides, requires divalent cations, particularly Ca2+ and Mg2+, is inhibited by Zn2+, and has a neutral pH optimum. Inside the cell, the enzyme is located in the cytoplasm (Peitsch et al., 1993). It has also been shown inside nuclei, but the mechanism of its introduction into nuclei has not been studied. Inhibition of DNase I by internalized nuclear anti-DNA antibodies was shown to provide protection of cells against apoptotic stimuli (Madaio et al., 1996). No known nuclear localization signal was identified in DNase I, and "leakage" through nuclear pores was suggested (Polzar et al., 1993). Among various organs and tissues, prostate, pancreas, salivatory glands and kidney tubular epithelium have the highest levels of DNase I activity (Lacks, 1981; Polzar et al., 1993). Little is known about DNase I regulation *in vivo*. An alternative pre-mRNA splicing both in 5'UTR and in coding region was shown to be a mechanism of DNase I regulation (Basnakian et al., 1998; Basnakian et al., 2002). It is known that some DNase I isoforms can be generated by post-translational modification,

Studies of endonucleases associated with prostate cancer are very limited. Usually neoplastic transformation is associated with the decrease of endonuclease expression and activity in various cancers, thus making them "immortal" (Banfalvi et al., 2007; Basnakian et al., 2006; Basnakian et al., 1991; Wang et al., 2008). The most profound decrease of endonuclease activity was observed in malignant invasive prostate and breast cancer cells (Basnakian et al., 2006; Wang et al., 2008). The decrease of endonuclease activity had been also observed in other cancers and models of carcinogenesis (Basnak'ian et al., 1989; Basnakian et al., 1991). Immortalization of rat fibroblasts with the S1A segment of SA7 adenovirus also led to a significant decrease of endonuclease activity (Basnak'ian et al.,

(Banerjee et al., 2000; Brandstrom et al., 1994; Kyprianou et al., 1988).

namely mannose-type glycosylation of the protein (Lacks, 1981).

considered in this review.

immediately prior to long after cell death.

NF-kappa B, serine/threonine protein kinase and others (Uzzo et al., 2008; Wang et al., 2004). Precisely, manipulating with sensitivity to cytotoxic agents to alter cancer progression has been suggested as a therapeutic approach for prostate cancer in some reports (McKenzie & Kyprianou, 2006; Watson & Fitzpatrick, 2005). However, for some reason, almost no attention was paid to apoptotic/cytotoxic endonucleases as potential targets.

## **2. Cytotoxic endonucleases in normal prostate and prostate cancer cells**

Cytotoxic endonucleases, also called "apoptotic endonucleases," are the initially recognized group of enzymes responsible for premortem and postmortem DNA fragmentation associated with cell death by apoptosis (Hengartner, 2001; Samejima & Earnshaw, 2005). Importantly, the same enzymes were later shown to provide DNA fragmentation that accompanies necrosis, autophagy, mitotic catastrophe and all other types of cell death. Therefore the term "apoptotic endonucleases" should be considered outdated.

Major representatives of cytotoxic endonucleases include: deoxyribonuclease I (DNase I) (Polzar et al., 1993), deoxyribonuclease II (DNase II) (Krieser & Eastman, 1998), Endonuclease G (EndoG) (Li et al., 2001), caspase-activated DNase (CAD) (Enari et al., 1998), and DNase gamma (Shiokawa et al., 1997). Some of these enzymes, for example DNases I and II had been known before 1960s. However the actual role of the cytotoxic endonucleases was clarified much later. Cytotoxic endonucleases were found in all studied cells and tissues, including the prostate (Koizumi, 1995; Napirei et al., 2004). The enzymes belong to the family of hydrolyses that cleave phosphodiether bonds in DNA. They differ in certain catalytic characteristics and DNA sequence specificity, and yet produce very similar type of DNA damage consisting of single-stranded or double-strand DNA breaks. Most harmful and hard to repair DNA breaks and double-stranded. They can be produced by so called "single hit" or "double hit" mechanism. In a "single hit" mode, both DNA strands are cut simultaneously at the same site. This mechanism is mainly characteristic of DNase II. The much more common mechanism is "double hit," in which strands are cleaved independently to result in a double-strand DNA break if the single-stranded breaks coincide with a 2-base or less shift between them. This mechanism is characteristic of DNase I and all other endonucleases, except DNase II.

Independently from the mechanism, endonuclease-generated breaks have been shown to strongly interfere with DNA synthesis in both normal and cancer cells (Nagata, 2000). That is why, while sometimes considered downstream effectors of apoptotic cascades, the endonucleases can cause DNA fragmentation and imminent and irreversible cell death when acting alone after overexpression or introduction into the cell (Enari et al., 1998; Krieser & Eastman, 1998; Polzar et al., 1993). The endonucleases are commonly found active during cell death; however, the overall link between these enzymes and apoptosis is weak. Some of the endonucleases seem to be dispensable in normal apoptosis (Davidson & Harper, 2005; Irvine et al., 2005; Napirei et al., 2000). On the other hand, their participation in cell death in general after tissue injury seems crucial and evidence of this is overwhelming. Recent studies demonstrated that inactivation of the endonucleases causes protection of normal and cancer cells against a variety of injuries *in vitro* and *in vivo* (Basnakian et al., 2006; Basnakian et al., 2005; Napirei et al., 2006; Yin et al., 2007), suggesting that the endonucleases are essential for and mechanistically linked to injuryrelated cell death. In addition to causing cell death itself, the endonuclease are certainly

NF-kappa B, serine/threonine protein kinase and others (Uzzo et al., 2008; Wang et al., 2004). Precisely, manipulating with sensitivity to cytotoxic agents to alter cancer progression has been suggested as a therapeutic approach for prostate cancer in some reports (McKenzie & Kyprianou, 2006; Watson & Fitzpatrick, 2005). However, for some reason, almost no

attention was paid to apoptotic/cytotoxic endonucleases as potential targets.

Therefore the term "apoptotic endonucleases" should be considered outdated.

other endonucleases, except DNase II.

**2. Cytotoxic endonucleases in normal prostate and prostate cancer cells** 

Cytotoxic endonucleases, also called "apoptotic endonucleases," are the initially recognized group of enzymes responsible for premortem and postmortem DNA fragmentation associated with cell death by apoptosis (Hengartner, 2001; Samejima & Earnshaw, 2005). Importantly, the same enzymes were later shown to provide DNA fragmentation that accompanies necrosis, autophagy, mitotic catastrophe and all other types of cell death.

Major representatives of cytotoxic endonucleases include: deoxyribonuclease I (DNase I) (Polzar et al., 1993), deoxyribonuclease II (DNase II) (Krieser & Eastman, 1998), Endonuclease G (EndoG) (Li et al., 2001), caspase-activated DNase (CAD) (Enari et al., 1998), and DNase gamma (Shiokawa et al., 1997). Some of these enzymes, for example DNases I and II had been known before 1960s. However the actual role of the cytotoxic endonucleases was clarified much later. Cytotoxic endonucleases were found in all studied cells and tissues, including the prostate (Koizumi, 1995; Napirei et al., 2004). The enzymes belong to the family of hydrolyses that cleave phosphodiether bonds in DNA. They differ in certain catalytic characteristics and DNA sequence specificity, and yet produce very similar type of DNA damage consisting of single-stranded or double-strand DNA breaks. Most harmful and hard to repair DNA breaks and double-stranded. They can be produced by so called "single hit" or "double hit" mechanism. In a "single hit" mode, both DNA strands are cut simultaneously at the same site. This mechanism is mainly characteristic of DNase II. The much more common mechanism is "double hit," in which strands are cleaved independently to result in a double-strand DNA break if the single-stranded breaks coincide with a 2-base or less shift between them. This mechanism is characteristic of DNase I and all

Independently from the mechanism, endonuclease-generated breaks have been shown to strongly interfere with DNA synthesis in both normal and cancer cells (Nagata, 2000). That is why, while sometimes considered downstream effectors of apoptotic cascades, the endonucleases can cause DNA fragmentation and imminent and irreversible cell death when acting alone after overexpression or introduction into the cell (Enari et al., 1998; Krieser & Eastman, 1998; Polzar et al., 1993). The endonucleases are commonly found active during cell death; however, the overall link between these enzymes and apoptosis is weak. Some of the endonucleases seem to be dispensable in normal apoptosis (Davidson & Harper, 2005; Irvine et al., 2005; Napirei et al., 2000). On the other hand, their participation in cell death in general after tissue injury seems crucial and evidence of this is overwhelming. Recent studies demonstrated that inactivation of the endonucleases causes protection of normal and cancer cells against a variety of injuries *in vitro* and *in vivo* (Basnakian et al., 2006; Basnakian et al., 2005; Napirei et al., 2006; Yin et al., 2007), suggesting that the endonucleases are essential for and mechanistically linked to injuryrelated cell death. In addition to causing cell death itself, the endonuclease are certainly essential for clean up after cell death, removal of DNA from blood plasma, and destroying "foreign" DNA from bacteria and viruses consumed by cells (Buzder et al., 2009). These roles of cytotoxic endonucleases are less relevant to prostate cancer and thus will not be considered in this review.

Although all cells and tissues seem to express all endonucleases, the spectrum of them differs between the tissues. The reason for such redundancy of the cytotoxic endonucleases is not known, which allows speculation about the importance of DNA destruction from immediately prior to long after cell death.

The most expressed and active endonuclease in normal prostate is DNase I, previously also known as Ca/Mg-dependent endonuclease (Kyprianou et al., 1988; Kyprianou & Isaacs, 1988). Ca/Mg-dependent endonuclease-mediated DNA fragmentation is used as a marker of apoptosis in prostate cancer. The degradation of genomic DNA into nucleosome-sized fragments is an early event in castration-induced androgen withdrawal that involves death of the androgen-dependent epithelial cells following an increase of endonuclease activity (Banerjee et al., 2000; Brandstrom et al., 1994; Kyprianou et al., 1988).

DNase I is found in all studied species and tissues (Lacks, 1981). It is expressed mainly in tissues of the digestive system, though the specific activity of the enzyme varies between the organs (Gonzalez et al., 2001; Jacob et al., 2002; Lacks, 1981). In digestive tissues (intestine, pancreas, salivary glands), it is a secreted enzyme intended to hydrolyze DNA in the alimentary tract. In non-digestive tissues (including prostate), the role of DNase I is not known. Bovine or mouse DNase I bind specifically to G-actin and blocks its polymerization (Lacks, 1981). The enzyme from all sources endonucleolytically cleaves double- or single-stranded DNA to 3'OH/5'P-end oligonucleotides, requires divalent cations, particularly Ca2+ and Mg2+, is inhibited by Zn2+, and has a neutral pH optimum. Inside the cell, the enzyme is located in the cytoplasm (Peitsch et al., 1993). It has also been shown inside nuclei, but the mechanism of its introduction into nuclei has not been studied. Inhibition of DNase I by internalized nuclear anti-DNA antibodies was shown to provide protection of cells against apoptotic stimuli (Madaio et al., 1996). No known nuclear localization signal was identified in DNase I, and "leakage" through nuclear pores was suggested (Polzar et al., 1993). Among various organs and tissues, prostate, pancreas, salivatory glands and kidney tubular epithelium have the highest levels of DNase I activity (Lacks, 1981; Polzar et al., 1993). Little is known about DNase I regulation *in vivo*. An alternative pre-mRNA splicing both in 5'UTR and in coding region was shown to be a mechanism of DNase I regulation (Basnakian et al., 1998; Basnakian et al., 2002). It is known that some DNase I isoforms can be generated by post-translational modification, namely mannose-type glycosylation of the protein (Lacks, 1981).

Studies of endonucleases associated with prostate cancer are very limited. Usually neoplastic transformation is associated with the decrease of endonuclease expression and activity in various cancers, thus making them "immortal" (Banfalvi et al., 2007; Basnakian et al., 2006; Basnakian et al., 1991; Wang et al., 2008). The most profound decrease of endonuclease activity was observed in malignant invasive prostate and breast cancer cells (Basnakian et al., 2006; Wang et al., 2008). The decrease of endonuclease activity had been also observed in other cancers and models of carcinogenesis (Basnak'ian et al., 1989; Basnakian et al., 1991). Immortalization of rat fibroblasts with the S1A segment of SA7 adenovirus also led to a significant decrease of endonuclease activity (Basnak'ian et al.,

Cytotoxic Endonucleases: New Targets for Prostate Cancer Chemotherapy 271

the anticancer therapy. However, delivery of endonucleases or modulation of endonuclease activity are not currently used for cancer therapy, in particular, for prostate cancer therapy.

