**Epidermal Growth Factor Receptor (EGFR) Phosphorylation, Signaling and Trafficking in Prostate Cancer**

Yao Huang and Yongchang Chang *St. Joseph's Hospital and Medical Center, Phoenix, Arizona USA* 

## **1. Introduction**

142 Prostate Cancer – From Bench to Bedside

Schwarz, E. C., Wissenbach, U., Niemeyer, B. A., Strauss, B., Philipp, S. E., Flockerzi, V., and

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trp, a novel mammalian gene family essential for agonist-activated capacitative

The molecular mechanisms of prostate cancer are still poorly understood, despite the threat that prostate cancer poses to the health of men worldwide. As prostate tumors are initially dependent on androgens for growth and survival, androgen deprivation therapy is the firstline treatment for prostate cancer patients. However, a hormonal-refractory (androgen independent) state often develops afterwards, and principal treatment options are palliative because of the tumor progression, which is characterized by uncontrolled growth and metastasis associated with androgen independence. To date, no effective therapy can abrogate prostate cancer progression to advanced, invasive forms. Recent evidence suggests that acquisition of androgen-independence may be due to upregulation of growth factor receptor signaling pathways, principally the epidermal growth factor receptor (EGFR)/ErbB/human epidermal receptor (HER) family (Craft et al., 1999), making it an attractive target for therapeutic intervention. EGFR/ErbB/HER signaling in cancer has been extensively studied for decades, and there have been a number of excellent reviews on the roles of ErbB receptors in the initiation and progression of a wide variety of cancers, including prostate cancer (Laskin & Sandler, 2004; Ratan et al., 2003; Yarden & Sliwkowski, 2001). Thus, this review chapter will focus more narrowly on EGFR phosphorylation, signaling, and trafficking, and their specific roles in prostate cancer development and progression (tumor growth and metastasis) given the growing literature in this area. Better understanding of the precise roles of divergent EGFR signaling pathways and their phenotypic consequences in prostate cancer (and normal prostate) will enable the development of more effective and selective therapies for this urologic disease.

### **2. Overview of the EGF/EGFR signaling system**

#### **2.1 The EGFR/ErbB/HER family and ligands**

EGFR/ErbB1/HER1 is the prototype of the EGFR or ErbB family, which also includes other three receptor tyrosine kinases, ErbB2/HER2/Neu, ErbB3/HER3, and ErbB4/HER4 (Figure 1). All four members have in common an extracellular ligand-binding domain, a single hydrophobic transmembrane domain, and a cytoplasmic region that contains a highly conserved tyrosine kinase domain and C-terminal tail (Wells, 1999). However, ErbB3 lacks intrinsic tyrosine kinase activity due to substitutions of critical amino acids within the kinase

Epidermal Growth Factor Receptor (EGFR)

**2.2 Major EGF/EGFR signaling pathways** 

Phosphorylation, Signaling, and Trafficking in Prostate Cancer 145

The repertoire of ErbB ligands and the combinatorial properties of ligand-induced receptor dimers give rise to the signaling diversity of the ErbB family. Ligand binding drives receptor homo- or hetero-dimerization, leading to activation of the intrinsic tyrosine kinase and subsequent auto- or trans-phosphorylation of specific tyrosine residues in the cytoplasmic tail (Citri & Yarden, 2006; Olayioye et al., 2000; Yarden & Sliwkowski, 2001), which provide the docking sites for proteins containing Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains (Shoelson, 1997; Sudol, 1998). These proteins generally include the adaptor proteins such as Src homology domain-containing adaptor protein C (Shc), Crk, growth factor receptorbound protein 2 (Grb2), Grb7, Grb2-associated binding protein 1 (Gab1), phospholipase C γ (PLCγ), Cbl, Esp15; the kinases such as Src, Chk and phosphatidylinositol-3-kinase (PI3K; via the p85 regulatory subunit); and the protein tyrosine phosphatases such as PTP1B, SHP1 and SHP2 (Olayioye et al., 2000; Sebastian et al., 2006), suggesting diversity and complexity of ErbB signaling networks. Among them, the signaling elicited by EGF-induced EGFR homodimers is

Adaptor proteins, kinases, and phosphatases recruited by the activated EGFR transmit signals from the receptor through different downstream signaling pathways to the nucleus to regulate various biological functions such as cell proliferation, differentiation, antiapoptosis (survival), adhesion, migration, and angiogenesis (Baselga & Hammond, 2002; Laskin & Sandler, 2004; Morandell et al., 2008; Yarden & Sliwkowski, 2001). So far with the numbers still growing, over one hundred EGFR interacting proteins have been described in the literature, of which many were discovered by proteomics approaches (Morandell et al., 2008). Approximately twenty phosphotyrosine residues located within the EGFR cytoplasmic tail have been identified as specific docking sites for above-mentioned EGFR interacting partners to engage various signaling cascades (Figure 2). The major EGF/EGFR signaling pathways include Ras/Raf/MAPK kinase (MEK)/extracellular-related kinase (ERK) and PI3K/Akt (Hirsch et al., 2003; Singh & Harris, 2005), although other pathways such as PLCγ/protein kinase C (PKC), signal transducer and activator of transcription (STAT) (Andl et al., 2004; Kloth et al., 2002), c-Jun terminal kinase (JNK) and p38 MAPKs (Johnson et al., 2005), and Ca2+-calmodulin-dependent protein kinase (CaMK) (Sengupta et al., 2009) have been reported. It is also known that upon EGF binding, EGFR undergoes a process of internalization, ubiquitination (via Cbl), destruction (namely EGFR endocytosis and trafficking), resulting in temporary EGFR downregulation (Citri & Yarden, 2006;

Both clinical and experimental data have established the importance of ErbBs, especially EGFR and ErbB2, in carcinogenesis and progression of various types of solid tumors including prostate cancer (Harari, 2004; Laskin & Sandler, 2004; Sebastian et al., 2006; Yarden & Sliwkowski, 2001). Increased expression and signaling of EGFR and/or ErbB2 are associated with a more aggressive clinical behavior of tumors, and correlate with a poor prognosis (Alroy & Yarden, 1997; Hatake et al., 2007; Lichtner, 2003; Nicholson et al., 2001). There are estimated 40-80% of prostate tumors with expressed EGFR (Kim et al., 1999; Sebastian et al., 2006). Studies mainly from breast cancer, lung cancer, and glioma have suggested many potential mechanisms related to aberrant EGFR signaling (quantitatively and/or qualitatively). These include elevated expression of ligands and/or receptors, enhanced autocrine signaling loop, constitutive activation of EGFR mutants, impaired endocytosis and trafficking of the ligand-

perhaps the best studied and has served as the prototype for other cases.

Sebastian et al., 2006; Wiley, 2003). This will be discussed in Section 3.

**2.3 EGFR signaling in prostate cancer development** 

domain (Guy et al., 1994). The extracellular domains are less conserved among the four, suggesting their ligand binding specificity (Yarden, 2001; Yarden & Sliwkowski, 2001).

Fig. 1. The four EGFR/ErbB family members and their ligands. TK, tyrosine kinase domain. See the text for more details.

ErbB receptors are activated by a number of ligands that belong to the EGF family of peptide growth factors (Citri & Yarden, 2006; Yarden, 2001). The EGF-related growth factors are characterized by the presence of an EGF-like domain consisting of three disulfide-bonded intramolecular groups conferring binding specificity, and additional structural motifs such as immunoglobulin-like domains, heparin-binding sites and glycosylation sites. They are produced as transmembrane precursors that are biologically active and able to interact with receptors expressed on adjacent cells, and the ectodomains are processed by proteolysis, resulting in the shedding of soluble growth factors (Massagué & Pandiella, 1993). Based on their affinity for one or more ErbBs, the EGFrelated growth factors are generally classified into three groups (Yarden & Sliwkowski, 2001) (Figure 1). The first group includes EGF, transforming growth factor-α (TGF-α) and amphiregulin (AR), which bind specifically to EGFR. The second group includes betacellulin (BTC), heparin-binding EGF (HB-EGF), and epiregulin (EPR), which exhibit dual specificity for both EGFR and ErbB4 (Yarden, 2001). The third group includes nuregulins (NRG, also called Neu differentiation factors (NDF) or heregulins (HRG)) that can be divided into two subgroups based on their binding specificity to both ErbB3 and ErbB4 (NRG-1 and NRG-2) or only ErbB4 (NRG-3 and NRG-4) (Harari et al., 1999; Zhang et al., 1997). Despite intensive efforts, no direct ligand for ErbB2 has yet been discovered. Increasing evidence suggests that ErbB2 primarily functions as a coreceptor for other ErbB family members (Graus-Porta et al., 1997; Tzahar et al., 1996).

## **2.2 Major EGF/EGFR signaling pathways**

144 Prostate Cancer – From Bench to Bedside

domain (Guy et al., 1994). The extracellular domains are less conserved among the four, suggesting their ligand binding specificity (Yarden, 2001; Yarden & Sliwkowski, 2001).

Fig. 1. The four EGFR/ErbB family members and their ligands. TK, tyrosine kinase domain.

ErbB receptors are activated by a number of ligands that belong to the EGF family of peptide growth factors (Citri & Yarden, 2006; Yarden, 2001). The EGF-related growth factors are characterized by the presence of an EGF-like domain consisting of three disulfide-bonded intramolecular groups conferring binding specificity, and additional structural motifs such as immunoglobulin-like domains, heparin-binding sites and glycosylation sites. They are produced as transmembrane precursors that are biologically active and able to interact with receptors expressed on adjacent cells, and the ectodomains are processed by proteolysis, resulting in the shedding of soluble growth factors (Massagué & Pandiella, 1993). Based on their affinity for one or more ErbBs, the EGFrelated growth factors are generally classified into three groups (Yarden & Sliwkowski, 2001) (Figure 1). The first group includes EGF, transforming growth factor-α (TGF-α) and amphiregulin (AR), which bind specifically to EGFR. The second group includes betacellulin (BTC), heparin-binding EGF (HB-EGF), and epiregulin (EPR), which exhibit dual specificity for both EGFR and ErbB4 (Yarden, 2001). The third group includes nuregulins (NRG, also called Neu differentiation factors (NDF) or heregulins (HRG)) that can be divided into two subgroups based on their binding specificity to both ErbB3 and ErbB4 (NRG-1 and NRG-2) or only ErbB4 (NRG-3 and NRG-4) (Harari et al., 1999; Zhang et al., 1997). Despite intensive efforts, no direct ligand for ErbB2 has yet been discovered. Increasing evidence suggests that ErbB2 primarily functions as a coreceptor for other ErbB

family members (Graus-Porta et al., 1997; Tzahar et al., 1996).

See the text for more details.

The repertoire of ErbB ligands and the combinatorial properties of ligand-induced receptor dimers give rise to the signaling diversity of the ErbB family. Ligand binding drives receptor homo- or hetero-dimerization, leading to activation of the intrinsic tyrosine kinase and subsequent auto- or trans-phosphorylation of specific tyrosine residues in the cytoplasmic tail (Citri & Yarden, 2006; Olayioye et al., 2000; Yarden & Sliwkowski, 2001), which provide the docking sites for proteins containing Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains (Shoelson, 1997; Sudol, 1998). These proteins generally include the adaptor proteins such as Src homology domain-containing adaptor protein C (Shc), Crk, growth factor receptorbound protein 2 (Grb2), Grb7, Grb2-associated binding protein 1 (Gab1), phospholipase C γ (PLCγ), Cbl, Esp15; the kinases such as Src, Chk and phosphatidylinositol-3-kinase (PI3K; via the p85 regulatory subunit); and the protein tyrosine phosphatases such as PTP1B, SHP1 and SHP2 (Olayioye et al., 2000; Sebastian et al., 2006), suggesting diversity and complexity of ErbB signaling networks. Among them, the signaling elicited by EGF-induced EGFR homodimers is perhaps the best studied and has served as the prototype for other cases.

Adaptor proteins, kinases, and phosphatases recruited by the activated EGFR transmit signals from the receptor through different downstream signaling pathways to the nucleus to regulate various biological functions such as cell proliferation, differentiation, antiapoptosis (survival), adhesion, migration, and angiogenesis (Baselga & Hammond, 2002; Laskin & Sandler, 2004; Morandell et al., 2008; Yarden & Sliwkowski, 2001). So far with the numbers still growing, over one hundred EGFR interacting proteins have been described in the literature, of which many were discovered by proteomics approaches (Morandell et al., 2008). Approximately twenty phosphotyrosine residues located within the EGFR cytoplasmic tail have been identified as specific docking sites for above-mentioned EGFR interacting partners to engage various signaling cascades (Figure 2). The major EGF/EGFR signaling pathways include Ras/Raf/MAPK kinase (MEK)/extracellular-related kinase (ERK) and PI3K/Akt (Hirsch et al., 2003; Singh & Harris, 2005), although other pathways such as PLCγ/protein kinase C (PKC), signal transducer and activator of transcription (STAT) (Andl et al., 2004; Kloth et al., 2002), c-Jun terminal kinase (JNK) and p38 MAPKs (Johnson et al., 2005), and Ca2+-calmodulin-dependent protein kinase (CaMK) (Sengupta et al., 2009) have been reported. It is also known that upon EGF binding, EGFR undergoes a process of internalization, ubiquitination (via Cbl), destruction (namely EGFR endocytosis and trafficking), resulting in temporary EGFR downregulation (Citri & Yarden, 2006; Sebastian et al., 2006; Wiley, 2003). This will be discussed in Section 3.

#### **2.3 EGFR signaling in prostate cancer development**

Both clinical and experimental data have established the importance of ErbBs, especially EGFR and ErbB2, in carcinogenesis and progression of various types of solid tumors including prostate cancer (Harari, 2004; Laskin & Sandler, 2004; Sebastian et al., 2006; Yarden & Sliwkowski, 2001). Increased expression and signaling of EGFR and/or ErbB2 are associated with a more aggressive clinical behavior of tumors, and correlate with a poor prognosis (Alroy & Yarden, 1997; Hatake et al., 2007; Lichtner, 2003; Nicholson et al., 2001). There are estimated 40-80% of prostate tumors with expressed EGFR (Kim et al., 1999; Sebastian et al., 2006). Studies mainly from breast cancer, lung cancer, and glioma have suggested many potential mechanisms related to aberrant EGFR signaling (quantitatively and/or qualitatively). These include elevated expression of ligands and/or receptors, enhanced autocrine signaling loop, constitutive activation of EGFR mutants, impaired endocytosis and trafficking of the ligand-

Epidermal Growth Factor Receptor (EGFR)

Phosphorylation, Signaling, and Trafficking in Prostate Cancer 147

upregulation of growth factor receptors and/or their ligands, and downregulation of tumor suppressor gene products (Djakiew, 2000; Ware, 1999). EGFR ligands, such as EGF, HB-EGF, and TGF-α, are expressed in the prostate and prostatic carcinomas (Elson et al., 1984; Freeman et al., 1998). In particular, the expression of TGF-α (signaling merely through EGFR) has been found to be greater in some higher grade and metastatic prostate cancers than in primary low grade tumors (Scher et al., 1995). It is now more widely believed that EGF is the predominant EGFR ligand in early, localized prostate cancer, and that TGF-α becomes more abundant than EGF at advanced, metastatic stages (Liu et al., 1993; Scher et al., 1995; Seth et al., 1999). This is the so-called EGFR ligand switch (DeHaan et al., 2009). Overexpression of EGFR and/or ErbB2 would have been expected in prostate carcinomas, as seen in breast cancer (Hatake et al., 2007; Lichtner, 2003; Nicholson et al., 2001). However, current data regarding ErbB receptor overexpression in prostate cancer appear to conflict with each other, possibly due to technical reasons and lack of standardized measurement and evaluation methods (Marks et al., 2008; Neto et al., 2010; Salomon et al., 1995; Schlomm et al., 2007; Sherwood & Lee, 1995). Nevertheless, several lines of evidence strongly support the important role of EGFR signaling in prostate cancer development. For example, autocrine activation of EGFR signaling by EGF and TGF-α most likely drives the autonomous growth of human prostate cancer (Hofer et al., 1991; Scher et al., 1995). Expression of mutant EGFRs also contributes to prostate carcinogenesis and malignant progression (Cai et al., 2008; Douglas et al., 2006; He & Young, 2009; Olapade-Olaopa et al., 2000). Taken together, studies over the years have suggested that both EGFR and ErbB2 signaling play important roles in prostate cancer development and, more specifically, in the

progression from an androgen-dependent to a hormone-refractory state.

as well as about EGFR endocytosis following binding of ligands other than EGF.

As just described, aberrant EGFR signaling is frequently associated with carcinogenesis and cancer progression. This can be the result of several unbalanced mechanisms controlling the quantitative and qualitative output of EGFR, such as elevated expression of receptors and ligands, activating receptor mutations, and impaired endocytic receptor downregulation. Proper endocytic uptake and endosomal sorting of signaling receptors have been considered as a crucial step for precisely controlling cellular processes such as growth, differentiation, and survival. Our current understanding of ligand-induced receptor endocytic downregulation is largely from the knowledge of EGFR trafficking routes following EGF binding, which has historically been and remains to be the most popular experimental system for studies in this field. In contrast, very little is known about endocytosis of ErbB2-4,

It is generally believed that EGFR is present at the plasma membrane as a monomer prior to activation. Ligand (EGF) binding triggers EGFR dimerization and activation of its intrinsic kinase, leading to signaling and relocation to invaginating clathrin-coated pits (CCPs) on the plasma membrane. The CCPs give rise to clathrin-coated endocytic vesicles. The vesicles are then released from the membrane and fuse with early endosomes. Thus, EGFR is delivered to this compartment by these sequential processes. From here the receptor is sorted for further transport, either back to the cell surface by recycling, or to the multivesicular bodies (MVBs), a pathway for eventual delivery of EGFR to late endosomes and lysosomes for degradation, which results in temporary EGFR downregulation (Figure 3). Under most physiological

**3. EGF/EGFR endocytosis and trafficking** 

receptor complex, hetero-dimerization with other ErbBs (Ciardiello & Tortora, 2003; Grandal & Madshus, 2008; Huang et al., 2009; Olayioye et al., 2000; Roepstorff et al., 2008; Sebastian et al., 2006; Sharma et al., 2007), as well as crosstalk with other receptor signaling systems such as type 1 insulin-like growth factor receptor (IGF-1R), G-protein-coupled receptors (GPCRs), and cytokine receptors (Adams et al., 2004; Gee et al., 2005; Prenzel et al., 2000).

Fig. 2. EGF/EGFR-mediated signaling pathways and cellular effects. Major tyrosine phosphorylation sites in the EGFR cytoplasmic tail, possible adaptors and signaling proteins, and signaling cascades are indicated. See the text for more details.

Progression from normal prostate epithelium to an androgen-responsive tumor, and finally to hormone-refractory carcinoma is a multistep process, usually accompanied by the

receptor complex, hetero-dimerization with other ErbBs (Ciardiello & Tortora, 2003; Grandal & Madshus, 2008; Huang et al., 2009; Olayioye et al., 2000; Roepstorff et al., 2008; Sebastian et al., 2006; Sharma et al., 2007), as well as crosstalk with other receptor signaling systems such as type 1 insulin-like growth factor receptor (IGF-1R), G-protein-coupled receptors (GPCRs), and

cytokine receptors (Adams et al., 2004; Gee et al., 2005; Prenzel et al., 2000).

Fig. 2. EGF/EGFR-mediated signaling pathways and cellular effects. Major tyrosine phosphorylation sites in the EGFR cytoplasmic tail, possible adaptors and signaling

Progression from normal prostate epithelium to an androgen-responsive tumor, and finally to hormone-refractory carcinoma is a multistep process, usually accompanied by the

proteins, and signaling cascades are indicated. See the text for more details.

upregulation of growth factor receptors and/or their ligands, and downregulation of tumor suppressor gene products (Djakiew, 2000; Ware, 1999). EGFR ligands, such as EGF, HB-EGF, and TGF-α, are expressed in the prostate and prostatic carcinomas (Elson et al., 1984; Freeman et al., 1998). In particular, the expression of TGF-α (signaling merely through EGFR) has been found to be greater in some higher grade and metastatic prostate cancers than in primary low grade tumors (Scher et al., 1995). It is now more widely believed that EGF is the predominant EGFR ligand in early, localized prostate cancer, and that TGF-α becomes more abundant than EGF at advanced, metastatic stages (Liu et al., 1993; Scher et al., 1995; Seth et al., 1999). This is the so-called EGFR ligand switch (DeHaan et al., 2009). Overexpression of EGFR and/or ErbB2 would have been expected in prostate carcinomas, as seen in breast cancer (Hatake et al., 2007; Lichtner, 2003; Nicholson et al., 2001). However, current data regarding ErbB receptor overexpression in prostate cancer appear to conflict with each other, possibly due to technical reasons and lack of standardized measurement and evaluation methods (Marks et al., 2008; Neto et al., 2010; Salomon et al., 1995; Schlomm et al., 2007; Sherwood & Lee, 1995). Nevertheless, several lines of evidence strongly support the important role of EGFR signaling in prostate cancer development. For example, autocrine activation of EGFR signaling by EGF and TGF-α most likely drives the autonomous growth of human prostate cancer (Hofer et al., 1991; Scher et al., 1995). Expression of mutant EGFRs also contributes to prostate carcinogenesis and malignant progression (Cai et al., 2008; Douglas et al., 2006; He & Young, 2009; Olapade-Olaopa et al., 2000). Taken together, studies over the years have suggested that both EGFR and ErbB2 signaling play important roles in prostate cancer development and, more specifically, in the progression from an androgen-dependent to a hormone-refractory state.