Epigenetic changes are believed to be the most common alteration at the DNA level in prostate cancer (Schulz & Hatina, 2006; Walton et al., 2008). Two types of DNA epigenetic changes that are known to occur in prostate cancer include regional DNA hypermethylation and regional/global DNA hypomethylation. Hypermethylation of the promoter region that contains CpG island occurs in a large number of genes and is usually associated with gene silencing in the vast majority of prostate cancer cases (Li et al., 2005; Perry et al., 2006; Rennie & Nelson, 1998). Studies have shown that hypermethylation of this region may be eventually used as a tumor biomarker for early diagnosis and risk assessment of prostate cancer. Furthermore, the prevalence of epigenetic changes in prostate cancer and the potential reversibility of DNA methylation alterations by DNA methylation inhibitors suggest that these changes are a viable target for cancer chemotherapy and chemoprevention strategies (Egger et al., 2004; Kopelovich

Mammalian genome contains patterns of methylated cytosines for normal function, but until recently the structural organization of the methylation landscape of the human genome was unclear (Rollins et al., 2006). It has been reported that the human genome consists of short (<4 kb) unmethylated domains enriched in promoters, CpG islands, and first exons, embedded in a matrix of long methylated domains (Rollins et al., 2006). Analysis of promoter sequences of all known human cytotoxic endonucleases – described below showed that EndoG is the only cytotoxic endonuclease that contains a CpG island, a segment of DNA with high G+C content and a site for methylation, in the promoter region

A large number of studies have shown that methylation of promoter CpG islands plays an important role in gene silencing (Ruchusatsawat et al., 2006; Taghavi & van Lohuizen, 2006). The broadly accepted definition of a CpG island as a 200-bp fragment of DNA with G + C content greater than 50% and observed CpG/expected CpG ratio higher than 0.6 failed to exclude many sequences (such as *Alu* repeats and unknown sequences) that are not associated with regulatory regions of genes (Takai & Jones, 2002). Recent studies indicate that the usage of a modified algorithm to search for CpG islands using a more stringent definition (G + C content higher than 55% and a length greater than 500 bp with observed CpG/expected CpG ratio 0.65) resulted in the exclusion of the majority of *Alu* repetitive and unknown sequences associated with the 5' region of genes (Takai & Jones, 2002). In view of these considerations, we applied this algorithm to the analysis of endonuclease genes, which could be regulated by DNA methylation. All known human cell death endonucleases and their sequence variants were analyzed using the CpG Island Searcher program (available at http://www.cpgislands.com (Takai & Jones, 2003)): DNase 1, DNase 1L1 variants 1, 2, 3 and 4; DNase 1L2, DNase 1L3 (DNase gamma), DNase 2α, DNase 2β variants 1 and 2, L-DNase II (LEI), CAD and EndoG. Surprisingly, this analysis showed that EndoG is the only gene that satisfied the criteria of containing a

long CpG island in the promoter and exon 1 of the gene.

**3. Modulation of EndoG by DNA methylation and histone deacetylation** 

et al., 2003; Yoo & Jones, 2006).

(Wang et al., 2008).

1989). Another report indicated that an endonuclease activity is decreased in diethylnitrosamine (DEN)-induced hepatomas in rats compared to normal liver tissue (Basnakian et al., 1991). The decrease was proportional to the degree of dedifferentiation and the activity was the lowest in poorly differentiated tumors.

With the decrease of main prostate endonuclease, DNase I, the endonuclease activity in human prostate cancer cells is provided by EndoG. This endonuclease has a unique siteselectivity, initially attacking poly(dG).poly(dC) sequences in double-stranded DNA, as denoted by this enzyme's name. The enzyme also has RNase activity. EndoG predominantly resides in the intermembrane space of mitochondrion (Ohsato et al., 2002). Mammalian EndoG is synthesized as a 32 kDa propeptide in the cytoplasm and imported into mitochondria through a process mediated by its amino-terminal mitochondriontargeting sequence (Cote & Ruiz-Carrillo, 1993; Ruiz-Carrillo & Renaud, 1987). The EndoG protein precursor is inactive (Ikeda & Kawasaki, 2001). The signal peptide is cleaved off after entering the mitochondria and the mature active 27 kDa EndoG is released from mitochondria during apoptosis, moves to the nuclei and cleaves nuclear DNA without sequence specificity (Li et al., 2001). EndoG expression varies in different tissues and in embryonic tissues the expression of EndoG is very low (Apostolov et al., 2007b). As opposed to DNase I, the enzyme has a greater activity on single-stranded nucleic acid substrates, single-stranded DNA and RNA. It preferentially cleaves non-canonical structures of DNA, damaged DNA, triplex DNA, and R-loops that appear non-specifically during transcription (Masse & Drolet, 1999). Cisplatin-treated DNA was shown to be preferentially cleaved by EndoG (Ikeda & Ozaki, 1997). EndoG requires either Mn2+ or Mg2+ ions, and is inhibited 15-fold at physiological ionic strengths (Widlak et al., 2001). Fe2+ and Zn2+ inhibit the enzyme activity. The EndoG gene in mice is a single copy gene, which consists of 3 exons (Prats et al., 1997). The loss of EndoG activity in C.elegans resulted in increased cell survival (Hengartner, 2001). However, EndoG knockout mouse is viable (Irvine et al., 2005). Reduction of EndoG in C.elegans using siRNA or genetic mutation affected normal DNA degradation, as revealed by staining with TUNEL assay, and resulted in the delayed appearance of cell corpses during development in C.elegans (Parrish et al., 2001). Thus in comparison to other endonucleases, EndoG is uniquely compartmentalized in mitochondria and it does not have known intracellular inhibitors (like DNase I or CAD). The EndoG location site may indicate that this enzyme is not an instrument of immediate response to cell injury.

EndoG seems to be particularly important in cancer cells because it regulates their sensitivity to chemotherapeutic agents (Basnakian et al., 2006). This report suggests the presence of EndoG in non-invasive breast cancer cells determines their sensitivity to apoptosis, which may be taken into consideration for developing the chemotherapeutic strategy for cancer treatment. In other cells, EndoG has been recognized as a key endonuclease in the caspase-independent apoptosis (Abbott et al., 2001; Bahi et al., 2006), mitotic catastrophe (Diener et al., ; Wang et al., 2008), and necrosis (Apostolov et al., 2007a; Jiang et al., 2006).

Because anticancer drugs induce apoptosis in cancer cells through endonuclease-mediated DNA fragmentation (Ploski & Aplan, 2001; Shrivastava et al., 2000), and the inhibition of endonucleases has a protective effect (Shrivastava et al., 2000), endonuclease should be considered as important mediators of cancer cell death and potential therapeutic targets for

1989). Another report indicated that an endonuclease activity is decreased in diethylnitrosamine (DEN)-induced hepatomas in rats compared to normal liver tissue (Basnakian et al., 1991). The decrease was proportional to the degree of dedifferentiation

With the decrease of main prostate endonuclease, DNase I, the endonuclease activity in human prostate cancer cells is provided by EndoG. This endonuclease has a unique siteselectivity, initially attacking poly(dG).poly(dC) sequences in double-stranded DNA, as denoted by this enzyme's name. The enzyme also has RNase activity. EndoG predominantly resides in the intermembrane space of mitochondrion (Ohsato et al., 2002). Mammalian EndoG is synthesized as a 32 kDa propeptide in the cytoplasm and imported into mitochondria through a process mediated by its amino-terminal mitochondriontargeting sequence (Cote & Ruiz-Carrillo, 1993; Ruiz-Carrillo & Renaud, 1987). The EndoG protein precursor is inactive (Ikeda & Kawasaki, 2001). The signal peptide is cleaved off after entering the mitochondria and the mature active 27 kDa EndoG is released from mitochondria during apoptosis, moves to the nuclei and cleaves nuclear DNA without sequence specificity (Li et al., 2001). EndoG expression varies in different tissues and in embryonic tissues the expression of EndoG is very low (Apostolov et al., 2007b). As opposed to DNase I, the enzyme has a greater activity on single-stranded nucleic acid substrates, single-stranded DNA and RNA. It preferentially cleaves non-canonical structures of DNA, damaged DNA, triplex DNA, and R-loops that appear non-specifically during transcription (Masse & Drolet, 1999). Cisplatin-treated DNA was shown to be preferentially cleaved by EndoG (Ikeda & Ozaki, 1997). EndoG requires either Mn2+ or Mg2+ ions, and is inhibited 15-fold at physiological ionic strengths (Widlak et al., 2001). Fe2+ and Zn2+ inhibit the enzyme activity. The EndoG gene in mice is a single copy gene, which consists of 3 exons (Prats et al., 1997). The loss of EndoG activity in C.elegans resulted in increased cell survival (Hengartner, 2001). However, EndoG knockout mouse is viable (Irvine et al., 2005). Reduction of EndoG in C.elegans using siRNA or genetic mutation affected normal DNA degradation, as revealed by staining with TUNEL assay, and resulted in the delayed appearance of cell corpses during development in C.elegans (Parrish et al., 2001). Thus in comparison to other endonucleases, EndoG is uniquely compartmentalized in mitochondria and it does not have known intracellular inhibitors (like DNase I or CAD). The EndoG location site may indicate that this enzyme is not an

EndoG seems to be particularly important in cancer cells because it regulates their sensitivity to chemotherapeutic agents (Basnakian et al., 2006). This report suggests the presence of EndoG in non-invasive breast cancer cells determines their sensitivity to apoptosis, which may be taken into consideration for developing the chemotherapeutic strategy for cancer treatment. In other cells, EndoG has been recognized as a key endonuclease in the caspase-independent apoptosis (Abbott et al., 2001; Bahi et al., 2006), mitotic catastrophe (Diener et al., ; Wang et al., 2008), and necrosis (Apostolov et al., 2007a;

Because anticancer drugs induce apoptosis in cancer cells through endonuclease-mediated DNA fragmentation (Ploski & Aplan, 2001; Shrivastava et al., 2000), and the inhibition of endonucleases has a protective effect (Shrivastava et al., 2000), endonuclease should be considered as important mediators of cancer cell death and potential therapeutic targets for

and the activity was the lowest in poorly differentiated tumors.

instrument of immediate response to cell injury.

Jiang et al., 2006).

the anticancer therapy. However, delivery of endonucleases or modulation of endonuclease activity are not currently used for cancer therapy, in particular, for prostate cancer therapy.

## **3. Modulation of EndoG by DNA methylation and histone deacetylation**

Epigenetic changes are believed to be the most common alteration at the DNA level in prostate cancer (Schulz & Hatina, 2006; Walton et al., 2008). Two types of DNA epigenetic changes that are known to occur in prostate cancer include regional DNA hypermethylation and regional/global DNA hypomethylation. Hypermethylation of the promoter region that contains CpG island occurs in a large number of genes and is usually associated with gene silencing in the vast majority of prostate cancer cases (Li et al., 2005; Perry et al., 2006; Rennie & Nelson, 1998). Studies have shown that hypermethylation of this region may be eventually used as a tumor biomarker for early diagnosis and risk assessment of prostate cancer. Furthermore, the prevalence of epigenetic changes in prostate cancer and the potential reversibility of DNA methylation alterations by DNA methylation inhibitors suggest that these changes are a viable target for cancer chemotherapy and chemoprevention strategies (Egger et al., 2004; Kopelovich et al., 2003; Yoo & Jones, 2006).

Mammalian genome contains patterns of methylated cytosines for normal function, but until recently the structural organization of the methylation landscape of the human genome was unclear (Rollins et al., 2006). It has been reported that the human genome consists of short (<4 kb) unmethylated domains enriched in promoters, CpG islands, and first exons, embedded in a matrix of long methylated domains (Rollins et al., 2006). Analysis of promoter sequences of all known human cytotoxic endonucleases – described below showed that EndoG is the only cytotoxic endonuclease that contains a CpG island, a segment of DNA with high G+C content and a site for methylation, in the promoter region (Wang et al., 2008).

A large number of studies have shown that methylation of promoter CpG islands plays an important role in gene silencing (Ruchusatsawat et al., 2006; Taghavi & van Lohuizen, 2006). The broadly accepted definition of a CpG island as a 200-bp fragment of DNA with G + C content greater than 50% and observed CpG/expected CpG ratio higher than 0.6 failed to exclude many sequences (such as *Alu* repeats and unknown sequences) that are not associated with regulatory regions of genes (Takai & Jones, 2002). Recent studies indicate that the usage of a modified algorithm to search for CpG islands using a more stringent definition (G + C content higher than 55% and a length greater than 500 bp with observed CpG/expected CpG ratio 0.65) resulted in the exclusion of the majority of *Alu* repetitive and unknown sequences associated with the 5' region of genes (Takai & Jones, 2002). In view of these considerations, we applied this algorithm to the analysis of endonuclease genes, which could be regulated by DNA methylation. All known human cell death endonucleases and their sequence variants were analyzed using the CpG Island Searcher program (available at http://www.cpgislands.com (Takai & Jones, 2003)): DNase 1, DNase 1L1 variants 1, 2, 3 and 4; DNase 1L2, DNase 1L3 (DNase gamma), DNase 2α, DNase 2β variants 1 and 2, L-DNase II (LEI), CAD and EndoG. Surprisingly, this analysis showed that EndoG is the only gene that satisfied the criteria of containing a long CpG island in the promoter and exon 1 of the gene.

Cytotoxic Endonucleases: New Targets for Prostate Cancer Chemotherapy 273

data indicated EndoG may be regulated by methylation of its gene promoter, and partially by histone acetylation, and that EndoG is essential for prostate cancer cell death in the used models. Histone modification, in particular, histone acetylation, is another epigenetic mechanism that is important in regulation of genes in prostate cancer (Das et al., 2006; Egger et al., 2004; Wang et al., 2008; Yoo & Jones, 2006). To determine whether and how histone acetylation regulates EndoG expression, two prostate cancer cell lines were treated with TSA, and EndoG protein expression was studied using Western blotting. The exposure of the cells to TSA induced high levels of EndoG expression in EndoG-positive 22Rv1 cells, whereas in EndoG-deficient PC3 cells, EndoG was not induced. Again, EndoG induction by TSA caused increased sensitivity to cisplatin. These data demonstrated that chromatin acetylation is important for EndoG expression. Taken together with the above methylation experiments, these data indicate that DNA methylation plays a primary role in EndoG regulation as compared to histone acetylation. In other words, the CpG island of the EndoG gene has to be hypomethylated in order to allow regulation of EndoG

Although these are attractive and potentially therapeutically useful approaches, modulations of endonuclease expression by DNA methylation or histone acetylation may not be a realistic approach because the specificity of epigenetic regulation is notoriously low. An alternative to these methods may be a gene delivery and overexpression in the target cancer cells. To determine whether overexpression of EndoG would make PC3 cells sensitive to the chemotherapy agents, the cells were transfected with human mature EndoG gene. To model chemotherapy in vitro, we used docetaxel, which is an FDAapproved the first line chemotherapeutic agent in castration-refractory prostate cancer (Oudard et al., 2007; Ryan et al., 2001). Despite survival benefits with docetaxel based chemotherapy, prognosis for castration-refractory prostate cancer patients usually is poor and patients typically show rapid progression (Oudard et al., 2005; Wang et al., 2008). Progressive prostate cancer is associated with the development, and subsequent expansion of tumor cells that are resistant to apoptotic triggers and dysregulation of apoptosis is often characterized by insufficient apoptosis (Kruslin, 2009; Mori et al., 1996). Therefore a delivery of EndoG gene was expected to increase sensitivity of prostate cancer

As described below, this genetic manipulation resulted in significant increase of PC3 cells sensitivity to docetaxel and cisplatin in vitro. Similar results were obtained when PC3 cells were transfected with EndoG precursor gene suggesting that the drugs induce speedy

PC3 cells were chosen because they have very low expression of EndoG (Wang et al., 2008). Human EndoG gene (NM 004435.2) was cloned in the mammalian expression vector pECFP.N1 to result in an expression of EndoG protein fused with the enhanced cyan fluorescence protein (CFP). The expression of the chimeric protein was confirmed by fluorescent microscopy. Cells were then treated with docetaxel and cell death was measured using lactate dehydrogenase (LDH) release assay. This experiment showed the sensitivity of the PC3 cells expressing EndoG-CFP to docetaxel was much higher than the cells expressing CFP alone (Figure 1). The same result was also observed in cisplatin-induced cells death:

**4. Endonuclease delivery to prostate cancer cells and tumors** 

expression by histone acetylation.

cells to docetaxel.

processing of the protein to mature endonuclease.

The methylation status of the EndoG promoter/exon 1 in prostate cancer cells was then determined by using the methylation-sensitive McrBC-PCR method. McrBC is a bacterial endonuclease, that does not act on unmethylated DNA, but cleaves DNA containing 5 methylcytosine in one or both strands and thus nullifies PCR amplification (Nakayama et al., 2004). This experiment showed that in three studied prostate cancer cell lines, LNCaP, 22Rv1 and PC3, EndoG promoter methylation was the lowest in 22Rv1 cells and highest in PC3 cells. Further comparison of the three prostate cancer cell lines showed that EndoG is highly expressed in 22Rv1 and LNCaP cells. In PC3 cells, EndoG was not expressed and the EndoG gene CpG island was hypermethylated (Wang et al., 2008).

The expression of EndoG correlated positively with sensitivity to docetaxel, cisplatin and etoposide, and the silencing of EndoG by siRNA decreased the sensitivity of the cells to the chemotherapeutic agents in the two EndoG-expressing cell lines. To determine whether the level of EndoG expression affects the sensitivity of prostate cancer cells to chemotherapeutic drugs, we exposed the three cell lines to two anticancer agents, cisplatin (0-100 µM) and etoposide (0-300 µM), which are known to induce cell death *in vitro* (Fang et al., 2004; Lee et al., 2006). As expected, the two cell lines that expressed EndoG, 22Rv1 and LNCaP, were highly sensitive to both chemotherapeutic agents. EndoG-deficient PC3 cells, in contrast, were insensitive to these drugs in the range of concentrations used.

Further study determined that cisplatin-induced death of prostate cancer cells can be prevented by EndoG silencing. Although EndoG is known to participate in cell death, it was necessary to determine whether the role of EndoG was the same in prostate cancer cells subjected to injury by cytotoxic agents as has been described in other cells. To test a causal relationship, EndoG was silenced in 22Rv1 cells by applying siRNA. To show that siRNA was delivered to the cells, fluorescent DY547-labeled siRNA was used. After DY547-siRNA transfection, 22Rv1 cells were exposed to 80 μM cisplatin, a concentration that had induced significant cell death in the above experiments. Next, TUNEL assay was conducted to measure DNA fragmentation. The assay showed the DNA fragmentation was decreased, indicating that silencing of EndoG leads to significant decrease of EndoG expression and protects cells from DNA fragmentation. As expected, EndoG silencing resulted in the increased viability of cisplatin-treated 22Rv1 cells as measured using clonogenic assay. The results suggest EndoG is responsible for cisplatin-induced death in prostate cancer cells.

It is interesting that inhibition of DNA methylation induced EndoG and increased sensitivity of PC3 cells to cisplatin and etoposide. 5-aza-2'-deoxycytidine (decitabine), which is a DNA methylation inhibitor, caused hypomethylation of the EndoG promoter in PC3 cells, induced EndoG mRNA and protein expression, and made the cells sensitive to the chemotherapy agents. Using McrBC-PCR method, we determined that the treatment of PC3 cells with decitabine inhibited methylation of the CpG island in the EndoG gene. The same concentration of decitabine also increased EndoG expression as determined by real-time RT-PCR and Western blotting. These data clearly suggested EndoG expression is regulated by DNA methylation. Importantly, the induction of EndoG by demethylation caused a significant increase in sensitivity to cisplatin and etoposide.

Finally, the acetylation of histones by trichostatin A (TSA), a histone deacetylase inhibitor, induced EndoG expression in 22Rv1 cells, while it had no such effect in PC3 cells. These

The methylation status of the EndoG promoter/exon 1 in prostate cancer cells was then determined by using the methylation-sensitive McrBC-PCR method. McrBC is a bacterial endonuclease, that does not act on unmethylated DNA, but cleaves DNA containing 5 methylcytosine in one or both strands and thus nullifies PCR amplification (Nakayama et al., 2004). This experiment showed that in three studied prostate cancer cell lines, LNCaP, 22Rv1 and PC3, EndoG promoter methylation was the lowest in 22Rv1 cells and highest in PC3 cells. Further comparison of the three prostate cancer cell lines showed that EndoG is highly expressed in 22Rv1 and LNCaP cells. In PC3 cells, EndoG was not expressed and the

The expression of EndoG correlated positively with sensitivity to docetaxel, cisplatin and etoposide, and the silencing of EndoG by siRNA decreased the sensitivity of the cells to the chemotherapeutic agents in the two EndoG-expressing cell lines. To determine whether the level of EndoG expression affects the sensitivity of prostate cancer cells to chemotherapeutic drugs, we exposed the three cell lines to two anticancer agents, cisplatin (0-100 µM) and etoposide (0-300 µM), which are known to induce cell death *in vitro* (Fang et al., 2004; Lee et al., 2006). As expected, the two cell lines that expressed EndoG, 22Rv1 and LNCaP, were highly sensitive to both chemotherapeutic agents. EndoG-deficient PC3 cells, in contrast, were insensitive to these drugs in the range of

Further study determined that cisplatin-induced death of prostate cancer cells can be prevented by EndoG silencing. Although EndoG is known to participate in cell death, it was necessary to determine whether the role of EndoG was the same in prostate cancer cells subjected to injury by cytotoxic agents as has been described in other cells. To test a causal relationship, EndoG was silenced in 22Rv1 cells by applying siRNA. To show that siRNA was delivered to the cells, fluorescent DY547-labeled siRNA was used. After DY547-siRNA transfection, 22Rv1 cells were exposed to 80 μM cisplatin, a concentration that had induced significant cell death in the above experiments. Next, TUNEL assay was conducted to measure DNA fragmentation. The assay showed the DNA fragmentation was decreased, indicating that silencing of EndoG leads to significant decrease of EndoG expression and protects cells from DNA fragmentation. As expected, EndoG silencing resulted in the increased viability of cisplatin-treated 22Rv1 cells as measured using clonogenic assay. The results suggest EndoG is responsible for cisplatin-induced death in

It is interesting that inhibition of DNA methylation induced EndoG and increased sensitivity of PC3 cells to cisplatin and etoposide. 5-aza-2'-deoxycytidine (decitabine), which is a DNA methylation inhibitor, caused hypomethylation of the EndoG promoter in PC3 cells, induced EndoG mRNA and protein expression, and made the cells sensitive to the chemotherapy agents. Using McrBC-PCR method, we determined that the treatment of PC3 cells with decitabine inhibited methylation of the CpG island in the EndoG gene. The same concentration of decitabine also increased EndoG expression as determined by real-time RT-PCR and Western blotting. These data clearly suggested EndoG expression is regulated by DNA methylation. Importantly, the induction of EndoG by demethylation caused a

Finally, the acetylation of histones by trichostatin A (TSA), a histone deacetylase inhibitor, induced EndoG expression in 22Rv1 cells, while it had no such effect in PC3 cells. These

significant increase in sensitivity to cisplatin and etoposide.

EndoG gene CpG island was hypermethylated (Wang et al., 2008).

concentrations used.

prostate cancer cells.

data indicated EndoG may be regulated by methylation of its gene promoter, and partially by histone acetylation, and that EndoG is essential for prostate cancer cell death in the used models. Histone modification, in particular, histone acetylation, is another epigenetic mechanism that is important in regulation of genes in prostate cancer (Das et al., 2006; Egger et al., 2004; Wang et al., 2008; Yoo & Jones, 2006). To determine whether and how histone acetylation regulates EndoG expression, two prostate cancer cell lines were treated with TSA, and EndoG protein expression was studied using Western blotting. The exposure of the cells to TSA induced high levels of EndoG expression in EndoG-positive 22Rv1 cells, whereas in EndoG-deficient PC3 cells, EndoG was not induced. Again, EndoG induction by TSA caused increased sensitivity to cisplatin. These data demonstrated that chromatin acetylation is important for EndoG expression. Taken together with the above methylation experiments, these data indicate that DNA methylation plays a primary role in EndoG regulation as compared to histone acetylation. In other words, the CpG island of the EndoG gene has to be hypomethylated in order to allow regulation of EndoG expression by histone acetylation.

## **4. Endonuclease delivery to prostate cancer cells and tumors**

Although these are attractive and potentially therapeutically useful approaches, modulations of endonuclease expression by DNA methylation or histone acetylation may not be a realistic approach because the specificity of epigenetic regulation is notoriously low. An alternative to these methods may be a gene delivery and overexpression in the target cancer cells. To determine whether overexpression of EndoG would make PC3 cells sensitive to the chemotherapy agents, the cells were transfected with human mature EndoG gene. To model chemotherapy in vitro, we used docetaxel, which is an FDAapproved the first line chemotherapeutic agent in castration-refractory prostate cancer (Oudard et al., 2007; Ryan et al., 2001). Despite survival benefits with docetaxel based chemotherapy, prognosis for castration-refractory prostate cancer patients usually is poor and patients typically show rapid progression (Oudard et al., 2005; Wang et al., 2008). Progressive prostate cancer is associated with the development, and subsequent expansion of tumor cells that are resistant to apoptotic triggers and dysregulation of apoptosis is often characterized by insufficient apoptosis (Kruslin, 2009; Mori et al., 1996). Therefore a delivery of EndoG gene was expected to increase sensitivity of prostate cancer cells to docetaxel.

As described below, this genetic manipulation resulted in significant increase of PC3 cells sensitivity to docetaxel and cisplatin in vitro. Similar results were obtained when PC3 cells were transfected with EndoG precursor gene suggesting that the drugs induce speedy processing of the protein to mature endonuclease.

PC3 cells were chosen because they have very low expression of EndoG (Wang et al., 2008). Human EndoG gene (NM 004435.2) was cloned in the mammalian expression vector pECFP.N1 to result in an expression of EndoG protein fused with the enhanced cyan fluorescence protein (CFP). The expression of the chimeric protein was confirmed by fluorescent microscopy. Cells were then treated with docetaxel and cell death was measured using lactate dehydrogenase (LDH) release assay. This experiment showed the sensitivity of the PC3 cells expressing EndoG-CFP to docetaxel was much higher than the cells expressing CFP alone (Figure 1). The same result was also observed in cisplatin-induced cells death:

Cytotoxic Endonucleases: New Targets for Prostate Cancer Chemotherapy 275

Fig. 2. EndoG expression facilitates docetaxel sensitivity of orthotopic PC3 xenograft tumors. Human prostate cancer PC3 cells were transfected with EndoG precursor gene. Parental EndoG-deficient cells or EndoG-expressing PC3 cells were implanted in ventral prostate. Docetaxel (10 mg/kg) was administrated at the 12th day after implantation. Tumor sizes were monitored by intravital ultrasound sonography using VisualSonics Vevo 770

instrument. Arrows indicate tumor edges.

EndoG overexpression resulted in an over 4-folds elevation of cisplatin-induced cell death (data not shown). We also have compared mature EndoG gene and precursor EndoG gene overexpression cytotoxicity and their effects on cisplatin-induced cell death, and found that both types of EndoG had familiar effect (data not shown).

Fig. 1. EndoG expression enhances prostate cancer cells' sensitivity to docetaxel in vitro. Left panel: Cell death measured by LDH release assay in EndoG-expressing 22Rv1 and EndoGnegative PC3 cells, which were exposed to varying concentrations of docetaxel for 24h (n=4, \*p<0.05). Right panel: Cell death measured by LDH release assay in PC3 cells with or without EndoG precursor overexpression 24 hrs after exposure to varying concentrations of docetaxel (n=4, \*p<0.05).

Finally, parental PC3 cells and PC3 cells overexpressing human EndoG precursor were implanted in prostates of SCID mice to produce orthotopic tumors. The animals with xenografts were subjected to the docetaxel chemotherapy and the tumor size progression was monitored by high frequency ultrasound visualization. This experiment showed that EndoG-expressing tumors shrink in response to chemotherapy, while control tumors made of EndoG-negative parental PC3 cells were chemoresistant. To produce orthotopic xenografts, 8-weeks old male SCID mice were injected with human prostate cancer PC3 cells or EndoG gene-transfected PC3 cells by surgical orthotopic implantation. 2x105 cells were mixed with matrigel at 1:1 ratio (v/v) in a total volume of 20μl were injected in the left ventral prostate lobes after surgical opening of the lower abdomen skin and peritoneal membrane. Ultrasound image could identify prostate tumor as early for 6 days after implantation. Monitoring of the tumor growth showed that the prostate lobe eventually was occupied by the tumor and lost its original shape. At the 12th day, the mice received docetaxel (10mg/kg) via peritoneal cavity injection while the control mice received saline injection. Ultrasound images were taken at the 6, 12, and 18 days after orthotopic implantation. By day 18, PC3 xenograft tumors significantly grew up regardless of the docetaxel treatment; EndoG-PC3 xenografts without docetaxel treatment grew up less, while the docetaxel-treated EndoG-PC3 xenografts did not grow in size and instead shrunk (Figure 2). Histology analysis confirmed the EndoG overexpression in tumors, which coincided with positive TUNEL staining, thus confirming EndoG overexpression made xenografts sensitive to the docetaxel treatment.