## **3. EGF/EGFR endocytosis and trafficking**

As just described, aberrant EGFR signaling is frequently associated with carcinogenesis and cancer progression. This can be the result of several unbalanced mechanisms controlling the quantitative and qualitative output of EGFR, such as elevated expression of receptors and ligands, activating receptor mutations, and impaired endocytic receptor downregulation. Proper endocytic uptake and endosomal sorting of signaling receptors have been considered as a crucial step for precisely controlling cellular processes such as growth, differentiation, and survival. Our current understanding of ligand-induced receptor endocytic downregulation is largely from the knowledge of EGFR trafficking routes following EGF binding, which has historically been and remains to be the most popular experimental system for studies in this field. In contrast, very little is known about endocytosis of ErbB2-4, as well as about EGFR endocytosis following binding of ligands other than EGF.

It is generally believed that EGFR is present at the plasma membrane as a monomer prior to activation. Ligand (EGF) binding triggers EGFR dimerization and activation of its intrinsic kinase, leading to signaling and relocation to invaginating clathrin-coated pits (CCPs) on the plasma membrane. The CCPs give rise to clathrin-coated endocytic vesicles. The vesicles are then released from the membrane and fuse with early endosomes. Thus, EGFR is delivered to this compartment by these sequential processes. From here the receptor is sorted for further transport, either back to the cell surface by recycling, or to the multivesicular bodies (MVBs), a pathway for eventual delivery of EGFR to late endosomes and lysosomes for degradation, which results in temporary EGFR downregulation (Figure 3). Under most physiological

Epidermal Growth Factor Receptor (EGFR)

into CCPs (Grandal & Madshus, 2008).

molecular mechanisms deserve further investigation.

**3.2 Ubiquitination and endosomal sorting of EGFR** 

Phosphorylation, Signaling, and Trafficking in Prostate Cancer 149

activated EGFR to mediate the receptor ubiquitination (Sebastian et al., 2006). Cbl can bind to EGFR either directly at phosphorylated tyrosine residue 1045 (Y1045) or indirectly via adaptor protein Grb2, which binds to phosphorylated EGFR at Y1068 and Y1086 (Levkowitz et al., 1999; Waterman et al., 2002). However, these two Cbl-EGFR interaction mechanisms have different effects on EGFR endocytosis. Direct binding via Y1045 may not be necessary for EGFR endocytosis, as the Y1045F mutation of EGFR results in impaired ubiquitination but does not affect receptor internalization (Grøvdal et al., 2004; Jiang et al., 2003). In contrast, Grb2-mediated binding is essential and sufficient for EGFR internalization. This is supported by the fact that Grb2 knockdown inhibits EGFR endocytosis and a chimeric protein consisting of the Y1068/Y1086-binding domain of Grb2 fused to Cbl can rescue the

In addition to acting as an E3 ubiquitin ligase, Cbl may have other functions in EGFR endocytic signaling. Phosphorylated Cbl can bind to the 85 kDa Cbl interacting protein (CIN85) that is constitutively associated with endophilin (Soubeyran et al., 2002), a known regulator of clathrin-mediated endocytosis (CME) (Reutens & Begley, 2002). The recruitment of CIN85 and endophilin to EGFR by Cbl plays an important role in EGF-induced EGFR internalization and downregulation (Soubeyran et al., 2002). Furthermore, Eps15 and epsin, the two adaptor proteins with ubiquitin binding capacity and known to localize to CCPs, are required for EGFR internalization and possibly form a complex with ubiquitinated EGFR (Polo et al., 2002; Roepstorff et al., 2008; Salcini et al., 1999). Interestingly, it has been reported that Esp15 localizes at the rim of CCPs (Stang et al., 2004; Tebar et al., 1996), while epsin localizes along the entire CCP curvature (Stang et al., 2004). These findings suggest that EGFR (ubiquitinated by Cbl) is captured by Eps15 and subsequently handed off to epsin deeper in the coated pits, which could be a more efficient way of EGFR progression

As shown above, many lines of evidence indicate that functional Cbl is a prerequisite for EGFR internalization and that Cbl ubiquitinates EGFR. However, the role of EGFR ubiquitination as an internalization signal remains controversial. One study reported that an ubiquitination-deficient mutant of EGFR with full kinase activity can still undergo normal internalization (Huang et al., 2007). Several studies also showed that siRNA depletion of epsin and/or Eps15 did not specifically affect the clathrin-mediated EGFR internalization (Chen & Zhuang, 2008; F. Huang et al., 2004; Sigismund et al., 2005; Vanden Broeck & De Wolf, 2006). A very recent study has demonstrated that CME of activated EGFR is regulated by four mechanisms, which function in a redundant and cooperative fashion (Goh et al., 2010). All these imply that the EGFR endocytosis is a rather complicated process whose

Upon EGF binding, activated EGFR undergoes CME at a much enhanced rate compared to the constitutive (ligand-independent) rate (Wiley, 2003). Immediately after internalization by CME, EGFR is delivered to early endosomes for sorting to either recycled back to the plasma membrane or transferred via MVBs to late endosomes/lysosomes for degradation (Figure 3). If not recycled back to the cell surface (as in the absence of EGF stimulation), EGFRs are sorted for lysosomal degradation. The latter is initiated by forming a complex with Esp15, signal transduction adaptor molecule (STAM), and hepatocyte growth factorregulated tyrosine kinase substrate (Hrs) (Bache et al., 2003). Hrs directs the receptors to

EGFR internalization in Grb2-depleted cells (Huang & Sorkin, 2005).

conditions, clathrin-dependent pathways are considered to be the main routes of EGFR internalization and downregulation. However, clathrin-independent pathways have also been reported and suggested as alternative mechanisms for EGFR endocytosis (Orth et al., 2006; Sigismund et al., 2005; Yamazaki et al., 2002), which will not be discussed here.

Fig. 3. EGFR endocytosis, trafficking, and turnover. EGF engagement results in EGFR activation and signaling from the cell surface. Upon EGF binding, EGFRs are internalized into clathrin-coated pits (CCP). Activated EGFR recruits the E3 ubiquitin (Ub) ligase Cbl, which ubiquitinates EGFR. EGFRs are delivered to early endosomes. From here, the receptors are sorted for either recycling back to the plasma membrane or transferring via multivesicular bodies (MVB) to late endosomes/lysosomes for degradation. The activated EGFR can continuously signal from endosomes or during its postendocytic trafficking. See the text for more details.

#### **3.1 Ubiquitination and internalization of EGFR**

Although the major steps of EGF/EGFR endocytosis and trafficking pathways are well established (Grandal & Madshus, 2008; Wiley, 2003), the molecular machinery controlling these processes remains poorly understood. It is believed that ubiquitination plays a key role in "tagging" or sorting EGFR for endocytosis and degradation (Hicke, 1999). Cbl is a ring-finger domain E3 ubiquitin ligase that is mainly responsible for EGFR ubiquitination (Levkowitz et al., 1998; Waterman et al., 1999). Upon EGF binding to EGFR, Cbl proteins are tyrosine phosphorylated by Src kinases (Feshchenko et al., 1998) and recruited rapidly to the

conditions, clathrin-dependent pathways are considered to be the main routes of EGFR internalization and downregulation. However, clathrin-independent pathways have also been reported and suggested as alternative mechanisms for EGFR endocytosis (Orth et al., 2006;

Sigismund et al., 2005; Yamazaki et al., 2002), which will not be discussed here.

Fig. 3. EGFR endocytosis, trafficking, and turnover. EGF engagement results in EGFR activation and signaling from the cell surface. Upon EGF binding, EGFRs are internalized into clathrin-coated pits (CCP). Activated EGFR recruits the E3 ubiquitin (Ub) ligase Cbl, which ubiquitinates EGFR. EGFRs are delivered to early endosomes. From here, the receptors are sorted for either recycling back to the plasma membrane or transferring via multivesicular bodies (MVB) to late endosomes/lysosomes for degradation. The activated EGFR can continuously signal from endosomes or during its postendocytic trafficking. See

Although the major steps of EGF/EGFR endocytosis and trafficking pathways are well established (Grandal & Madshus, 2008; Wiley, 2003), the molecular machinery controlling these processes remains poorly understood. It is believed that ubiquitination plays a key role in "tagging" or sorting EGFR for endocytosis and degradation (Hicke, 1999). Cbl is a ring-finger domain E3 ubiquitin ligase that is mainly responsible for EGFR ubiquitination (Levkowitz et al., 1998; Waterman et al., 1999). Upon EGF binding to EGFR, Cbl proteins are tyrosine phosphorylated by Src kinases (Feshchenko et al., 1998) and recruited rapidly to the

the text for more details.

**3.1 Ubiquitination and internalization of EGFR** 

activated EGFR to mediate the receptor ubiquitination (Sebastian et al., 2006). Cbl can bind to EGFR either directly at phosphorylated tyrosine residue 1045 (Y1045) or indirectly via adaptor protein Grb2, which binds to phosphorylated EGFR at Y1068 and Y1086 (Levkowitz et al., 1999; Waterman et al., 2002). However, these two Cbl-EGFR interaction mechanisms have different effects on EGFR endocytosis. Direct binding via Y1045 may not be necessary for EGFR endocytosis, as the Y1045F mutation of EGFR results in impaired ubiquitination but does not affect receptor internalization (Grøvdal et al., 2004; Jiang et al., 2003). In contrast, Grb2-mediated binding is essential and sufficient for EGFR internalization. This is supported by the fact that Grb2 knockdown inhibits EGFR endocytosis and a chimeric protein consisting of the Y1068/Y1086-binding domain of Grb2 fused to Cbl can rescue the EGFR internalization in Grb2-depleted cells (Huang & Sorkin, 2005).

In addition to acting as an E3 ubiquitin ligase, Cbl may have other functions in EGFR endocytic signaling. Phosphorylated Cbl can bind to the 85 kDa Cbl interacting protein (CIN85) that is constitutively associated with endophilin (Soubeyran et al., 2002), a known regulator of clathrin-mediated endocytosis (CME) (Reutens & Begley, 2002). The recruitment of CIN85 and endophilin to EGFR by Cbl plays an important role in EGF-induced EGFR internalization and downregulation (Soubeyran et al., 2002). Furthermore, Eps15 and epsin, the two adaptor proteins with ubiquitin binding capacity and known to localize to CCPs, are required for EGFR internalization and possibly form a complex with ubiquitinated EGFR (Polo et al., 2002; Roepstorff et al., 2008; Salcini et al., 1999). Interestingly, it has been reported that Esp15 localizes at the rim of CCPs (Stang et al., 2004; Tebar et al., 1996), while epsin localizes along the entire CCP curvature (Stang et al., 2004). These findings suggest that EGFR (ubiquitinated by Cbl) is captured by Eps15 and subsequently handed off to epsin deeper in the coated pits, which could be a more efficient way of EGFR progression into CCPs (Grandal & Madshus, 2008).