EndoG overexpression resulted in an over 4-folds elevation of cisplatin-induced cell death (data not shown). We also have compared mature EndoG gene and precursor EndoG gene overexpression cytotoxicity and their effects on cisplatin-induced cell death, and found that

Fig. 1. EndoG expression enhances prostate cancer cells' sensitivity to docetaxel in vitro. Left panel: Cell death measured by LDH release assay in EndoG-expressing 22Rv1 and EndoGnegative PC3 cells, which were exposed to varying concentrations of docetaxel for 24h (n=4, \*p<0.05). Right panel: Cell death measured by LDH release assay in PC3 cells with or without EndoG precursor overexpression 24 hrs after exposure to varying concentrations of

Finally, parental PC3 cells and PC3 cells overexpressing human EndoG precursor were implanted in prostates of SCID mice to produce orthotopic tumors. The animals with xenografts were subjected to the docetaxel chemotherapy and the tumor size progression was monitored by high frequency ultrasound visualization. This experiment showed that EndoG-expressing tumors shrink in response to chemotherapy, while control tumors made of EndoG-negative parental PC3 cells were chemoresistant. To produce orthotopic xenografts, 8-weeks old male SCID mice were injected with human prostate cancer PC3 cells or EndoG gene-transfected PC3 cells by surgical orthotopic implantation. 2x105 cells were mixed with matrigel at 1:1 ratio (v/v) in a total volume of 20μl were injected in the left ventral prostate lobes after surgical opening of the lower abdomen skin and peritoneal membrane. Ultrasound image could identify prostate tumor as early for 6 days after implantation. Monitoring of the tumor growth showed that the prostate lobe eventually was occupied by the tumor and lost its original shape. At the 12th day, the mice received docetaxel (10mg/kg) via peritoneal cavity injection while the control mice received saline injection. Ultrasound images were taken at the 6, 12, and 18 days after orthotopic implantation. By day 18, PC3 xenograft tumors significantly grew up regardless of the docetaxel treatment; EndoG-PC3 xenografts without docetaxel treatment grew up less, while the docetaxel-treated EndoG-PC3 xenografts did not grow in size and instead shrunk (Figure 2). Histology analysis confirmed the EndoG overexpression in tumors, which coincided with positive TUNEL staining, thus confirming EndoG overexpression made

both types of EndoG had familiar effect (data not shown).

docetaxel (n=4, \*p<0.05).

xenografts sensitive to the docetaxel treatment.

Fig. 2. EndoG expression facilitates docetaxel sensitivity of orthotopic PC3 xenograft tumors. Human prostate cancer PC3 cells were transfected with EndoG precursor gene. Parental EndoG-deficient cells or EndoG-expressing PC3 cells were implanted in ventral prostate. Docetaxel (10 mg/kg) was administrated at the 12th day after implantation. Tumor sizes were monitored by intravital ultrasound sonography using VisualSonics Vevo 770 instrument. Arrows indicate tumor edges.

Cytotoxic Endonucleases: New Targets for Prostate Cancer Chemotherapy 277

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## **5. Conclusive remarks**

Overall, our studies demonstrated that the expression and activity of the cytotoxic endonucleases are decreased in prostate cancer cells that are resistant to chemotherapy (Wang et al., 2008). This is consistent with previous studies of breast cancer, which also showed disappearance of DNase I in immortalized breast epithelial cells, and decrease of EndoG that coincided with dedifferentiation and invasiveness of breast cancer (Basnakian et al., 2006). EndoG is shown essential for prostate cancer cell death induced by chemotherapy. Expression of EndoG positively correlated with the sensitivity to chemotherapeutic agents cisplatin and etoposide, while the silencing of EndoG by siRNA in two cancer lines, 22Rv1 and LNCaP, decreased the sensitivity of the cells to the chemotherapeutic agents. In PC3 cell line, which does not express EndoG, the chemotherapeutic agent 5-aza-2'-deoxycytidine caused hypomethylation of the EndoG promoter, induced EndoG expression, and made the cells sensitive to both cisplatin and etoposide. In our latest studies described above, the overexpression of EndoG in PC3 cells made them also sensitive to docetaxel *in vitro* and *in vivo*. Therefore these studies demonstrate the first application of endonucleases as a helper drug for the chemotherapy of prostate cancer.

Because the mechanisms of chemo- and radiosensitivity of cells are very similar, these observations may be easily extrapolated to the radiotherapy of prostate cancer. Future studies may be necessary to determine the role of other epigenetic mechanisms in regulation of EndoG and their role in chemoresistance to prostate cancer and cancers of other organs. Chemotherapy is currently one of the frequently used therapeutic strategies for prostate cancer (Dyrstad et al., 2006; Kaku et al., 2006; Nakabayashi & Oh, 2006), and measurement of EndoG may be a potentially useful approach to evaluate chemosensitivity of cancer cells to determine optimal conditions for chemotherapy prior to the therapy.

If further *in vivo* studies confirm our observation that EndoG is a potential key mediator of prostate cancer cell death regulated by the methylation of *EndoG* gene promoter, future epigenetic therapeutics will need to be targeted to EndoG. A development of this approach may lead to similar therapeutic strategies for cancer of other organs.

Recent study determined that DNase I and EndoG, which represent most of DNase activity in prostate epithelial and many other cells and are linked in a single pathway, in which DNase I expression positively modulates EndoG expression (Yin et al., 2007). DNase I has the highest specific activity (per mg protein) among all known endonucleases, and it is the only endonuclease that can be directly incorporated into cells. The mechanisms of DNA destruction and the role in cell death are same between the two endonucleases. Therefore, it may not be necessary to deliver EndoG gene to prostate tumors and instead deliver DNase I protein packed in liposomes, which are attractive as vehicles because they have low toxicity. The only example of an endonuclease being applied for a therapy is human recombinant DNase I is used in complex therapy of cystic fibrosis. Future studies may lead to the first application of endonucleases as a helper drug for chemotherapy of prostate cancer.

#### **6. References**

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Overall, our studies demonstrated that the expression and activity of the cytotoxic endonucleases are decreased in prostate cancer cells that are resistant to chemotherapy (Wang et al., 2008). This is consistent with previous studies of breast cancer, which also showed disappearance of DNase I in immortalized breast epithelial cells, and decrease of EndoG that coincided with dedifferentiation and invasiveness of breast cancer (Basnakian et al., 2006). EndoG is shown essential for prostate cancer cell death induced by chemotherapy. Expression of EndoG positively correlated with the sensitivity to chemotherapeutic agents cisplatin and etoposide, while the silencing of EndoG by siRNA in two cancer lines, 22Rv1 and LNCaP, decreased the sensitivity of the cells to the chemotherapeutic agents. In PC3 cell line, which does not express EndoG, the chemotherapeutic agent 5-aza-2'-deoxycytidine caused hypomethylation of the EndoG promoter, induced EndoG expression, and made the cells sensitive to both cisplatin and etoposide. In our latest studies described above, the overexpression of EndoG in PC3 cells made them also sensitive to docetaxel *in vitro* and *in vivo*. Therefore these studies demonstrate the first application of endonucleases as a helper

Because the mechanisms of chemo- and radiosensitivity of cells are very similar, these observations may be easily extrapolated to the radiotherapy of prostate cancer. Future studies may be necessary to determine the role of other epigenetic mechanisms in regulation of EndoG and their role in chemoresistance to prostate cancer and cancers of other organs. Chemotherapy is currently one of the frequently used therapeutic strategies for prostate cancer (Dyrstad et al., 2006; Kaku et al., 2006; Nakabayashi & Oh, 2006), and measurement of EndoG may be a potentially useful approach to evaluate chemosensitivity of cancer cells

If further *in vivo* studies confirm our observation that EndoG is a potential key mediator of prostate cancer cell death regulated by the methylation of *EndoG* gene promoter, future epigenetic therapeutics will need to be targeted to EndoG. A development of this approach

Recent study determined that DNase I and EndoG, which represent most of DNase activity in prostate epithelial and many other cells and are linked in a single pathway, in which DNase I expression positively modulates EndoG expression (Yin et al., 2007). DNase I has the highest specific activity (per mg protein) among all known endonucleases, and it is the only endonuclease that can be directly incorporated into cells. The mechanisms of DNA destruction and the role in cell death are same between the two endonucleases. Therefore, it may not be necessary to deliver EndoG gene to prostate tumors and instead deliver DNase I protein packed in liposomes, which are attractive as vehicles because they have low toxicity. The only example of an endonuclease being applied for a therapy is human recombinant DNase I is used in complex therapy of cystic fibrosis. Future studies may lead to the first

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**5. Conclusive remarks** 

**6. References** 

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**13** 

*Spain* 

**MAP Kinases and Prostate Cancer** 

Ricardo Paniagua and Mar Royuela

Gonzalo Rodríguez-Berriguete, Benito Fraile, Laura Galvis,

*Department of Cell Biology and Genetics. University of Alcalá, Madrid* 

One of the most relevant aspects in cell death regulation is the signaling of apoptosis by serine/threonine kinases, a broad category of kinases that includes, among others, the mitogen-activated protein kinases (MAPKs) (Cross et al., 2000; Khlodenko & Birtwistle, 2009). The three main members that integrate the MAPK family in mammalian cells are: the stressactivated protein kinase c-Jun NH2-terminal kinases (JNK), the stress-activated protein kinase 2 (SAPK2, p38), and the extracellular signal-regulated protein kinases (ERK1/2, p44/p42) (Fig. 1). In addition, other less well-characterized MAPK pathways exist, such as the extracellular regulated kinase 5 (ERK5) pathway (Hayashi &Lee, 2004; Junttila & Li, 2008) (Fig. 1). Albeit with multiple exceptions, JNK and ERK5 are generally associated with apoptosis induction; while ERK1/2 are generally associated to mitogenesis, and therefore inversely related to apoptosis (Hayashi &Lee, 2004; Junttila & Li, 2008); and contradictory effects on cell death have been described to p38 (Chang et al., 2008; Joo & Yoo, 2009; Khwaja et al., 2008; Ricote et

ERK is a threonine-glutamic acid-tyrosine (Thr-Glu-Tyr) motif (Hunter, 2000; Liu et al., 2010) that play a central role in stimulation of cell proliferation (Marais & Marshall, 1990; Peng et al., 2010). Two isoforms of ERK, referred as ERK1 (or p44) and ERK2 (or p42), are ubiquitously expressed and represent a convergence point for mitogenic signaling from a diverse array of pathways (Cullen &Lockyer, 2002; Eisinger &Ammer, 2008; Gao et al., 2010). Both are ubiquitously expressed, although their relative abundance in tissues is variable. For example, in many immune cells ERK2 is the predominant species, while in several cells of neuroendocrine origin they may be equally expressed (Zebisch et al., 2007). ERK 1/2 is activated by MEK1/2 specifically by phosphorylating a tyrosine and a threonine residue, separated by a glutamate residue (TEY) (Zebisch et al., 2007). Activated ERK1 and ERK2 can translocate to the nucleus, where it activates several transcription factors such as ATF-2, Elk-1, c-Fos, c-myc or Ets-1 (Junttila & Li, 2008). At the same time, it can also phosphorylate cytoplasmic and nuclear kinases, such as MNK1, MNK2, MPKAP-2, RSK or MSK1 (Zebisch et al., 2007). The ERK1/2 cascade is triggered by growth factors and cytokines acting through receptor tyrosine kinases, G-protein-coupled receptors, and nonnuclear activated steroid hormone receptors. The biological consequences of ERK1/2 substrate phosphorylation include pro-proliferative (Pearson et al., 2001), pro-differentiation (Pearson et al., 2001), pro-survival (Pearson et al., 2001), pro-angiogenic (Pàges et al., 2000),

al., 2006a; Shimada et al., 2006; Vayalil et al., 2004; Zhang &Kong, 2008).

pro-motility (Joslin et al., 2007) and pro-invasive effects (Price et al., 2002).

**1. Introduction** 


## **MAP Kinases and Prostate Cancer**

Gonzalo Rodríguez-Berriguete, Benito Fraile, Laura Galvis, Ricardo Paniagua and Mar Royuela *Department of Cell Biology and Genetics. University of Alcalá, Madrid Spain* 

#### **1. Introduction**

282 Prostate Cancer – From Bench to Bedside

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One of the most relevant aspects in cell death regulation is the signaling of apoptosis by serine/threonine kinases, a broad category of kinases that includes, among others, the mitogen-activated protein kinases (MAPKs) (Cross et al., 2000; Khlodenko & Birtwistle, 2009). The three main members that integrate the MAPK family in mammalian cells are: the stressactivated protein kinase c-Jun NH2-terminal kinases (JNK), the stress-activated protein kinase 2 (SAPK2, p38), and the extracellular signal-regulated protein kinases (ERK1/2, p44/p42) (Fig. 1). In addition, other less well-characterized MAPK pathways exist, such as the extracellular regulated kinase 5 (ERK5) pathway (Hayashi &Lee, 2004; Junttila & Li, 2008) (Fig. 1). Albeit with multiple exceptions, JNK and ERK5 are generally associated with apoptosis induction; while ERK1/2 are generally associated to mitogenesis, and therefore inversely related to apoptosis (Hayashi &Lee, 2004; Junttila & Li, 2008); and contradictory effects on cell death have been described to p38 (Chang et al., 2008; Joo & Yoo, 2009; Khwaja et al., 2008; Ricote et al., 2006a; Shimada et al., 2006; Vayalil et al., 2004; Zhang &Kong, 2008).

ERK is a threonine-glutamic acid-tyrosine (Thr-Glu-Tyr) motif (Hunter, 2000; Liu et al., 2010) that play a central role in stimulation of cell proliferation (Marais & Marshall, 1990; Peng et al., 2010). Two isoforms of ERK, referred as ERK1 (or p44) and ERK2 (or p42), are ubiquitously expressed and represent a convergence point for mitogenic signaling from a diverse array of pathways (Cullen &Lockyer, 2002; Eisinger &Ammer, 2008; Gao et al., 2010). Both are ubiquitously expressed, although their relative abundance in tissues is variable. For example, in many immune cells ERK2 is the predominant species, while in several cells of neuroendocrine origin they may be equally expressed (Zebisch et al., 2007). ERK 1/2 is activated by MEK1/2 specifically by phosphorylating a tyrosine and a threonine residue, separated by a glutamate residue (TEY) (Zebisch et al., 2007). Activated ERK1 and ERK2 can translocate to the nucleus, where it activates several transcription factors such as ATF-2, Elk-1, c-Fos, c-myc or Ets-1 (Junttila & Li, 2008). At the same time, it can also phosphorylate cytoplasmic and nuclear kinases, such as MNK1, MNK2, MPKAP-2, RSK or MSK1 (Zebisch et al., 2007). The ERK1/2 cascade is triggered by growth factors and cytokines acting through receptor tyrosine kinases, G-protein-coupled receptors, and nonnuclear activated steroid hormone receptors. The biological consequences of ERK1/2 substrate phosphorylation include pro-proliferative (Pearson et al., 2001), pro-differentiation (Pearson et al., 2001), pro-survival (Pearson et al., 2001), pro-angiogenic (Pàges et al., 2000), pro-motility (Joslin et al., 2007) and pro-invasive effects (Price et al., 2002).

MAP Kinases and Prostate Cancer 285

JNK proteins, also called stress activated protein kinases (SAPKs), are activated in response to a variety of extracellular stimuli, including UV irradiation, mitogens and cytokines (De Graeve et al., 1999). Notably, the earliest discoveries included the identification of the three mammalian JNK genes called JNK1, JNK2, and JNK3 (also termed stress-activated protein kinase (SAPK)-γ, SAPK-α and SAPK-β, respectively) which can be subdivided into 10 isoforms by alternative splicing (Bogoyevitch et al., 2010; Dérijard et al., 1994). Alternative splicing further increases the diversity of JNK proteins, however apart from early biochemical studies on these splice forms (Gupta et al., 1996) their functional significance *in vivo* has remained largely unexplored (Bogoyevitch et al., 2010). The products of JNK1 and JNK2 are ubiquitously expressed in every cells and tissues, whereas JNK3 is localized primarily in brain, heart and testis. Due to the specificity of tissue, JNK3 presents different functions than JNK1 and JNK2. In addition, several authors believe that JNK1 and JNK2 present redundant functions. Several studies suggest that JNK are involved in regulation of the cell cycle (Bode & Dong, 2007.). JNK signaling contributes to the ability of p53 to mediate apoptosis through stabilization and activation of p53 (Bode & Dong, 2007; Fuchs et al., 1998). The fourth MAPK of interest in this review is ERK5. ERK5 is a large molecular size kinase (Lee et al., 1995) identified independently by two groups. One used a two hybrid screen with an upstream activator MEK5 as the bait; the other used a degenerate PCR strategy to clone novel MAPK (Lee et al., 1995; Zhou et al., 2005). ERK5 is activated by growth factors (Kato et al., 1998), integrin engagement (Sawhney et al., 2009) and cell stress (Pi et al., 2004), and its important molecular targets would seem to include the induction of transcription of components of the transcription factor Ap1 (cJun (Kayahama et al., 2005) and Fos (Kamakura et al., 1999) and activation of transcription factors of the myocyte enhancer family group (for example, MEF2C, a well characterized target (Kato et al., 1997)), and cMyc

In an *in vitro* study directed using androgen-dependent PC3 cells, McCracken et al. (2008) described ERK5-overexpresion related with proliferative, migrative and invasive capabilities, establishing the potential importance of ERK5 in aggressive prostate cancer. In similar studies Sawhney et al. (Sawhney et al., 2009) hypothesized that ERK5 activation

In mammalian cells, ERK, p38 and JNK activities are respectively regulated by three different MAPK cascades, which provide a link between transmembrane signaling and changes in transcription and are activated in response to different environmental or developmental signals (Junttila & Li, 2008) (Fig. 1). Depending on the cell type, a particular MAPK cascade may be involved in different cellular responses. The JNK and p38 signaling pathways are activated by pro-inflammatory (TNFα, IL-6 or IL-1) or anti-inflammatory (EGF, TGF-β) cytokines, but also in response to cellular stresses such as genotoxic, osmotic, hypoxic, or oxidative stress. The JNK pathway consists of JNK, a MAPKK such as SEK1 (also known as MEK4) or MEK7, and a MAPKKK such as ASK1, MEKK1, mixed-lineage kinase (MLK), or transforming growth factor-β-activated kinase 1 (TAK1) (Davis, 2000; Kim & Choi, 2010). In the p38 signaling pathway, distinct MAPKKs such as MEK3 and MEK6 activate p38 and these are activated by the same MAPKKKs (such as ASK1 and TAK1) that function in the JNK pathway. In the ERK signaling pathway, ERK1 or ERK2 (ERK1/2) is activated by MEK1/2, which in turn is activated by a Raf isoform such as A-Raf, B-Raf, or Raf-1 (also known as C-Raf) but also by TRAF-2 and TRAF-6. The kinase Raf-1 is activated by the small Ras-like GTPase, whose activation is mediated by the receptor tyrosine kinase

(English et al., 1998).

could promote cancer metastasis.

P38 plays roles in cell differentiation, growth inhibition and apoptosis, proliferation and cell survival (Hui et al., 2007; Raingeaud et al., 1995; Thornton & Rincon, 2009). p38 is activated in cells in response to stress signals, growth factors, inflammatory cytokines, UV, heat and osmotic shock (Raingeaud et al., 1995; Whyte et al., 2009). Four isoforms of p38 exist (p38α, β, γ and δ), although p38α is the most widely expressed. MKK3/6 (MAPKKK) and SEK (MAPKK) activate p38. A great number MAPKKs and MAPKKKs (e.g. Mlk1-3, MEKK1-4, TAK, ASK1/2) upstream of p38 have been identified. Both MAPKKs and MAPKKKs are generally activated by G small proteins as Rac1, Cdc42, RhoA and RhoB (Fenf et al., 2009). Activated p38 phosphorylates and regulates many transcription factors (including activating transcription factor-2, NF-kB, Elk-1, Max, myocyte enhancer factor-2, Mac, p53 or Stat1) (Royuela et al., 2008; Whyte et al., 2009; Zhao et al., 1999), and other cell cycle and apoptosis mediators (e.g. Cdc25A, Bcl-2) (Thornton & Rincon, 2009). p38 has been defined as tumor suppressor and generally exert a pro-apoptotic role. However, it has been also shown to enhance cell survival in response to stress stimuli, for instance, in response to DNA damage (Thornton& Rincon., 2009; Whyte et al., 2009; Jiang et al., 1997; Wang XS et al., 1997; Feng et al., 2009; Zhao et al., 1999; Royuela et al., 2008; Wood et al., 2009). Triggering of pro- or antiapoptotic p38-mediated response seems to depend on the stimuli, the cell system and the involved p38 isoform (Feng et al., 2009).

Fig. 1. Mitogen activated protein kinase (MAPK) signaling. MAP kinases are activated by upstream kinases such as MAP kinase kinase (MAPKK), that include MEKs 1, 2, 3, 4, 5, 6 and 7. In turn, MAPKKs are activated by several different MAP kinase kinase kinases (MAPKKKs). Numerous stimulatory factors such as cytokines, mitogens or death receptors, can activate MAPKKKs. Each MAPK, depending on the stimulus and cell type, can phosphorylate different transcription factors.

P38 plays roles in cell differentiation, growth inhibition and apoptosis, proliferation and cell survival (Hui et al., 2007; Raingeaud et al., 1995; Thornton & Rincon, 2009). p38 is activated in cells in response to stress signals, growth factors, inflammatory cytokines, UV, heat and osmotic shock (Raingeaud et al., 1995; Whyte et al., 2009). Four isoforms of p38 exist (p38α, β, γ and δ), although p38α is the most widely expressed. MKK3/6 (MAPKKK) and SEK (MAPKK) activate p38. A great number MAPKKs and MAPKKKs (e.g. Mlk1-3, MEKK1-4, TAK, ASK1/2) upstream of p38 have been identified. Both MAPKKs and MAPKKKs are generally activated by G small proteins as Rac1, Cdc42, RhoA and RhoB (Fenf et al., 2009). Activated p38 phosphorylates and regulates many transcription factors (including activating transcription factor-2, NF-kB, Elk-1, Max, myocyte enhancer factor-2, Mac, p53 or Stat1) (Royuela et al., 2008; Whyte et al., 2009; Zhao et al., 1999), and other cell cycle and apoptosis mediators (e.g. Cdc25A, Bcl-2) (Thornton & Rincon, 2009). p38 has been defined as tumor suppressor and generally exert a pro-apoptotic role. However, it has been also shown to enhance cell survival in response to stress stimuli, for instance, in response to DNA damage (Thornton& Rincon., 2009; Whyte et al., 2009; Jiang et al., 1997; Wang XS et al., 1997; Feng et al., 2009; Zhao et al., 1999; Royuela et al., 2008; Wood et al., 2009). Triggering of pro- or antiapoptotic p38-mediated response seems to depend on the stimuli, the cell system and the

Fig. 1. Mitogen activated protein kinase (MAPK) signaling. MAP kinases are activated by upstream kinases such as MAP kinase kinase (MAPKK), that include MEKs 1, 2, 3, 4, 5, 6 and 7. In turn, MAPKKs are activated by several different MAP kinase kinase kinases (MAPKKKs). Numerous stimulatory factors such as cytokines, mitogens or death receptors,

can activate MAPKKKs. Each MAPK, depending on the stimulus and cell type, can

involved p38 isoform (Feng et al., 2009).

phosphorylate different transcription factors.

JNK proteins, also called stress activated protein kinases (SAPKs), are activated in response to a variety of extracellular stimuli, including UV irradiation, mitogens and cytokines (De Graeve et al., 1999). Notably, the earliest discoveries included the identification of the three mammalian JNK genes called JNK1, JNK2, and JNK3 (also termed stress-activated protein kinase (SAPK)-γ, SAPK-α and SAPK-β, respectively) which can be subdivided into 10 isoforms by alternative splicing (Bogoyevitch et al., 2010; Dérijard et al., 1994). Alternative splicing further increases the diversity of JNK proteins, however apart from early biochemical studies on these splice forms (Gupta et al., 1996) their functional significance *in vivo* has remained largely unexplored (Bogoyevitch et al., 2010). The products of JNK1 and JNK2 are ubiquitously expressed in every cells and tissues, whereas JNK3 is localized primarily in brain, heart and testis. Due to the specificity of tissue, JNK3 presents different functions than JNK1 and JNK2. In addition, several authors believe that JNK1 and JNK2 present redundant functions. Several studies suggest that JNK are involved in regulation of the cell cycle (Bode & Dong, 2007.). JNK signaling contributes to the ability of p53 to mediate apoptosis through stabilization and activation of p53 (Bode & Dong, 2007; Fuchs et al., 1998). The fourth MAPK of interest in this review is ERK5. ERK5 is a large molecular size kinase (Lee et al., 1995) identified independently by two groups. One used a two hybrid screen with an upstream activator MEK5 as the bait; the other used a degenerate PCR strategy to clone novel MAPK (Lee et al., 1995; Zhou et al., 2005). ERK5 is activated by growth factors (Kato et al., 1998), integrin engagement (Sawhney et al., 2009) and cell stress (Pi et al., 2004), and its important molecular targets would seem to include the induction of transcription of components of the transcription factor Ap1 (cJun (Kayahama et al., 2005) and Fos (Kamakura et al., 1999) and activation of transcription factors of the myocyte enhancer family group (for example, MEF2C, a well characterized target (Kato et al., 1997)), and cMyc (English et al., 1998).

In an *in vitro* study directed using androgen-dependent PC3 cells, McCracken et al. (2008) described ERK5-overexpresion related with proliferative, migrative and invasive capabilities, establishing the potential importance of ERK5 in aggressive prostate cancer. In similar studies Sawhney et al. (Sawhney et al., 2009) hypothesized that ERK5 activation could promote cancer metastasis.