As shown above, many lines of evidence indicate that functional Cbl is a prerequisite for EGFR internalization and that Cbl ubiquitinates EGFR. However, the role of EGFR ubiquitination as an internalization signal remains controversial. One study reported that an ubiquitination-deficient mutant of EGFR with full kinase activity can still undergo normal internalization (Huang et al., 2007). Several studies also showed that siRNA depletion of epsin and/or Eps15 did not specifically affect the clathrin-mediated EGFR internalization (Chen & Zhuang, 2008; F. Huang et al., 2004; Sigismund et al., 2005; Vanden Broeck & De Wolf, 2006). A very recent study has demonstrated that CME of activated EGFR is regulated by four mechanisms, which function in a redundant and cooperative fashion (Goh et al., 2010). All these imply that the EGFR endocytosis is a rather complicated process whose molecular mechanisms deserve further investigation.

#### **3.2 Ubiquitination and endosomal sorting of EGFR**

Upon EGF binding, activated EGFR undergoes CME at a much enhanced rate compared to the constitutive (ligand-independent) rate (Wiley, 2003). Immediately after internalization by CME, EGFR is delivered to early endosomes for sorting to either recycled back to the plasma membrane or transferred via MVBs to late endosomes/lysosomes for degradation (Figure 3). If not recycled back to the cell surface (as in the absence of EGF stimulation), EGFRs are sorted for lysosomal degradation. The latter is initiated by forming a complex with Esp15, signal transduction adaptor molecule (STAM), and hepatocyte growth factorregulated tyrosine kinase substrate (Hrs) (Bache et al., 2003). Hrs directs the receptors to

Epidermal Growth Factor Receptor (EGFR)

**4.1 Ras/Raf/MEK/ERK signaling pathway** 

Phosphorylation, Signaling, and Trafficking in Prostate Cancer 151

**4. ERK-dependent EGFR phosphorylation and its impact on EGFR trafficking** 

The Ras/Raf/MEK/ERK cascade is one of the major and best studied EGFR downstream pathways, which links extracellular signals to the machinery that can regulate diverse and fundamentally important cellular processes such as cell proliferation, differentiation, migration, apoptosis, angiogenesis, and chromatin remodeling (Dunn et al., 2005; Yoon & Seger, 2006). Upon ligand binding, receptor dimerization, and EGFR intrinsic kinase activation and auto-phosphorylation, activation of the ERK pathway is triggered by Grb2 binding directly to EGFR at Y1068 and Y1086 and indirectly through Shc binding at Y1148 and Y1173 (Batzer et al., 1994; Lowenstein et al., 1992) (Figure 2). Grb2 recruits Sos guanine nucleotide exchange factor to the receptor complex and Sos mediates the route of activation of Ras proteins (H-Ras, K-Ras, and N-Ras) at the plasma membrane (Downward, 1996; Quilliam et al., 1995). Ras activation induces the activation of Raf family kinases including A-Raf, B-Raf, and C-Raf (Raf-1) (Marais et al., 1997; Marais & Marshall, 1996). The active Raf then activates MEK1 and MEK2 by phosphorylating serines 218 and 222 in the activation loop, which further phosphorylate and activate ERK1 and ERK2 (Dhillon et al., 2007; McKay & Morrison, 2007). These active ERKs phosphorylate numerous cytoplasmic and nuclear targets including kinases, phosphatases, transcription factors, and cytoskeletal proteins (Yoon & Seger, 2006). We have recently uncovered ERK activation-dependent phosphorylation of EGFR in several cell systems including human prostate cancer cells, which can have profound feedback to EGFR signaling and trafficking and EGFR-driven cell

migration. These previously understudied aspects are further discussed below.

As illustrated in Figure 2, EGF binding to EGFR causes activation of the receptor tyrosine kinase and phosphorylation at multiple tyrosine residues in the cytoplasmic tail. Besides the tyrosine phosphorylation events, EGFR can be phosphorylated at several serine and threonine residues (Bao et al., 2000; Countaway et al., 1990; Theroux et al., 1992a), which may influence the EGFR kinase activity (Countaway et al., 1992; Theroux et al., 1992b). We have recently uncovered a previously unappreciated type of EGFR phosphorylation induced by EGF stimulation in several cell types. By employing a state-specific monoclonal antibody (mAb), PTP101, which specifically recognizes phosphorylation of the consensus site(s) (serine or threonine residues) in the substrates for proline-directed protein kinases such as ERKs (Pearson & Kemp, 1991), we initially observed that upon EGF stimulation, both EGFR and ErbB2 undergo PTP101-reactive phosphorylation in addition to tyrosine phosphorylation in murine 3T3-F442A preadipocytes (Huang et al., 2003). Such PTP101 reactive phosphorylation seems to correlate well with EGF-induced ERK activation, as the phosphorylation can be specifically inhibited by pretreatment of the cells with two separate MEK1 inhibitors, PD98059 and UO126 (Huang et al., 2003). Furthermore, we found that peptide hormones, such as growth hormone (GH) and prolactin, can activate ERKs and cause PTP101-reactive phosphorylation of both EGFR and ErbB2 in 3T3-F442A (Huang et al., 2003) and human T47D breast cancer cells (Y. Huang et al., 2006), respectively. Previous studies suggested that serine/threonine phosphorylation of EGFR and ErbB2/Neu induced by the phorbol ester (PMA) and platelet-derived growth factor (PDGF) are attributable to the activation of PKC (Bao et al., 2000; Davis & Czech, 1985; Davis & Czech, 1987; Epstein et al., 1990; Hunter et al., 1984; Lund et al., 1990). Our data showed that neither GH-induced

**4.2 ERK activity-dependent phosphorylation of EGFR at threonine-669** 

tumor susceptibility gene-101 (TSG101). The endosomal sorting complex required for transport (ESCRT) complexes (ESCRT-I to III) are sequentially recruited. These processes eventually lead to the translocation of EGFRs into the intralumenal vesicles (ILVs) of MVBs and MVS fusion with lysosomes for receptor degradation and signal termination (Bache et al., 2006; Katzmann et al., 2002; Q. Lu et al., 2003; Williams & Urbé, 2007).

In contrast to its controversial role in EGFR internalization (see above), it is clear that Cblmediated EGFR ubiquitination plays a pivotal role at the early endosome to the late endosome/lysosome sorting step of EGFR downregulation (Duan et al., 2003). EGFR mutants with reduced ubiquitination display impaired downregulation or degradation (Grøvdal et al., 2004; Huang et al., 2007; F. Huang et al., 2006; Jiang & Sorkin, 2003; Levkowitz et al., 1999), and the Y1045F mutant can not translocate to ILVs (Grøvdal et al., 2004). Thus, it can be concluded from these studies that Cbl-associated ubiquitination is the signal for EGFR downregulation.

#### **3.3 EGFR signaling from the endosome**

EGF binding leads to and accelerates internalization and lysosomal degradation of EGFR. The most obvious function of receptor endocytosis is to remove activated EGF/EGFR complexes from the cell surface to achieve consumption of ligand and activated receptors and to prevent excessive signaling. Thus, the canonical view holds that endocytosis is a mechanism to attenuate receptor signaling via receptor downregulation. On the other hand, it has been known for many years that activated EGFR following EGF stimulation remains at the cell surface only briefly (5-10 min), and the majority of activated receptors are located in endosomes for a much longer time (1 h) (Lai et al., 1989; Sebastian et al., 2006; Wiley, 2003). Accumulating evidence indicates that the activated EGFR can continuously signal from endosomes or during its postendocytic trafficking (Baass et al., 1995; Carpenter, 2000; Pennock & Wang, 2003; Wang et al., 2002a).

Studies of EGFR signaling in the context of endocytosis have uncovered that endosomeassociated EGFR is linked to many, if not all, of its downstream signaling cascades, suggesting the complex and multifaceted effects of EGFR endocytosis on its signaling. Early work done in rat liver parenchyma (in vivo) has demonstrated that, shortly after EGF administration (1 min), internalized activated EGFR recruits a protein complex of Shc, Grb2, and the son of sevenless (Sos) to endosomes, leading to endosomally localized activation of the Ras/Raf/MEK/ERK pathway (Di Guglielmo et al., 1994). In mice, the MEK1 binding partner (MP1), adaptor protein p14, and MEK1 form a complex in endosomes. Such endosomal p14-MP1-MEK1 signaling plays an important role in cell proliferation during tissue homeostasis (Teis et al., 2006; Teis et al., 2002). Further, appropriate trafficking of activated EGFRs through endosomes ensures spatial and temporal fidelity of MAPK signaling (Taub et al., 2007). An elegant work in which EGFR is specifically activated when it is endocytosed into endosomes, has established that internalized EGFR can exert signals from endosomes to control cell survival (Wang et al., 2002a; Wang et al., 2002b), possibly by stimulating the PI3K/Akt pathway (Haugh & Meyer, 2002; Sorkin & von Zastrow, 2009). Finally, it has been reported that EGFR endocytosis is essential for STAT3 nuclear translocation and STAT3-dependent gene regulation, suggesting that endocytosis is the transport machinery for STAT3 translocation through the cytoplasm to the nucleus (Bild et al., 2002). Collectively, ligand-activated EGFR has been demonstrated to continue to signal along the endocytic pathway, which contributes to the spatio-temporal regulation of signaling, i.e. determining the specificity of signals and controlling the strength and duration of signaling.

## **4. ERK-dependent EGFR phosphorylation and its impact on EGFR trafficking**

#### **4.1 Ras/Raf/MEK/ERK signaling pathway**

150 Prostate Cancer – From Bench to Bedside

tumor susceptibility gene-101 (TSG101). The endosomal sorting complex required for transport (ESCRT) complexes (ESCRT-I to III) are sequentially recruited. These processes eventually lead to the translocation of EGFRs into the intralumenal vesicles (ILVs) of MVBs and MVS fusion with lysosomes for receptor degradation and signal termination (Bache et

In contrast to its controversial role in EGFR internalization (see above), it is clear that Cblmediated EGFR ubiquitination plays a pivotal role at the early endosome to the late endosome/lysosome sorting step of EGFR downregulation (Duan et al., 2003). EGFR mutants with reduced ubiquitination display impaired downregulation or degradation (Grøvdal et al., 2004; Huang et al., 2007; F. Huang et al., 2006; Jiang & Sorkin, 2003; Levkowitz et al., 1999), and the Y1045F mutant can not translocate to ILVs (Grøvdal et al., 2004). Thus, it can be concluded from these studies that Cbl-associated ubiquitination is the

EGF binding leads to and accelerates internalization and lysosomal degradation of EGFR. The most obvious function of receptor endocytosis is to remove activated EGF/EGFR complexes from the cell surface to achieve consumption of ligand and activated receptors and to prevent excessive signaling. Thus, the canonical view holds that endocytosis is a mechanism to attenuate receptor signaling via receptor downregulation. On the other hand, it has been known for many years that activated EGFR following EGF stimulation remains at the cell surface only briefly (5-10 min), and the majority of activated receptors are located in endosomes for a much longer time (1 h) (Lai et al., 1989; Sebastian et al., 2006; Wiley, 2003). Accumulating evidence indicates that the activated EGFR can continuously signal from endosomes or during its postendocytic trafficking (Baass et al., 1995; Carpenter, 2000;

Studies of EGFR signaling in the context of endocytosis have uncovered that endosomeassociated EGFR is linked to many, if not all, of its downstream signaling cascades, suggesting the complex and multifaceted effects of EGFR endocytosis on its signaling. Early work done in rat liver parenchyma (in vivo) has demonstrated that, shortly after EGF administration (1 min), internalized activated EGFR recruits a protein complex of Shc, Grb2, and the son of sevenless (Sos) to endosomes, leading to endosomally localized activation of the Ras/Raf/MEK/ERK pathway (Di Guglielmo et al., 1994). In mice, the MEK1 binding partner (MP1), adaptor protein p14, and MEK1 form a complex in endosomes. Such endosomal p14-MP1-MEK1 signaling plays an important role in cell proliferation during tissue homeostasis (Teis et al., 2006; Teis et al., 2002). Further, appropriate trafficking of activated EGFRs through endosomes ensures spatial and temporal fidelity of MAPK signaling (Taub et al., 2007). An elegant work in which EGFR is specifically activated when it is endocytosed into endosomes, has established that internalized EGFR can exert signals from endosomes to control cell survival (Wang et al., 2002a; Wang et al., 2002b), possibly by stimulating the PI3K/Akt pathway (Haugh & Meyer, 2002; Sorkin & von Zastrow, 2009). Finally, it has been reported that EGFR endocytosis is essential for STAT3 nuclear translocation and STAT3-dependent gene regulation, suggesting that endocytosis is the transport machinery for STAT3 translocation through the cytoplasm to the nucleus (Bild et al., 2002). Collectively, ligand-activated EGFR has been demonstrated to continue to signal along the endocytic pathway, which contributes to the spatio-temporal regulation of signaling, i.e. determining the specificity of signals and

al., 2006; Katzmann et al., 2002; Q. Lu et al., 2003; Williams & Urbé, 2007).

signal for EGFR downregulation.

**3.3 EGFR signaling from the endosome** 

Pennock & Wang, 2003; Wang et al., 2002a).

controlling the strength and duration of signaling.

The Ras/Raf/MEK/ERK cascade is one of the major and best studied EGFR downstream pathways, which links extracellular signals to the machinery that can regulate diverse and fundamentally important cellular processes such as cell proliferation, differentiation, migration, apoptosis, angiogenesis, and chromatin remodeling (Dunn et al., 2005; Yoon & Seger, 2006). Upon ligand binding, receptor dimerization, and EGFR intrinsic kinase activation and auto-phosphorylation, activation of the ERK pathway is triggered by Grb2 binding directly to EGFR at Y1068 and Y1086 and indirectly through Shc binding at Y1148 and Y1173 (Batzer et al., 1994; Lowenstein et al., 1992) (Figure 2). Grb2 recruits Sos guanine nucleotide exchange factor to the receptor complex and Sos mediates the route of activation of Ras proteins (H-Ras, K-Ras, and N-Ras) at the plasma membrane (Downward, 1996; Quilliam et al., 1995). Ras activation induces the activation of Raf family kinases including A-Raf, B-Raf, and C-Raf (Raf-1) (Marais et al., 1997; Marais & Marshall, 1996). The active Raf then activates MEK1 and MEK2 by phosphorylating serines 218 and 222 in the activation loop, which further phosphorylate and activate ERK1 and ERK2 (Dhillon et al., 2007; McKay & Morrison, 2007). These active ERKs phosphorylate numerous cytoplasmic and nuclear targets including kinases, phosphatases, transcription factors, and cytoskeletal proteins (Yoon & Seger, 2006). We have recently uncovered ERK activation-dependent phosphorylation of EGFR in several cell systems including human prostate cancer cells, which can have profound feedback to EGFR signaling and trafficking and EGFR-driven cell migration. These previously understudied aspects are further discussed below.

#### **4.2 ERK activity-dependent phosphorylation of EGFR at threonine-669**

As illustrated in Figure 2, EGF binding to EGFR causes activation of the receptor tyrosine kinase and phosphorylation at multiple tyrosine residues in the cytoplasmic tail. Besides the tyrosine phosphorylation events, EGFR can be phosphorylated at several serine and threonine residues (Bao et al., 2000; Countaway et al., 1990; Theroux et al., 1992a), which may influence the EGFR kinase activity (Countaway et al., 1992; Theroux et al., 1992b). We have recently uncovered a previously unappreciated type of EGFR phosphorylation induced by EGF stimulation in several cell types. By employing a state-specific monoclonal antibody (mAb), PTP101, which specifically recognizes phosphorylation of the consensus site(s) (serine or threonine residues) in the substrates for proline-directed protein kinases such as ERKs (Pearson & Kemp, 1991), we initially observed that upon EGF stimulation, both EGFR and ErbB2 undergo PTP101-reactive phosphorylation in addition to tyrosine phosphorylation in murine 3T3-F442A preadipocytes (Huang et al., 2003). Such PTP101 reactive phosphorylation seems to correlate well with EGF-induced ERK activation, as the phosphorylation can be specifically inhibited by pretreatment of the cells with two separate MEK1 inhibitors, PD98059 and UO126 (Huang et al., 2003). Furthermore, we found that peptide hormones, such as growth hormone (GH) and prolactin, can activate ERKs and cause PTP101-reactive phosphorylation of both EGFR and ErbB2 in 3T3-F442A (Huang et al., 2003) and human T47D breast cancer cells (Y. Huang et al., 2006), respectively. Previous studies suggested that serine/threonine phosphorylation of EGFR and ErbB2/Neu induced by the phorbol ester (PMA) and platelet-derived growth factor (PDGF) are attributable to the activation of PKC (Bao et al., 2000; Davis & Czech, 1985; Davis & Czech, 1987; Epstein et al., 1990; Hunter et al., 1984; Lund et al., 1990). Our data showed that neither GH-induced

Epidermal Growth Factor Receptor (EGFR)

reconstitution cell system (Li et al., 2008).

**and trafficking** 

Phosphorylation, Signaling, and Trafficking in Prostate Cancer 153

**4.3 Impact of Thr-669 phosphorylation on EGFR tyrosine phosphorylation (activation)** 

EGF binding triggers EGFR kinase activation and phosphorylation, and also initiates the process of EGFR endocytosis and degradation, leading to temporary downregulation of EGFR (Wiley, 2003; Wiley et al., 2003). Previous views held that signaling emanated only from activated cell-surface EGFRs and that internalization terminated signaling (Wells et al., 1990). However, it is now more widely believed that signaling can also emanate from the EGFR in the process of postendocytic trafficking and thus, altered postendocytic trafficking of the activated EGFR may quantitatively and/or qualitatively influence its net signaling (Burke et al., 2001; Ceresa & Schmid, 2000; Di Fiore & De Camilli, 2001; Sebastian et al., 2006; Wiley, 2003). We previously reported that GH pretreatment lessens EGF-induced EGFR downregulation in murine 3T3-F442A preadipocytes (Huang et al., 2003). Further, GH-mediated attenuation of EGF-induced EGFR downregulation is ERK pathway-dependent, correlating with GHinduced threonine phosphorylation of EGFR and signaling synergy of GH and EGF (Y. Huang et al., 2004; Huang et al., 2003). Similarly, in human T47D breast carcinoma cells, prolactininduced, ERK activation-dependent, PTP101-reactive phosphorylation of EGFR retards subsequent EGF-induced receptor downregulation and potentiates acute EGF/EGFR signaling (Y. Huang et al., 2006). Furthermore, in T47D cells, EGF itself causes PTP101-reactive threonine phosphorylation of EGFR, and inhibition of the MEK/ERK pathway enhances EGF-induced EGFR downregulation (Y. Huang et al., 2006). Similar results were obtained in a human fibrosarcoma cell line that harbors an activating Ras mutation and subsequent basal activation of ERK and ERK-dependent PTP101-reactive EGFR phosphorylation (Li et al., 2008). Recently, we have demonstrated that in two human prostate cancer cell lines, DU145 and PC-3, pharmacological blockade of MEK/ERK pathway, but not PI3K/Akt pathway, results in accelerated EGF-induced EGFR downregulation (Figure 5), which negatively correlates with ligand-induced ERK-dependent threonine phosphorylation of EGFR (Figure 4) (Gan et al., 2010). Taken together, these results strongly suggest that ERK-mediated threonine phosphorylation of EGFR, whether accomplished by GH or prolactin (via crosstalk), or as a result of EGF-induced ERK activation, may serve as a "brake" on ligand-induced EGFR downregulation. Indeed, elimination of EGFR phosphorylation at threonine-669 by a point mutation (threonine to alanine) resulted in accelerated EGF-induced EGFR loss in CHO

Fig. 5. Inhibition of ERK pathway but not Akt pathway accelerates EGF-induced EGFR downregulation. (*A*) Serum-starved DU145 cells were pretreated with vehicle (DMSO), PD98059 or LY294002 for 1 h prior to stimulation with EGF for 0-30 min. Protein extracts were subjected to immunoblotting (IB) with anti-EGFR or anti-β-actin. (*B*) Statistical analysis of pooled data from five independent experiments indicated that PD98059 significantly enhances EGF-induced EGFR downregulation at 15 and 30 min (\*\*, *P* < 0.01). For more

details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

ERK activation nor EGFR and ErbB2 PTP101 reactivity are affected by the PKC inhibitor (GF109203X), though the MEK1 inhibitors (PD98059 and UO126) are indeed inhibitory (Huang et al., 2003). Similar results have been obtained for prolactin-induced ERK activation and PTP101-reactivity of EGFR in T47D cells (Y. Huang et al., 2006). Collectively, our data suggests that the mAb PTP101 detects ERK-dependent, rather than PKC-dependent, serine/threonine phosphorylation of EGFR and ErbB2, and that EGF/GH/prolactin-induced and PMA-induced phosphorylation may have distinct mechanisms (Huang et al., 2003; Y. Huang et al., 2006). Interestingly, we have recently demonstrated that such an EGF-induced serine/threonine phosphorylation of EGFR also occurs in human prostate cancer cells, which requires activation of ERK pathway but not Akt pathway (Gan et al., 2010) (Figure 4).