In mammalian cells, ERK, p38 and JNK activities are respectively regulated by three different MAPK cascades, which provide a link between transmembrane signaling and changes in transcription and are activated in response to different environmental or developmental signals (Junttila & Li, 2008) (Fig. 1). Depending on the cell type, a particular MAPK cascade may be involved in different cellular responses. The JNK and p38 signaling pathways are activated by pro-inflammatory (TNFα, IL-6 or IL-1) or anti-inflammatory (EGF, TGF-β) cytokines, but also in response to cellular stresses such as genotoxic, osmotic, hypoxic, or oxidative stress. The JNK pathway consists of JNK, a MAPKK such as SEK1 (also known as MEK4) or MEK7, and a MAPKKK such as ASK1, MEKK1, mixed-lineage kinase (MLK), or transforming growth factor-β-activated kinase 1 (TAK1) (Davis, 2000; Kim & Choi, 2010). In the p38 signaling pathway, distinct MAPKKs such as MEK3 and MEK6 activate p38 and these are activated by the same MAPKKKs (such as ASK1 and TAK1) that function in the JNK pathway. In the ERK signaling pathway, ERK1 or ERK2 (ERK1/2) is activated by MEK1/2, which in turn is activated by a Raf isoform such as A-Raf, B-Raf, or Raf-1 (also known as C-Raf) but also by TRAF-2 and TRAF-6. The kinase Raf-1 is activated by the small Ras-like GTPase, whose activation is mediated by the receptor tyrosine kinase

MAP Kinases and Prostate Cancer 287

O'Hayer, 2008). Raf-1 activation might stimulate two different pathways. One pathway is initiated by MEK1/2 and the other with the activation of JNK. In prostate cancer expression of to Raf-1, MEK-1 and p-MEK were increased with Gleason grade (Rodruguez et al., 2010a). TNF-α is a 17 kDa polypeptide that has been implicated in skin carcinogenesis and in metastatic tumor spread of a variety of carcinomas and sarcomas. The action of TNF- α is mediated by two distinct receptors named TNF-receptor I (55 kDa, TNFRI) and receptor II (75 kDa, TNFRII) with similar affinity for TNF- α in human tissues (Loetscher et al., 1990; Smith et al., 1990). The domains of these receptors are different (Tartaglia et al., 1991). TNFRI is the major mediator of most TNF-α activity (Wiegmann et al., 1992). The expression and action of TNF- α and its receptors has been reported in several tumors such as esophageal (Hubel et al., 2000), prostate (De Miguel et al., 2000; Meshki et al., 2010), follicular thyroid (Zubelewicz et al., 2002), skin (Arnott et al., 2004), ovarian (Qiu et al., 2010;

In human prostate cancer, TNF cascade seems to be over-stimulated since TNF receptors (TNFRI and TNFRII) present high immunoexpression (Ricote et al., 2003). Binding of TNFα/TNFRI complex to TNF receptor associated death domain proteins (TRADD) activates TRAF-2 (1 of the 6 members of the TNF receptor associated factor), which represents an integration point for pro-apoptotic and antiapoptotic signals (Wajant & Scheurich, 2001). TRAF-2 activation might stimulate two different pathways. One pathway is initiated by the interaction of TRAF-2 with the activation of NF-kB inducing kinase termed NIK, which is a MAP3K-related kinase that activates the IKK complex composed of IKK- and IKK- (Wu & Kral, 2005). In prostate cancer, NIK seems to be triggered by TNF/TRAF-2 or IL-1/IRAK/TRAF-6, since the presence of TNF, TNFRI and TRAF-2 has been described (De Miguel et al., 2000; Ricote et al., 2003), but also the presence of IL-1 family members (Nuñez et al., 2008; Ricote et al., 2004). NIK stimulate IKK-, which induces IKK- degradation. IKK complex phosphorylates IkB, following its ubiquitination and rapid degradation causing the nuclear translocation of NF-kB, which in turn, activates target genes involved in carcinogenesis: tumor initiation, malignant transformation and metastasis (Wu & Kral., 2005; Chengedza & Benbrook, 2010). In PC, TRAF-2 might be involved in the NIK activation pathway, although immunoexpression to TRAF-2 was detected in a low number of cases (decrease with Gleason grade), at the same time that the most of these patients were positives to NF-kB/p50 and NF-kB/p65 (Nuñez et al., 2008). These data, in addition with the elevated immunoexpressions to IL-1, IRAK, TRAF-6 and NIK observed in the same samples, suggest that NIK is stimulated by IL-1. Using the prostate carcinoma cell lines LNCaP, DU45 and PC3, Gasparian et al. (2009) found that increased IKK activation leads to the activation of NF-kB. A potential role of NF-kB in the development of different tumors as breast (Miller et al., 2000; Wu & Kral, 2005), colon (Dejardin et al., 1999; Wang S et al., 2009), pancreas (Wang W et al 1999; Eldor et al., 2009), thyroid (Visconti et al., 1997) or prostate

Rzymski et al., 2005) and breast (García-Tuñon et al., 2006) cancers.

(Nuñez et al., 2008; Domingo-Domenech et al., 2005) have been reported.

The other pathway activates the cascade ASK-1 (signal regulating kinase), MEK-4 (mitogen activated protein kinase-kinase 4) and Jun N-terminal kinase (JNK) (Royuela et al., 2008). When JNK is translocated to the nucleus is phosphorylated and activates transcription factors such as AP-1 or ATF-2. In all normal human prostates, positive immunoreactions to TRAF-2 and ASK1 (cytoplasm localization) MEK-4 (cytoplasm and nucleus localization) and JNK were found. Although in prostate cancer the transduction pathway from TRAF-2 to AP-1 seems to be inanctive, since immunoreaction to TRAF-2, ASK-1 And MEK-4 decreased and

(RTK)-Grb2-SOS signaling axis (Dhillon et al., 2007). Members of the Ras family of proteins, including K-Ras, H-Ras, and N-Ras, play a key role in transmission of extracellular signals into cells (Ancrile et al., 2008) (Fig. 1).

The aim of this review was to focus on the possible involvement of MAPKs in several transduction pathways related with prostate cancer development as well as the possible functional role of MAPKs in cell death/ survival/ proliferation decisions depending on the cell type, stage and cell stimulus. We also discuss the possible use of some members of this pathway as a potential therapeutic target.

## **2. IL-6/TNF/JNK pathway**

Depending on the stimulus and cell type, JNKs can phosphorylate different substrates such as Ap1, ATF-2, Elk-1, c-Myc, p53, MLK2 and several members of the Bcl-2 family. JNKs are implicated in development, morphogenesis and cell differentiation (Heasley & Han, 2006). Several studies suggest that in apoptosis JNKs have opposite functions depending on the cellular stimulus. In this way, JNKs can induce apoptosis, but also can enhance cell survival and proliferation. JNKs are also involved in regulation of the cell cycle (Bode & Dong, 2007). JNK signaling contributes to the ability of p53 to mediate apoptosis through stabilization and activation of p53 (Bode & Dong, 2007; Fuchs et al., 1998). Several authors suggest that JNK activity is chronically altered in various cancer types such as prostate (Meshki et al., 2010; Royuela et al., 2002), breast (Wang HY et al., 2003; Wang J et al., 2010), pancreatic or lung (Lee et al., 2010; Su et al., 1998) carcinomas.

Investigations of JNKs have focused on their activation in response to diverse stresses including ultraviolet and gamma radiation, inflammatory cytokines and cytotoxic drugs. In this way, pro inflammatory cytokines such as IL-6 or TNF activate different transduction pathway (Khalaf et al., 2010).

IL-6 exerts its effects through a membrane receptor complex composed by IL-6 receptor a (IL-6Ra) and glycoprotein 130 (gp130). Silver and Hunter (Silver & Hunter, 2010) described the role of gp130 in promoting or preventing the development of autoimmunity and cancer, two processes that are associated with aberrant inflammatory responses. In addition to an immunological role, IL-6 is involved in cell proliferation in other tissues such as bone (Kurihara et al., 1990), testis (spermatogenesis) (Huleihel & Lunenfeld, 2004), skin (Krueguer et al., 1990) or nervous system (Hama et al., 1989). It has been shown that IL-6 also stimulates the development of many tumors, including melanoma, renal cell carcinoma, Kaposi's sarcoma, ovarian carcinoma, lymphoma and leukemia, multiple myeloma, prostate carcinoma and breast carcinoma (García-Tuñon et al., 2005; Hong et al., 2007; Rabinovich et al., 2007; Royuela et al., 2004).

First, IL-6 binds to IL-6Ra, which is unable to initiate signal transduction, and this complex attracts gp130 molecules, which dimerize leading to the intracellular signal by the activation of constituvely-associated gp130 Jak proteins (Heinrich et al., 1998; Hong et al., 2007; Silver & Hunter, 2010). In PC (prostate cancer) immunoreaction to IL-6 and gp-130 were increased. IL-6 signalling could be enhanced not only due to increased autocrine production but also increasing levels of this receptor (Rodriguez-Berriguete et al., 2010a; Royuela et al., 2004). Jak proteins can simultaneously trigger functionally distinct and even contradictory signaling pathways. One of them leads to the recruitment at the complex receptor of SHP2, Sos and Grb2, which in turn activates Ras by stimulating the exchange of GDP bound to Ras for GTP. Then, Ras initiates a MAPK cascade by phosphorylation of Raf-1 (Ancrile &

(RTK)-Grb2-SOS signaling axis (Dhillon et al., 2007). Members of the Ras family of proteins, including K-Ras, H-Ras, and N-Ras, play a key role in transmission of extracellular signals

The aim of this review was to focus on the possible involvement of MAPKs in several transduction pathways related with prostate cancer development as well as the possible functional role of MAPKs in cell death/ survival/ proliferation decisions depending on the cell type, stage and cell stimulus. We also discuss the possible use of some members of this

Depending on the stimulus and cell type, JNKs can phosphorylate different substrates such as Ap1, ATF-2, Elk-1, c-Myc, p53, MLK2 and several members of the Bcl-2 family. JNKs are implicated in development, morphogenesis and cell differentiation (Heasley & Han, 2006). Several studies suggest that in apoptosis JNKs have opposite functions depending on the cellular stimulus. In this way, JNKs can induce apoptosis, but also can enhance cell survival and proliferation. JNKs are also involved in regulation of the cell cycle (Bode & Dong, 2007). JNK signaling contributes to the ability of p53 to mediate apoptosis through stabilization and activation of p53 (Bode & Dong, 2007; Fuchs et al., 1998). Several authors suggest that JNK activity is chronically altered in various cancer types such as prostate (Meshki et al., 2010; Royuela et al., 2002), breast (Wang HY et al., 2003; Wang J et al., 2010), pancreatic or

Investigations of JNKs have focused on their activation in response to diverse stresses including ultraviolet and gamma radiation, inflammatory cytokines and cytotoxic drugs. In this way, pro inflammatory cytokines such as IL-6 or TNF activate different transduction

IL-6 exerts its effects through a membrane receptor complex composed by IL-6 receptor a (IL-6Ra) and glycoprotein 130 (gp130). Silver and Hunter (Silver & Hunter, 2010) described the role of gp130 in promoting or preventing the development of autoimmunity and cancer, two processes that are associated with aberrant inflammatory responses. In addition to an immunological role, IL-6 is involved in cell proliferation in other tissues such as bone (Kurihara et al., 1990), testis (spermatogenesis) (Huleihel & Lunenfeld, 2004), skin (Krueguer et al., 1990) or nervous system (Hama et al., 1989). It has been shown that IL-6 also stimulates the development of many tumors, including melanoma, renal cell carcinoma, Kaposi's sarcoma, ovarian carcinoma, lymphoma and leukemia, multiple myeloma, prostate carcinoma and breast carcinoma (García-Tuñon et al., 2005; Hong et al., 2007; Rabinovich et

First, IL-6 binds to IL-6Ra, which is unable to initiate signal transduction, and this complex attracts gp130 molecules, which dimerize leading to the intracellular signal by the activation of constituvely-associated gp130 Jak proteins (Heinrich et al., 1998; Hong et al., 2007; Silver & Hunter, 2010). In PC (prostate cancer) immunoreaction to IL-6 and gp-130 were increased. IL-6 signalling could be enhanced not only due to increased autocrine production but also increasing levels of this receptor (Rodriguez-Berriguete et al., 2010a; Royuela et al., 2004). Jak proteins can simultaneously trigger functionally distinct and even contradictory signaling pathways. One of them leads to the recruitment at the complex receptor of SHP2, Sos and Grb2, which in turn activates Ras by stimulating the exchange of GDP bound to Ras for GTP. Then, Ras initiates a MAPK cascade by phosphorylation of Raf-1 (Ancrile &

into cells (Ancrile et al., 2008) (Fig. 1).

pathway as a potential therapeutic target.

lung (Lee et al., 2010; Su et al., 1998) carcinomas.

**2. IL-6/TNF/JNK pathway** 

pathway (Khalaf et al., 2010).

al., 2007; Royuela et al., 2004).

O'Hayer, 2008). Raf-1 activation might stimulate two different pathways. One pathway is initiated by MEK1/2 and the other with the activation of JNK. In prostate cancer expression of to Raf-1, MEK-1 and p-MEK were increased with Gleason grade (Rodruguez et al., 2010a). TNF-α is a 17 kDa polypeptide that has been implicated in skin carcinogenesis and in metastatic tumor spread of a variety of carcinomas and sarcomas. The action of TNF- α is mediated by two distinct receptors named TNF-receptor I (55 kDa, TNFRI) and receptor II (75 kDa, TNFRII) with similar affinity for TNF- α in human tissues (Loetscher et al., 1990; Smith et al., 1990). The domains of these receptors are different (Tartaglia et al., 1991). TNFRI is the major mediator of most TNF-α activity (Wiegmann et al., 1992). The expression and action of TNF- α and its receptors has been reported in several tumors such as esophageal (Hubel et al., 2000), prostate (De Miguel et al., 2000; Meshki et al., 2010), follicular thyroid (Zubelewicz et al., 2002), skin (Arnott et al., 2004), ovarian (Qiu et al., 2010; Rzymski et al., 2005) and breast (García-Tuñon et al., 2006) cancers.