Fig. 4. EGF-induced PTP101-reactive threonine phosphorylation of EGFR is ERK pathway dependent. Serum-starved DU145 cells were pretreated with vehicle control, MEK/ERK pathway inhibitors (PD98059 or UO126) or PI3K/Akt pathway inhibitor (LY294002) for 1 h prior to stimulation with EGF for 15 min. Protein extracts were either immunoprecipitated (IP) with anti-EGFR antibody, followed by immunoblotting (IB) with PTP101 (*A*) or anti-EGFR (*B*), or directly immunoblotted with anti-phospho-ERK (*C*) or anti-total ERK (*D*). For more details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

Two major threonine phosphorylation sites are known in the EGFR juxtamembrane cytoplasmic domain, Thr-654 and Thr-669 (Davis & Czech, 1985; Heisermann & Gill, 1988; Hunter et al., 1984; Takishima et al., 1988). PKC may directly mediate Thr-654 phosphorylation (Davis & Czech, 1985; Hunter et al., 1984), whereas Thr-669 is thought to be phosphorylated by ERKs (Northwood et al., 1991; Takishima et al., 1991). The human EGFR cytoplasmic tail contains only one ERK consensus phosphorylation site [PX(S/T)P], i.e. PL669TP (Li et al., 2008). In reconstituted Chinese hamster ovary (CHO) cells, we showed that only wild-type EGFR, but not EGFR mutant (EGFR-T669A in which Thr-669 is mutated to alanine), underwent PTP101-reactive phosphorylation upon EGF stimulation, although EGF can cause tyrosine phosphorylation of both forms of receptors (Li et al., 2008). In a comparison experiment, in distinction to EGFR-T669A, a different EGFR mutant (EGFR-T654A in which Thr-654 is mutated to alanine) can undergo PTP101-reactive phosphorylation after EGF treatment, which was abolished by the MEK/ERK pathway inhibitor PD98059 (Li et al., 2008). These findings indicate that Thr-669, but not Thr-654, is required for EGF-induced, ERK activity-dependent PTP101-reactive (threonine) phosphorylation of EGFR.

ERK activation nor EGFR and ErbB2 PTP101 reactivity are affected by the PKC inhibitor (GF109203X), though the MEK1 inhibitors (PD98059 and UO126) are indeed inhibitory (Huang et al., 2003). Similar results have been obtained for prolactin-induced ERK activation and PTP101-reactivity of EGFR in T47D cells (Y. Huang et al., 2006). Collectively, our data suggests that the mAb PTP101 detects ERK-dependent, rather than PKC-dependent, serine/threonine phosphorylation of EGFR and ErbB2, and that EGF/GH/prolactin-induced and PMA-induced phosphorylation may have distinct mechanisms (Huang et al., 2003; Y. Huang et al., 2006). Interestingly, we have recently demonstrated that such an EGF-induced serine/threonine phosphorylation of EGFR also occurs in human prostate cancer cells, which requires activation

Fig. 4. EGF-induced PTP101-reactive threonine phosphorylation of EGFR is ERK pathway dependent. Serum-starved DU145 cells were pretreated with vehicle control, MEK/ERK pathway inhibitors (PD98059 or UO126) or PI3K/Akt pathway inhibitor (LY294002) for 1 h prior to stimulation with EGF for 15 min. Protein extracts were either immunoprecipitated (IP) with anti-EGFR antibody, followed by immunoblotting (IB) with PTP101 (*A*) or anti-EGFR (*B*), or directly immunoblotted with anti-phospho-ERK (*C*) or anti-total ERK (*D*). For

Two major threonine phosphorylation sites are known in the EGFR juxtamembrane cytoplasmic domain, Thr-654 and Thr-669 (Davis & Czech, 1985; Heisermann & Gill, 1988; Hunter et al., 1984; Takishima et al., 1988). PKC may directly mediate Thr-654 phosphorylation (Davis & Czech, 1985; Hunter et al., 1984), whereas Thr-669 is thought to be phosphorylated by ERKs (Northwood et al., 1991; Takishima et al., 1991). The human EGFR cytoplasmic tail contains only one ERK consensus phosphorylation site [PX(S/T)P], i.e. PL669TP (Li et al., 2008). In reconstituted Chinese hamster ovary (CHO) cells, we showed that only wild-type EGFR, but not EGFR mutant (EGFR-T669A in which Thr-669 is mutated to alanine), underwent PTP101-reactive phosphorylation upon EGF stimulation, although EGF can cause tyrosine phosphorylation of both forms of receptors (Li et al., 2008). In a comparison experiment, in distinction to EGFR-T669A, a different EGFR mutant (EGFR-T654A in which Thr-654 is mutated to alanine) can undergo PTP101-reactive phosphorylation after EGF treatment, which was abolished by the MEK/ERK pathway inhibitor PD98059 (Li et al., 2008). These findings indicate that Thr-669, but not Thr-654, is required for EGF-induced, ERK activity-dependent PTP101-reactive (threonine)

more details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

phosphorylation of EGFR.

of ERK pathway but not Akt pathway (Gan et al., 2010) (Figure 4).

#### **4.3 Impact of Thr-669 phosphorylation on EGFR tyrosine phosphorylation (activation) and trafficking**

EGF binding triggers EGFR kinase activation and phosphorylation, and also initiates the process of EGFR endocytosis and degradation, leading to temporary downregulation of EGFR (Wiley, 2003; Wiley et al., 2003). Previous views held that signaling emanated only from activated cell-surface EGFRs and that internalization terminated signaling (Wells et al., 1990). However, it is now more widely believed that signaling can also emanate from the EGFR in the process of postendocytic trafficking and thus, altered postendocytic trafficking of the activated EGFR may quantitatively and/or qualitatively influence its net signaling (Burke et al., 2001; Ceresa & Schmid, 2000; Di Fiore & De Camilli, 2001; Sebastian et al., 2006; Wiley, 2003). We previously reported that GH pretreatment lessens EGF-induced EGFR downregulation in murine 3T3-F442A preadipocytes (Huang et al., 2003). Further, GH-mediated attenuation of EGF-induced EGFR downregulation is ERK pathway-dependent, correlating with GHinduced threonine phosphorylation of EGFR and signaling synergy of GH and EGF (Y. Huang et al., 2004; Huang et al., 2003). Similarly, in human T47D breast carcinoma cells, prolactininduced, ERK activation-dependent, PTP101-reactive phosphorylation of EGFR retards subsequent EGF-induced receptor downregulation and potentiates acute EGF/EGFR signaling (Y. Huang et al., 2006). Furthermore, in T47D cells, EGF itself causes PTP101-reactive threonine phosphorylation of EGFR, and inhibition of the MEK/ERK pathway enhances EGF-induced EGFR downregulation (Y. Huang et al., 2006). Similar results were obtained in a human fibrosarcoma cell line that harbors an activating Ras mutation and subsequent basal activation of ERK and ERK-dependent PTP101-reactive EGFR phosphorylation (Li et al., 2008). Recently, we have demonstrated that in two human prostate cancer cell lines, DU145 and PC-3, pharmacological blockade of MEK/ERK pathway, but not PI3K/Akt pathway, results in accelerated EGF-induced EGFR downregulation (Figure 5), which negatively correlates with ligand-induced ERK-dependent threonine phosphorylation of EGFR (Figure 4) (Gan et al., 2010). Taken together, these results strongly suggest that ERK-mediated threonine phosphorylation of EGFR, whether accomplished by GH or prolactin (via crosstalk), or as a result of EGF-induced ERK activation, may serve as a "brake" on ligand-induced EGFR downregulation. Indeed, elimination of EGFR phosphorylation at threonine-669 by a point mutation (threonine to alanine) resulted in accelerated EGF-induced EGFR loss in CHO reconstitution cell system (Li et al., 2008).

Fig. 5. Inhibition of ERK pathway but not Akt pathway accelerates EGF-induced EGFR downregulation. (*A*) Serum-starved DU145 cells were pretreated with vehicle (DMSO), PD98059 or LY294002 for 1 h prior to stimulation with EGF for 0-30 min. Protein extracts were subjected to immunoblotting (IB) with anti-EGFR or anti-β-actin. (*B*) Statistical analysis of pooled data from five independent experiments indicated that PD98059 significantly enhances EGF-induced EGFR downregulation at 15 and 30 min (\*\*, *P* < 0.01). For more details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

Epidermal Growth Factor Receptor (EGFR)

Phosphorylation, Signaling, and Trafficking in Prostate Cancer 155

Fig. 6. Schematic model of how ERK activity-dependent threonine phosphorylation of EGFR modulates EGF-induced EGFR ubiquitination and downregulation. Based on our published data (Gan et al., 2010; Huang et al., 2003; Y. Huang et al., 2006; Li et al., 2008), ERK activation

phosphorylation of EGFR, which releases the "brake", resulting in enhanced EGF-induced

**5. Akt signaling, EGF/EGFR-driven epithelial-mesenchymal transition (EMT)** 

The PI3K/Akt pathway plays an important role in human cancers including prostate carcinoma (Chin & Toker, 2009; de Souza et al., 2009; Morgan et al., 2009; Qiao et al., 2008). Akt was initially identified as an oncogene within the murine leukemia virus AKT8 (Staal, 1987; Staal & Hartley, 1998). It is a serine/threonine kinase and also called protein kinase B (PKB) because its catalytic domain is related to PKA and PKC family members (Jones et al., 1991). In humans, there are three highly homologous isoforms of Akt (Akt1, Akt2, and Akt3) (Nicholson & Anderson, 2002). However, it remains controversial whether all three are equally important in human malignancies (Chin & Toker, 2009; Le Page et al., 2006; Maroulakou et al., 2008). PI3K and the tumor suppressor, phosphatase and tensin homolog

results in PTP101-reactive phosphorylation of EGFR at Thr-669. Such threonine phosphorylation serves as a "brake" on EGF-induced EGFR tyrosine phosphorylation (kinase activation), ubiquitination, and downregulation (*A*). Mutation of Thr-669 to alanine

(*B*), or blockade of the ERK pathway by PD98059 (*C*) abolishes the threonine

**and tumor metastasis** 

**5.1 PI3K/Akt signaling pathway** 

EGFR tyrosine phosphorylation/activation, ubiquitination, and downregulation.