In human prostate cancer, TNF cascade seems to be over-stimulated since TNF receptors (TNFRI and TNFRII) present high immunoexpression (Ricote et al., 2003). Binding of TNFα/TNFRI complex to TNF receptor associated death domain proteins (TRADD) activates TRAF-2 (1 of the 6 members of the TNF receptor associated factor), which represents an integration point for pro-apoptotic and antiapoptotic signals (Wajant & Scheurich, 2001). TRAF-2 activation might stimulate two different pathways. One pathway is initiated by the interaction of TRAF-2 with the activation of NF-kB inducing kinase termed NIK, which is a MAP3K-related kinase that activates the IKK complex composed of IKK- and IKK- (Wu & Kral, 2005). In prostate cancer, NIK seems to be triggered by TNF/TRAF-2 or IL-1/IRAK/TRAF-6, since the presence of TNF, TNFRI and TRAF-2 has been described (De Miguel et al., 2000; Ricote et al., 2003), but also the presence of IL-1 family members (Nuñez et al., 2008; Ricote et al., 2004). NIK stimulate IKK-, which induces IKK- degradation. IKK complex phosphorylates IkB, following its ubiquitination and rapid degradation causing the nuclear translocation of NF-kB, which in turn, activates target genes involved in carcinogenesis: tumor initiation, malignant transformation and metastasis (Wu & Kral., 2005; Chengedza & Benbrook, 2010). In PC, TRAF-2 might be involved in the NIK activation pathway, although immunoexpression to TRAF-2 was detected in a low number of cases (decrease with Gleason grade), at the same time that the most of these patients were positives to NF-kB/p50 and NF-kB/p65 (Nuñez et al., 2008). These data, in addition with the elevated immunoexpressions to IL-1, IRAK, TRAF-6 and NIK observed in the same samples, suggest that NIK is stimulated by IL-1. Using the prostate carcinoma cell lines LNCaP, DU45 and PC3, Gasparian et al. (2009) found that increased IKK activation leads to the activation of NF-kB. A potential role of NF-kB in the development of different tumors as breast (Miller et al., 2000; Wu & Kral, 2005), colon (Dejardin et al., 1999; Wang S et al., 2009), pancreas (Wang W et al 1999; Eldor et al., 2009), thyroid (Visconti et al., 1997) or prostate (Nuñez et al., 2008; Domingo-Domenech et al., 2005) have been reported.

The other pathway activates the cascade ASK-1 (signal regulating kinase), MEK-4 (mitogen activated protein kinase-kinase 4) and Jun N-terminal kinase (JNK) (Royuela et al., 2008). When JNK is translocated to the nucleus is phosphorylated and activates transcription factors such as AP-1 or ATF-2. In all normal human prostates, positive immunoreactions to TRAF-2 and ASK1 (cytoplasm localization) MEK-4 (cytoplasm and nucleus localization) and JNK were found. Although in prostate cancer the transduction pathway from TRAF-2 to AP-1 seems to be inanctive, since immunoreaction to TRAF-2, ASK-1 And MEK-4 decreased and

MAP Kinases and Prostate Cancer 289

levels of cytokines and JNK in these patients but to other factors including the increased p38 levels mentioned above. Therefore, the most probable action of JNK in PC would be cell

Several studies suggested that p38 play an important role in leukemia (Feng et al., 2009); lymphomas (Zheng et al., 2003) or tumor such as breast (Ancrile et al., 2008), prostate (Ricote

Fig. 3. p38 immunostaining appeared in normal (A), BPH (B) and PC (B) samples. Scale bars:

In addition to TNF/AP1 pathway (by ASK-1 or MEK-4), Interleukin-1 (IL-1) is another physiological regulator of p38. IL-1 activates PAK-1 through its binding to two GTPases, called Cdc42 and Rac. These ones activate PAK-1, which induces MEK-6 activation that in

Several reports about IL-1 family in cancer have been reported. IL-1, IL-1 and IL-1Ra have been detected in human breast cancer, and have been related to protumorigenic activity (Miller et al., 2010). The number of men showing IL-1 immunoexpression is lower in prostate cancer group than in normal prostate group but most cancer patients studied presented immunoreaction to IL-1, IL-1RI, IL-1RII and IL-1Ra [60]. The interaction between IL-1 and IL-RI would be involved in the high proliferation degree of these tumors. No association between IL-1 and IL-1Ra has also been reported in premalignant gastric

In human prostate cancer, intense immnoreaction to PAK-1, MEK-6 and p38 were found but also to p-Elk-1 and p-ATF-2 whose location change from the nucleus to the cytoplasm (Ricote et al., 2006a; Rodriguez-Berriguete et al., 2010b). This fact may be related with its biological function. In mammalian cells, endogenous p38 is present in the nucleus but it can be exported to the cytoplasm upon activation (Ricote et al., 2006). Recently, Wood et al. (2009) described nuclear localization of p38 in response to DNA damage. In the nucleus, p38 phosphorylates Elk-1, ATF-2 and also NF-kB (Junttila et al., 2008; Raingeaud et al., 1995; Royuela et al., 2008). ATF-2 (Li & Wicks, 2001) and Elk-1 (Amorino & Parsons, 2004) are not only a target of p38 but also a target for JNK. Since immunoreaction to JNK was found in normal human prostate, but not in prostate cancer, is reasonable to suggest that the activation of ATF-2 and Elk-1 are the consequence of p38 pathway activation (Ricote et al.,

proliferation stimulation rather than apoptosis.

et al., 2006a), gastrical (Guo et al., 2008) or lung (Zhang et al., 2010).

**3. IL-1/ TNF/ p38** 

25 m (B) 30 m (A, C).

turns activates p38 (Raingeaud et al., 1995).

conditions (Kupcinskas et al., 2010).

no immunoreaction to AP-1 was even found (Ricote et al., 2003; Royuela et al., 2008). The mechanism that accounts for the nuclear location MEK-4 is unclear, since this protein is activated by a cytoplasmic protein and phosphorylates JNK in the cytoplasm. However, MEK-4 function may not be restricted to the JNK signal transduction pathway because MEK-4 also phosphorylate and activates p38, and this latter is prelocalized in the nucleus and is rapidly exported to the cytoplasm upon activation (Taylor et al., 2008).

Fig. 2. JNK immunostaining appeared in normal (A), BPH (B) and PC (B) samples. Scale bars: 20 m (A-B) and 30 m (C).

JNK immunoreactiveness is increased in the glandular epithelium of PC specimens (Royuela et al., 2002; Shimada et al., 2006). With these data, Ricote et al. (2003) suggest that MEK-4 is not involved in JNK/AP-1 pathway, although it might be involved in p38 activation pathway. This hypothesis agrees with the high p38 levels found in normal prostate in our laboratory (Royuela et al., 2002). In this pathology there must be several extracellular or intracellular factors that are blocking the activation of this transduction pathway in different steps. ASK1 might be a critical blockage point of this transduction pathway. P21 has been reported as an ASK1 inhibitor and has been found significantly associated with a high Gleason score (Aaltomaa et al., 1999; Royuela et al., 2001). Bcl-2 has been postulated as a potential modulator of JNK activation in fibroblasts. Since an increase of bcl-2 has been reported in prostate cancer specimens, bcl-2 might be another potential inhibitor of JNK in prostate cancer (Haeusgen et al., 2010; Royuela et al., 2000). Ricote et al. (2006) reported in an *in vitro* study that JNK phosphorylation was found to be increased by TNF- dosedependent manner in LNCaP cells (but not in PC3 cells), and the rate of apoptosis was reduced by the administration of a specific JNK inhibitor, suggesting that JNK positively regulates apoptosis induction by TNF- in this cell model.

Two opposite roles in the cell cycle control have been reported for JNK: cell proliferation and apoptosis. In contrast, JNK activation by some cytokines, such as TNF- and IL-6, stimulates apoptosis. Since these two cytokines have been found increased in the prostatic epithelium of PC patients (Rodriguez-Berriguete et al., 2010a; Royuela et al., 2008), it might be that the increased apoptotic indexes in PC are related to the elevated levels of TNF-, IL-6 and JNK. Nevertheless, the apoptotic mechanism stimulated by JNK is via p53 (Fuchs et al., 1998), but the p53 present in PC patients (Lee y cols., 2008), as occurs in most cancers is a mutant form with deletions or mutations which obstruct its association to JNK (Fuchs et al., 1998). Therefore, the elevated apoptotic rates in PC does not seem to be related to the high levels of cytokines and JNK in these patients but to other factors including the increased p38 levels mentioned above. Therefore, the most probable action of JNK in PC would be cell proliferation stimulation rather than apoptosis.

## **3. IL-1/ TNF/ p38**

288 Prostate Cancer – From Bench to Bedside

no immunoreaction to AP-1 was even found (Ricote et al., 2003; Royuela et al., 2008). The mechanism that accounts for the nuclear location MEK-4 is unclear, since this protein is activated by a cytoplasmic protein and phosphorylates JNK in the cytoplasm. However, MEK-4 function may not be restricted to the JNK signal transduction pathway because MEK-4 also phosphorylate and activates p38, and this latter is prelocalized in the nucleus

and is rapidly exported to the cytoplasm upon activation (Taylor et al., 2008).

Fig. 2. JNK immunostaining appeared in normal (A), BPH (B) and PC (B) samples. Scale

JNK immunoreactiveness is increased in the glandular epithelium of PC specimens (Royuela et al., 2002; Shimada et al., 2006). With these data, Ricote et al. (2003) suggest that MEK-4 is not involved in JNK/AP-1 pathway, although it might be involved in p38 activation pathway. This hypothesis agrees with the high p38 levels found in normal prostate in our laboratory (Royuela et al., 2002). In this pathology there must be several extracellular or intracellular factors that are blocking the activation of this transduction pathway in different steps. ASK1 might be a critical blockage point of this transduction pathway. P21 has been reported as an ASK1 inhibitor and has been found significantly associated with a high Gleason score (Aaltomaa et al., 1999; Royuela et al., 2001). Bcl-2 has been postulated as a potential modulator of JNK activation in fibroblasts. Since an increase of bcl-2 has been reported in prostate cancer specimens, bcl-2 might be another potential inhibitor of JNK in prostate cancer (Haeusgen et al., 2010; Royuela et al., 2000). Ricote et al. (2006) reported in an *in vitro* study that JNK phosphorylation was found to be increased by TNF- dosedependent manner in LNCaP cells (but not in PC3 cells), and the rate of apoptosis was reduced by the administration of a specific JNK inhibitor, suggesting that JNK positively

Two opposite roles in the cell cycle control have been reported for JNK: cell proliferation and apoptosis. In contrast, JNK activation by some cytokines, such as TNF- and IL-6, stimulates apoptosis. Since these two cytokines have been found increased in the prostatic epithelium of PC patients (Rodriguez-Berriguete et al., 2010a; Royuela et al., 2008), it might be that the increased apoptotic indexes in PC are related to the elevated levels of TNF-, IL-6 and JNK. Nevertheless, the apoptotic mechanism stimulated by JNK is via p53 (Fuchs et al., 1998), but the p53 present in PC patients (Lee y cols., 2008), as occurs in most cancers is a mutant form with deletions or mutations which obstruct its association to JNK (Fuchs et al., 1998). Therefore, the elevated apoptotic rates in PC does not seem to be related to the high

bars: 20 m (A-B) and 30 m (C).

regulates apoptosis induction by TNF- in this cell model.

Several studies suggested that p38 play an important role in leukemia (Feng et al., 2009); lymphomas (Zheng et al., 2003) or tumor such as breast (Ancrile et al., 2008), prostate (Ricote et al., 2006a), gastrical (Guo et al., 2008) or lung (Zhang et al., 2010).

Fig. 3. p38 immunostaining appeared in normal (A), BPH (B) and PC (B) samples. Scale bars: 25 m (B) 30 m (A, C).

In addition to TNF/AP1 pathway (by ASK-1 or MEK-4), Interleukin-1 (IL-1) is another physiological regulator of p38. IL-1 activates PAK-1 through its binding to two GTPases, called Cdc42 and Rac. These ones activate PAK-1, which induces MEK-6 activation that in turns activates p38 (Raingeaud et al., 1995).

Several reports about IL-1 family in cancer have been reported. IL-1, IL-1 and IL-1Ra have been detected in human breast cancer, and have been related to protumorigenic activity (Miller et al., 2010). The number of men showing IL-1 immunoexpression is lower in prostate cancer group than in normal prostate group but most cancer patients studied presented immunoreaction to IL-1, IL-1RI, IL-1RII and IL-1Ra [60]. The interaction between IL-1 and IL-RI would be involved in the high proliferation degree of these tumors. No association between IL-1 and IL-1Ra has also been reported in premalignant gastric conditions (Kupcinskas et al., 2010).

In human prostate cancer, intense immnoreaction to PAK-1, MEK-6 and p38 were found but also to p-Elk-1 and p-ATF-2 whose location change from the nucleus to the cytoplasm (Ricote et al., 2006a; Rodriguez-Berriguete et al., 2010b). This fact may be related with its biological function. In mammalian cells, endogenous p38 is present in the nucleus but it can be exported to the cytoplasm upon activation (Ricote et al., 2006). Recently, Wood et al. (2009) described nuclear localization of p38 in response to DNA damage. In the nucleus, p38 phosphorylates Elk-1, ATF-2 and also NF-kB (Junttila et al., 2008; Raingeaud et al., 1995; Royuela et al., 2008). ATF-2 (Li & Wicks, 2001) and Elk-1 (Amorino & Parsons, 2004) are not only a target of p38 but also a target for JNK. Since immunoreaction to JNK was found in normal human prostate, but not in prostate cancer, is reasonable to suggest that the activation of ATF-2 and Elk-1 are the consequence of p38 pathway activation (Ricote et al.,

MAP Kinases and Prostate Cancer 291

GDP bound to Ras for GTP (Silver & Hunter, 2010). Then, Ras phosphorylate of Raf-1. In this way is iniciate a MAPK cascade when Raf-1 (via IL-6 pathway), TRAF-2 (via TNF pathway) or TRAF-6 (via Il-6 pathway) phosphorylate sequentially MEK1/2 and ERK1/2, in a process that culminates in modulation of gene transcription through the activation of several transcription factors such as c-Myc, Elk-1 (Werlen et al., 2003) or NF-kB (Turjanski et

Fig. 5. P50 was scantly in the cytoplasm epithelial cells of normal (A) but in PC

Scale bars: 20 m (A), 25 m (B-C, E-F) and 30 m (D).

hematological malignaces (Grant, 2008; McCubrey et al., 2007).

immunostaining also was nuclear, increasing the expression in medium (B) and after, in high (C) Gleason. No immunoreaction was found to p65 in normal prostate but was

localized in the cytoplasm of epithelial cells in BPH (D) and PC (E-F) samples; but in PC was also localized in the nuclei of epithelial increasing nuclear localization with Gleason grade.

Some components of the Raf-MEK-ERK pathway are activated in solid tumors and

In approximately 30% of human breast cancers, mutations are found in the ERK1/2 MAPK pathway (Whyte et al., 2009). ERK1/2 and downstream ERK1/2 targets are hyperphosphorylated in a large subset of mammary tumors (Mueller et al., 2000). Increased expressions of Raf pathway has been associated with advance prostate cancer, hormonal independence, metastasis and a poor prognosis (Keller et al., 2004). Moreover, prostate cancer cell lines isolated from advanced cancer patients (LNCaP, PC3, DU145) expressed low levels of active Raf kinase inhibitors (McCubrey et al., 2007). TNFα acts as an ERK activator in some cases related to inflammation and cell proliferation. In this way, Ricote et al. (2006b) showed that ERK phosphorylation was notably increased by TNFα dose dependent manner in LNCaP cells. In prostate cancer, presence of Raf-1 and MEK-1 in

al., 2007).

Fig. 4. Nuclear immunoreaction to p-Elk-1 appeared in the epithelial basal cells of normal prostate (A) or all epithelial cells in BPH (B). In PC samples p-Elk-1 (C) was observed in the cytoplasm. p-ATF-2 immunostaining was localised in the nuclei of epithelial cells in normal prostate (D) but more intense in BPH (E) and PC (F). Scale bar: 20 m (B, E) and 25 m (A, C-D, F).

2006a). However, the TNF-α signal may be diverted from the Ap-1 pathway towards the p38 pathway, because MEK-4 may also phosphorylate and activate p38 and ASK-1 may activate MEK-6, which, in turn, phosphorylates p38 (Stein et al., 1996). Proapoptotic effects of TNF/AP-1 pathway decrease, because this pathway is inhibited by p21 at ASK1 step (Ricote et al., 2003). Cell proliferation stimulation triggered by TNF via p38 occurs, since intense immunoreaction to PAK-1 and MEK-6 was found (Ricote et al., 2006a), but previous studies have shown elevated levels of IL-1 (Ricote et al., 2004) and p38 (Royuela et al., 2002). Ricote et al. (2006)b using LNCaP cells suggest that p38 plays an important role in prostatic tumor promotion by TNFα stimulation, and hence may represent a target for the treatment of prostatic cancer. Treatment with the p38 inhibitor SB203580 caused a notable increase in the frequency of apoptosis in LNCaP cell cultures, indicating that p38 exerts an antiapoptotic action in this cell line (Ricote et al., 2006). Noted that LNCaP cells represent a good model of well-differentiated tumor and as such its behavior is more comparable to the in vivo tumor condition. In this way, Thornton and Rincon (2009) considered the potential use of pharmacological inhibitors of p38 in therapeutic treatment for several diseases.

#### **4. TNF/IL-1/IL-6/ERK**

When IL-6 and IL-6Rα induces dimerization of gp130, and subsequently the activation of constituvely-associated gp130 Jak proteins, simultaneously trigger functionally distinct and even contradictory signaling pathways. One of them leads to the recruitment at the complex receptor of SHP2, Sos and Grb2, which in turn activates Ras by stimulating the exchange of

Fig. 4. Nuclear immunoreaction to p-Elk-1 appeared in the epithelial basal cells of normal prostate (A) or all epithelial cells in BPH (B). In PC samples p-Elk-1 (C) was observed in the cytoplasm. p-ATF-2 immunostaining was localised in the nuclei of epithelial cells in normal prostate (D) but more intense in BPH (E) and PC (F). Scale bar: 20 m (B, E) and 25 m (A,

2006a). However, the TNF-α signal may be diverted from the Ap-1 pathway towards the p38 pathway, because MEK-4 may also phosphorylate and activate p38 and ASK-1 may activate MEK-6, which, in turn, phosphorylates p38 (Stein et al., 1996). Proapoptotic effects of TNF/AP-1 pathway decrease, because this pathway is inhibited by p21 at ASK1 step (Ricote et al., 2003). Cell proliferation stimulation triggered by TNF via p38 occurs, since intense immunoreaction to PAK-1 and MEK-6 was found (Ricote et al., 2006a), but previous studies have shown elevated levels of IL-1 (Ricote et al., 2004) and p38 (Royuela et al., 2002). Ricote et al. (2006)b using LNCaP cells suggest that p38 plays an important role in prostatic tumor promotion by TNFα stimulation, and hence may represent a target for the treatment of prostatic cancer. Treatment with the p38 inhibitor SB203580 caused a notable increase in the frequency of apoptosis in LNCaP cell cultures, indicating that p38 exerts an antiapoptotic action in this cell line (Ricote et al., 2006). Noted that LNCaP cells represent a good model of well-differentiated tumor and as such its behavior is more comparable to the in vivo tumor condition. In this way, Thornton and Rincon (2009) considered the potential use

of pharmacological inhibitors of p38 in therapeutic treatment for several diseases.

When IL-6 and IL-6Rα induces dimerization of gp130, and subsequently the activation of constituvely-associated gp130 Jak proteins, simultaneously trigger functionally distinct and even contradictory signaling pathways. One of them leads to the recruitment at the complex receptor of SHP2, Sos and Grb2, which in turn activates Ras by stimulating the exchange of

C-D, F).

**4. TNF/IL-1/IL-6/ERK** 

GDP bound to Ras for GTP (Silver & Hunter, 2010). Then, Ras phosphorylate of Raf-1. In this way is iniciate a MAPK cascade when Raf-1 (via IL-6 pathway), TRAF-2 (via TNF pathway) or TRAF-6 (via Il-6 pathway) phosphorylate sequentially MEK1/2 and ERK1/2, in a process that culminates in modulation of gene transcription through the activation of several transcription factors such as c-Myc, Elk-1 (Werlen et al., 2003) or NF-kB (Turjanski et al., 2007).

Fig. 5. P50 was scantly in the cytoplasm epithelial cells of normal (A) but in PC immunostaining also was nuclear, increasing the expression in medium (B) and after, in high (C) Gleason. No immunoreaction was found to p65 in normal prostate but was localized in the cytoplasm of epithelial cells in BPH (D) and PC (E-F) samples; but in PC was also localized in the nuclei of epithelial increasing nuclear localization with Gleason grade. Scale bars: 20 m (A), 25 m (B-C, E-F) and 30 m (D).

Some components of the Raf-MEK-ERK pathway are activated in solid tumors and hematological malignaces (Grant, 2008; McCubrey et al., 2007).

In approximately 30% of human breast cancers, mutations are found in the ERK1/2 MAPK pathway (Whyte et al., 2009). ERK1/2 and downstream ERK1/2 targets are hyperphosphorylated in a large subset of mammary tumors (Mueller et al., 2000). Increased expressions of Raf pathway has been associated with advance prostate cancer, hormonal independence, metastasis and a poor prognosis (Keller et al., 2004). Moreover, prostate cancer cell lines isolated from advanced cancer patients (LNCaP, PC3, DU145) expressed low levels of active Raf kinase inhibitors (McCubrey et al., 2007). TNFα acts as an ERK activator in some cases related to inflammation and cell proliferation. In this way, Ricote et al. (2006b) showed that ERK phosphorylation was notably increased by TNFα dose dependent manner in LNCaP cells. In prostate cancer, presence of Raf-1 and MEK-1 in

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conjunction with elevated ERK-1 and ERK-2 suggest that stimulation of cell proliferation could be triggered by IL-6 via the ERK pathway (Rodriguez-Berriguete et al., 2010a). In this way, Ricote et al. (2006b) in *in vitro* studies with LNCaP cells, showed that the use of specific ERK inhibitor minimally affected apoptosis, suggesting that ERK activation does not play a significant role in apoptosis regulation.

Moreover, ERK may also induce the phosphorilation of apoptotic regulatory molecules including bcl-2 family members (e.g. Bad, Bim and controversially Bcl-2) and caspase 9 (McCubrey et al., 2007). There are evidences suggesting a protective effect in cells by NF-kB activation via ERK (Chu et al., 2008; Zhu et al., 2004). This transcription factor in a basal state is retained in the cytoplasm by binding to specific inhibitors, the inhibitors of NF-kB (IkBs). Upon cell stimulation IkBs are degradated and consequently NF-kB is translocated into the nucleus (Karin, 2006), where it promotes the expression of several anti-apoptotic genes such as inhibitors of apoptosis proteins (IAPs) (Rodriguez-Berriguete et al., 2010) and bcl-2 family members (Aggarwal, 2000).

## **5. New perspectives**

In summary, it is reasonable to speculate that MAPK could be involved in prostate cancer development, maintenance and/or progression, since are involucrate in several transduction pathway related with prostate cancer development. These transduction pathways were interrelated and activated by pro-inflammatory (IL-6, IL-1 and TNF). At the end are activated several transcription factor such as NF-kB, Elk-1, ATF-2, p53, or mcl-1. Translocation of NF-kB to the nucleus in PC might be due to the overactivation of several transduction pathways triggered by pro-inflammatory cytokines (IL-1, IL-6 and TNF-). NFkB has been considered a marker of predicting PC since nuclear localization was only observed in PC, but another transcription factor activate by these pro-inflammatory cytokines relate with cell proliferation such as Elk-1, ATF-2 or c-myc were also increased in PC. For this, might be that overexpression of MAPKs might be secondary to overexpression of these cytokines and, subsequently, MAPKs also might be involved in the development of prostatic hyperplasia and neoplasia. Therefore, since PC is a heterogeneous disease in which multiple transduction pathways may contribute to uncontrolled apoptosis/cell proliferation balance, we concluded that significant attention would be focused to the rational combination of novel agents directed toward the inactivation of pro-inflammatory cytokines, because could be disrupt complementary tumor cell proliferation pathways.

#### **6. Acknowledgements**

Supported by grants from the "Ministerio de Educación y Ciencia", Spain (SAF2007-61928) and the "Fundación Mutua Madrileña, 2010" (Spain). Gonzalo Rodríguez-Berriguete had a predoctoral fellowship from the Alcalá University (Madrid, Spain) during the course of this work.

#### **7. References**

Aaltomaa, S.; Lipponen, P.; Eskelinen, M.; Ala-Opas, M.; Kosma, V.M. (1999). Prognostic value and expression of p21 (waf1/cip1) protein in prostate cancer. *The Prostate*, Vol. 39, No. 1 (Apr 1), pp. 8-15, ISSN: 0270-4137.

conjunction with elevated ERK-1 and ERK-2 suggest that stimulation of cell proliferation could be triggered by IL-6 via the ERK pathway (Rodriguez-Berriguete et al., 2010a). In this way, Ricote et al. (2006b) in *in vitro* studies with LNCaP cells, showed that the use of specific ERK inhibitor minimally affected apoptosis, suggesting that ERK activation does not play a

Moreover, ERK may also induce the phosphorilation of apoptotic regulatory molecules including bcl-2 family members (e.g. Bad, Bim and controversially Bcl-2) and caspase 9 (McCubrey et al., 2007). There are evidences suggesting a protective effect in cells by NF-kB activation via ERK (Chu et al., 2008; Zhu et al., 2004). This transcription factor in a basal state is retained in the cytoplasm by binding to specific inhibitors, the inhibitors of NF-kB (IkBs). Upon cell stimulation IkBs are degradated and consequently NF-kB is translocated into the nucleus (Karin, 2006), where it promotes the expression of several anti-apoptotic genes such as inhibitors of apoptosis proteins (IAPs) (Rodriguez-Berriguete et al., 2010) and bcl-2 family

In summary, it is reasonable to speculate that MAPK could be involved in prostate cancer development, maintenance and/or progression, since are involucrate in several transduction pathway related with prostate cancer development. These transduction pathways were interrelated and activated by pro-inflammatory (IL-6, IL-1 and TNF). At the end are activated several transcription factor such as NF-kB, Elk-1, ATF-2, p53, or mcl-1. Translocation of NF-kB to the nucleus in PC might be due to the overactivation of several transduction pathways triggered by pro-inflammatory cytokines (IL-1, IL-6 and TNF-). NFkB has been considered a marker of predicting PC since nuclear localization was only observed in PC, but another transcription factor activate by these pro-inflammatory cytokines relate with cell proliferation such as Elk-1, ATF-2 or c-myc were also increased in PC. For this, might be that overexpression of MAPKs might be secondary to overexpression of these cytokines and, subsequently, MAPKs also might be involved in the development of prostatic hyperplasia and neoplasia. Therefore, since PC is a heterogeneous disease in which multiple transduction pathways may contribute to uncontrolled apoptosis/cell proliferation balance, we concluded that significant attention would be focused to the rational combination of novel agents directed toward the inactivation of pro-inflammatory cytokines, because could be disrupt complementary tumor cell proliferation pathways.

Supported by grants from the "Ministerio de Educación y Ciencia", Spain (SAF2007-61928) and the "Fundación Mutua Madrileña, 2010" (Spain). Gonzalo Rodríguez-Berriguete had a predoctoral fellowship from the Alcalá University (Madrid, Spain) during the course of this

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**5. New perspectives** 

**6. Acknowledgements** 

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**Part 4** 

**Drug Therapeutics/Biological Agents** 

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