Early studies of EGFR phosphorylation at serine and threonine sites, including serine-1046, serine 1047, and threonine-654, revealed that mutations at these sites can modulate EGFR signaling and downregulation (Bao et al., 2000; Countaway et al., 1990; Countaway et al., 1992; Theroux et al., 1992a). When examining the impact of ERK-mediated EGFR phosphorylation at threonine-669 on EGFR signaling, we found that in the CHO cell reconstitution system, the mutant EGFR-T669A exhibits enhanced tyrosine phosphorylation (reflecting EGFR kinase activation) compared to wild-type EGFR upon EGF stimulation (Li et al., 2008). Interestingly, coexpression of wild-type EGFR and EGFR-T669A, presumably resulting in a hybridimer of wild-type and mutant EGFR, does not dampen the propensity of EGFR-T669A to enhance EGF sensitivity (reflected in enhanced EGFR kinase activation) (Li et al., 2008). This led us to conclude that, in the hybridimer, the mutant EGFR-T669A exerts dominance regarding the EGF-induced EGFR activation (Li et al., 2008). More recently, in human prostate cancer cells (DU145 and PC-3) where the endogenous EGFR level is high, we have shown that pharmacological inhibition of the MEK/ERK pathway, but not the PI3K/Akt pathway, significantly augments the EGF-induced EGFR phosphorylation at multiple tyrosine residues including Y845, Y1045, and Y1068 (Gan et al., 2010).

The EGF-induced downregulation of EGFR is a complex, tightly regulated process, and impaired endocytic downregulation is often associated with malignancy (Grandal & Madshus, 2008; Polo et al., 2004; Roepstorff et al., 2008). The molecular machinery controlling ligandinduced EGFR endocytic trafficking remains poorly understood. It is believed that ubiquitination plays a key role in "tagging" EGFR for endocytosis. Subsequent to EGF binding to EGFR, the activated receptor is rapidly ubiquitinated by Cbl, an ubiquitin ligase that binds to phosphorylated EGFR, promoting post-internalization EGFR sorting to lysosomes for degradation (see Sections 3.1 and 3.2 for details). In human prostate cancer cells, we uncovered that blockade of the MEK/ERK pathway, but not the PI3K/Akt pathway, significantly enhanced EGF-induced ubiquitination of EGFR, correlating with increased Cbl tyrosine phosphorylation level and degree of physical association between tyrosine phosphorylated Cbl and activated EGFR (Gan et al., 2010). This phenomenon in prostate cancer cells resembles the effects of mutant EGFR-T669A in the CHO reconstitution system, in which EGFR-T669A underwent more robust ubiquitination than wild-type EGFR did upon EGF stimulation, due to the loss of phosphorylation at Thr-669 in EGFR-T669A cells (Li et al., 2008).

Emerging evidence suggests that Cbl can bind to EGFR directly at phosphorylated Y1045 or indirectly through Grb2, which binds to phosphorylated Y1068 and Y1086 in the EGFR cytoplasmic tail (Levkowitz et al., 1999; Waterman et al., 2002). As described above, our data in prostate cancer cells indicated that inhibition of ERK activity enhances the EGF-induced tyrosine phosphorylation of EGFR at multiple sites, at least including Y1045 and Y1068 (Gan et al., 2010). This raises several interesting questions, such as through which site(s) or tyrosine residue(s) within the EGFR cytoplasmic domain is the effect of the ERK activation-dependent Thr-669 phosphorylation exerted; whether Cbl is the sole factor in EGFR ubiquitination or are there any other contributors, such as CIN85, Grb2, Eps15, epsin, Hrs, and ESCRT complexes (see Sections 3.1 and 3.2 for details); and finally whether two completely different types of EGFR phosphorylation (tyrosine versus threonine phosphorylation) exist and how they are balanced under physiological and pathological conditions. More detailed studies are required to decipher these mechanisms. Taken together, our recent experimental data from multiple cell systems strongly support the notion that ERK-mediated Thr-669 phosphorylation of EGFR may serve as a "brake" on EGF-induced EGFR activation, signaling, and trafficking (ubiquitination and downregulation) (Figure 6).

Early studies of EGFR phosphorylation at serine and threonine sites, including serine-1046, serine 1047, and threonine-654, revealed that mutations at these sites can modulate EGFR signaling and downregulation (Bao et al., 2000; Countaway et al., 1990; Countaway et al., 1992; Theroux et al., 1992a). When examining the impact of ERK-mediated EGFR phosphorylation at threonine-669 on EGFR signaling, we found that in the CHO cell reconstitution system, the mutant EGFR-T669A exhibits enhanced tyrosine phosphorylation (reflecting EGFR kinase activation) compared to wild-type EGFR upon EGF stimulation (Li et al., 2008). Interestingly, coexpression of wild-type EGFR and EGFR-T669A, presumably resulting in a hybridimer of wild-type and mutant EGFR, does not dampen the propensity of EGFR-T669A to enhance EGF sensitivity (reflected in enhanced EGFR kinase activation) (Li et al., 2008). This led us to conclude that, in the hybridimer, the mutant EGFR-T669A exerts dominance regarding the EGF-induced EGFR activation (Li et al., 2008). More recently, in human prostate cancer cells (DU145 and PC-3) where the endogenous EGFR level is high, we have shown that pharmacological inhibition of the MEK/ERK pathway, but not the PI3K/Akt pathway, significantly augments the EGF-induced EGFR phosphorylation at multiple tyrosine residues

The EGF-induced downregulation of EGFR is a complex, tightly regulated process, and impaired endocytic downregulation is often associated with malignancy (Grandal & Madshus, 2008; Polo et al., 2004; Roepstorff et al., 2008). The molecular machinery controlling ligandinduced EGFR endocytic trafficking remains poorly understood. It is believed that ubiquitination plays a key role in "tagging" EGFR for endocytosis. Subsequent to EGF binding to EGFR, the activated receptor is rapidly ubiquitinated by Cbl, an ubiquitin ligase that binds to phosphorylated EGFR, promoting post-internalization EGFR sorting to lysosomes for degradation (see Sections 3.1 and 3.2 for details). In human prostate cancer cells, we uncovered that blockade of the MEK/ERK pathway, but not the PI3K/Akt pathway, significantly enhanced EGF-induced ubiquitination of EGFR, correlating with increased Cbl tyrosine phosphorylation level and degree of physical association between tyrosine phosphorylated Cbl and activated EGFR (Gan et al., 2010). This phenomenon in prostate cancer cells resembles the effects of mutant EGFR-T669A in the CHO reconstitution system, in which EGFR-T669A underwent more robust ubiquitination than wild-type EGFR did upon EGF stimulation, due

Emerging evidence suggests that Cbl can bind to EGFR directly at phosphorylated Y1045 or indirectly through Grb2, which binds to phosphorylated Y1068 and Y1086 in the EGFR cytoplasmic tail (Levkowitz et al., 1999; Waterman et al., 2002). As described above, our data in prostate cancer cells indicated that inhibition of ERK activity enhances the EGF-induced tyrosine phosphorylation of EGFR at multiple sites, at least including Y1045 and Y1068 (Gan et al., 2010). This raises several interesting questions, such as through which site(s) or tyrosine residue(s) within the EGFR cytoplasmic domain is the effect of the ERK activation-dependent Thr-669 phosphorylation exerted; whether Cbl is the sole factor in EGFR ubiquitination or are there any other contributors, such as CIN85, Grb2, Eps15, epsin, Hrs, and ESCRT complexes (see Sections 3.1 and 3.2 for details); and finally whether two completely different types of EGFR phosphorylation (tyrosine versus threonine phosphorylation) exist and how they are balanced under physiological and pathological conditions. More detailed studies are required to decipher these mechanisms. Taken together, our recent experimental data from multiple cell systems strongly support the notion that ERK-mediated Thr-669 phosphorylation of EGFR may serve as a "brake" on EGF-induced EGFR activation, signaling, and trafficking

to the loss of phosphorylation at Thr-669 in EGFR-T669A cells (Li et al., 2008).

including Y845, Y1045, and Y1068 (Gan et al., 2010).

(ubiquitination and downregulation) (Figure 6).

Fig. 6. Schematic model of how ERK activity-dependent threonine phosphorylation of EGFR modulates EGF-induced EGFR ubiquitination and downregulation. Based on our published data (Gan et al., 2010; Huang et al., 2003; Y. Huang et al., 2006; Li et al., 2008), ERK activation results in PTP101-reactive phosphorylation of EGFR at Thr-669. Such threonine phosphorylation serves as a "brake" on EGF-induced EGFR tyrosine phosphorylation (kinase activation), ubiquitination, and downregulation (*A*). Mutation of Thr-669 to alanine (*B*), or blockade of the ERK pathway by PD98059 (*C*) abolishes the threonine phosphorylation of EGFR, which releases the "brake", resulting in enhanced EGF-induced EGFR tyrosine phosphorylation/activation, ubiquitination, and downregulation.

## **5. Akt signaling, EGF/EGFR-driven epithelial-mesenchymal transition (EMT) and tumor metastasis**

#### **5.1 PI3K/Akt signaling pathway**

The PI3K/Akt pathway plays an important role in human cancers including prostate carcinoma (Chin & Toker, 2009; de Souza et al., 2009; Morgan et al., 2009; Qiao et al., 2008). Akt was initially identified as an oncogene within the murine leukemia virus AKT8 (Staal, 1987; Staal & Hartley, 1998). It is a serine/threonine kinase and also called protein kinase B (PKB) because its catalytic domain is related to PKA and PKC family members (Jones et al., 1991). In humans, there are three highly homologous isoforms of Akt (Akt1, Akt2, and Akt3) (Nicholson & Anderson, 2002). However, it remains controversial whether all three are equally important in human malignancies (Chin & Toker, 2009; Le Page et al., 2006; Maroulakou et al., 2008). PI3K and the tumor suppressor, phosphatase and tensin homolog

Epidermal Growth Factor Receptor (EGFR)

Phosphorylation, Signaling, and Trafficking in Prostate Cancer 157

both EGFR and ErbB-2 were expressed (Gan et al., 2010). EGF activated EGFR and its downstream ERK and Akt pathways, and markedly promoted cell migration in both DU145 and PC-3. Using pharmacological inhibitors, LY294002 and PD98059, to specifically block PI3K/Akt and MEK/ERK pathways, respectively, we further demonstrated that LY29004, but not PD98059, significantly inhibited EGF/EGFR-driven cell motility. In parallel, we observed that DU145 cells expressing constitutively activated (myristoylated) Akt (Myr-Akt) migrated much faster than control cells (Gan et al., 2010). Taken together, our data suggests

As described above, tumors of epithelial origin, as they transform to malignancy, appear to exploit the innate plasticity of epithelial cells, with EMT conferring increased invasiveness and metastatic potential. Previous studies have implicated the involvement of ErbBs in EMT and E-cadherin downregulation in breast, lung, and cervical cancer cells (Lee et al., 2008; Lu et al., 2009; Z. Lu et al., 2003). Our recent work has clearly demonstrated that prostate cancer cells undergo EMT-like morphological changes after EGF treatment, accompanied by the loss of E-cadherin at cell-cell junctions (Gan et al., 2010). Interestingly, these EGF-induced phenomena were markedly prevented when the cells were exposed to the PI3K/Akt pathway inhibitor LY294002 (Figure 7). Consistent with downregulation of E-cadherin (an epithelial marker), we further showed an upregulation of vimentin (a mesenchymal marker) induced by EGF treatment. Similarly, LY294002 pretreatment abolished the EGF-induced quantitative (mass) changes of both E-cadherin and vimentin (Figure 8) (Gan et al., 2010).

that Akt activation is critical for EGFR-mediated prostate cancer cell migration.

All these findings suggest that Akt activation is required for EGFR-driven EMT.

Fig. 7. Effect of inhibition of Akt pathway on EGF-induced EMT and loss of E-cadherin at cell-cell junction. Serum-starved DU145 cells were treated with vehicle (-) or EGF for 24 h in the presence or absence of LY294002. Inhibition of the Akt pathway by LY294002 prevents EGF-induced EMT (*A*) and loss of E-cadherin expression at cell-cell adherens junctions (*B*).

For details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

deleted on chromosome 10 (PTEN), are two well-known upstream components of Akt. Receptor tyrosine kinases such as EGFR and IGF-1R can activate PI3K at the cell membrane, initiating the PI3K/Akt signaling cascade. Once activated, PI3K phosphorylates phosphatidylinositol-4,5-diphosphate (PIP2), leading to accumulation of phosphatidylinositol-3,4,5-triphosphate (PIP3) (Morgan et al., 2009). PIP3 recruits Akt and phosphoinositide dependent protein kinase 1 (PDK1) to the cell membrane, where Akt is phosphorylated at Thr-308 by PDK1 and at Ser-473 via an unknown mechanism (de Souza et al., 2009). Activated Akt translocates to the nucleus, resulting in downstream effects, such as cell survival (anti-apoptosis), cell motility, angiogenesis, proliferation, and metabolism (Chin & Toker, 2009; de Souza et al., 2009; Morgan et al., 2009). PTEN is the primary negative regulator of Akt (Li et al., 1997). Loss of PTEN or PTEN mutation is the most common cause of hyperactivation of the PI3K/Akt pathway in many human cancers (Sansal & Sellers, 2004). Most recently, we have demonstrated that the Akt pathway plays a central role in EGFR-driven prostate cancer cell migration by activating epithelial-mesenchymal transition (EMT) (Gan et al., 2010), which is discussed in detail below.

#### **5.2 EMT and tumor metastasis**

EMT is a pivotal physiological process involved in embryogenesis, wound healing, and tissue remodeling (Thiery, 2003), and is regulated by complex signaling networks (Thiery & Sleeman, 2006). It is now recognized that EMT may be an important mechanism for carcinoma progression given EMT-like phenotypes of epithelial cancers (Klymkowsky & Savagner, 2009; Thiery, 2002). Acquisition of migratory properties is a prerequisite for cancer progression and for invasive migration of tumor cells into surrounding tissue. Within carcinoma (cancer of epithelial origin) cells, acquisition of invasiveness requires a dramatic morphological alteration similar to EMT, wherein carcinoma cells lose their epithelial characteristics of cell polarity and cell-cell adhesion and switch to a motile mesenchymal phenotype (Thiery, 2002; Thiery, 2003; Thiery & Sleeman, 2006). Disruption of cell-cell adherens junctions mediated by E-cadherin (one of the epithelial markers) is considered a crucial step in EMT and the downregulation of E-cadherin is common in metastatic carcinomas (Cavallaro & Christofori, 2004). Reduced E-cadherin expression has been found in high-grade prostate cancers and is associated with poor prognosis (Umbas et al., 1994; Umbas et al., 1992), reflective of its critical role in tumor progression. It is widely believed that downregulation of E-cadherin occurs via transcriptional repression mediated by the protein, Snail (Cano et al., 2000; Moreno-Bueno et al., 2008; Peinado et al., 2007). Accumulating evidence indicates that the EGFR family and PI3K/Akt signaling pathway can regulate Snail expression (Hipp et al., 2009; Lee et al., 2008; Qiao et al., 2008), suggesting that inhibition of the EGFR signaling pathways may prevent the loss of E-cadherin function and thereby acquisition of invasive motility (metastasis).

#### **5.3 Role of Akt signaling in EGF/EGFR-driven EMT and prostate cancer cell migration**

To understand which pathway(s) may have significant impact on EGFR-driven migration, we have recently probed this issue in human prostate cancer cells. The two cell lines, DU145 and PC-3, are both androgen insensitive (van Bokhoven et al., 2003), and are excellent models for studying EGFR signaling in hormonal-refractory prostate cancer. We showed that the two cell lines predominantly expressed EGFR but not ErbB-2 when compared to an androgen-responsive prostate cancer cell line, LnCap (van Bokhoven et al., 2003), in which

deleted on chromosome 10 (PTEN), are two well-known upstream components of Akt. Receptor tyrosine kinases such as EGFR and IGF-1R can activate PI3K at the cell membrane, initiating the PI3K/Akt signaling cascade. Once activated, PI3K phosphorylates phosphatidylinositol-4,5-diphosphate (PIP2), leading to accumulation of phosphatidylinositol-3,4,5-triphosphate (PIP3) (Morgan et al., 2009). PIP3 recruits Akt and phosphoinositide dependent protein kinase 1 (PDK1) to the cell membrane, where Akt is phosphorylated at Thr-308 by PDK1 and at Ser-473 via an unknown mechanism (de Souza et al., 2009). Activated Akt translocates to the nucleus, resulting in downstream effects, such as cell survival (anti-apoptosis), cell motility, angiogenesis, proliferation, and metabolism (Chin & Toker, 2009; de Souza et al., 2009; Morgan et al., 2009). PTEN is the primary negative regulator of Akt (Li et al., 1997). Loss of PTEN or PTEN mutation is the most common cause of hyperactivation of the PI3K/Akt pathway in many human cancers (Sansal & Sellers, 2004). Most recently, we have demonstrated that the Akt pathway plays a central role in EGFR-driven prostate cancer cell migration by activating epithelial-mesenchymal

EMT is a pivotal physiological process involved in embryogenesis, wound healing, and tissue remodeling (Thiery, 2003), and is regulated by complex signaling networks (Thiery & Sleeman, 2006). It is now recognized that EMT may be an important mechanism for carcinoma progression given EMT-like phenotypes of epithelial cancers (Klymkowsky & Savagner, 2009; Thiery, 2002). Acquisition of migratory properties is a prerequisite for cancer progression and for invasive migration of tumor cells into surrounding tissue. Within carcinoma (cancer of epithelial origin) cells, acquisition of invasiveness requires a dramatic morphological alteration similar to EMT, wherein carcinoma cells lose their epithelial characteristics of cell polarity and cell-cell adhesion and switch to a motile mesenchymal phenotype (Thiery, 2002; Thiery, 2003; Thiery & Sleeman, 2006). Disruption of cell-cell adherens junctions mediated by E-cadherin (one of the epithelial markers) is considered a crucial step in EMT and the downregulation of E-cadherin is common in metastatic carcinomas (Cavallaro & Christofori, 2004). Reduced E-cadherin expression has been found in high-grade prostate cancers and is associated with poor prognosis (Umbas et al., 1994; Umbas et al., 1992), reflective of its critical role in tumor progression. It is widely believed that downregulation of E-cadherin occurs via transcriptional repression mediated by the protein, Snail (Cano et al., 2000; Moreno-Bueno et al., 2008; Peinado et al., 2007). Accumulating evidence indicates that the EGFR family and PI3K/Akt signaling pathway can regulate Snail expression (Hipp et al., 2009; Lee et al., 2008; Qiao et al., 2008), suggesting that inhibition of the EGFR signaling pathways may prevent the loss of E-cadherin function

**5.3 Role of Akt signaling in EGF/EGFR-driven EMT and prostate cancer cell migration**  To understand which pathway(s) may have significant impact on EGFR-driven migration, we have recently probed this issue in human prostate cancer cells. The two cell lines, DU145 and PC-3, are both androgen insensitive (van Bokhoven et al., 2003), and are excellent models for studying EGFR signaling in hormonal-refractory prostate cancer. We showed that the two cell lines predominantly expressed EGFR but not ErbB-2 when compared to an androgen-responsive prostate cancer cell line, LnCap (van Bokhoven et al., 2003), in which

transition (EMT) (Gan et al., 2010), which is discussed in detail below.

and thereby acquisition of invasive motility (metastasis).

**5.2 EMT and tumor metastasis** 

both EGFR and ErbB-2 were expressed (Gan et al., 2010). EGF activated EGFR and its downstream ERK and Akt pathways, and markedly promoted cell migration in both DU145 and PC-3. Using pharmacological inhibitors, LY294002 and PD98059, to specifically block PI3K/Akt and MEK/ERK pathways, respectively, we further demonstrated that LY29004, but not PD98059, significantly inhibited EGF/EGFR-driven cell motility. In parallel, we observed that DU145 cells expressing constitutively activated (myristoylated) Akt (Myr-Akt) migrated much faster than control cells (Gan et al., 2010). Taken together, our data suggests that Akt activation is critical for EGFR-mediated prostate cancer cell migration.

As described above, tumors of epithelial origin, as they transform to malignancy, appear to exploit the innate plasticity of epithelial cells, with EMT conferring increased invasiveness and metastatic potential. Previous studies have implicated the involvement of ErbBs in EMT and E-cadherin downregulation in breast, lung, and cervical cancer cells (Lee et al., 2008; Lu et al., 2009; Z. Lu et al., 2003). Our recent work has clearly demonstrated that prostate cancer cells undergo EMT-like morphological changes after EGF treatment, accompanied by the loss of E-cadherin at cell-cell junctions (Gan et al., 2010). Interestingly, these EGF-induced phenomena were markedly prevented when the cells were exposed to the PI3K/Akt pathway inhibitor LY294002 (Figure 7). Consistent with downregulation of E-cadherin (an epithelial marker), we further showed an upregulation of vimentin (a mesenchymal marker) induced by EGF treatment. Similarly, LY294002 pretreatment abolished the EGF-induced quantitative (mass) changes of both E-cadherin and vimentin (Figure 8) (Gan et al., 2010). All these findings suggest that Akt activation is required for EGFR-driven EMT.

Fig. 7. Effect of inhibition of Akt pathway on EGF-induced EMT and loss of E-cadherin at cell-cell junction. Serum-starved DU145 cells were treated with vehicle (-) or EGF for 24 h in the presence or absence of LY294002. Inhibition of the Akt pathway by LY294002 prevents EGF-induced EMT (*A*) and loss of E-cadherin expression at cell-cell adherens junctions (*B*). For details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

Epidermal Growth Factor Receptor (EGFR)

al., 2010). Reprinted with permission from *Oncogene*.

2004). The underlying mechanisms remain poorly understood.

Phosphorylation, Signaling, and Trafficking in Prostate Cancer 159

Fig. 9. Knockdown of endogenous Snail prevents EGF-induced E-cadherin loss, EMT, and cell migration. (*A*) Knockdown of Snail in DU145 cells. (*B*) Knockdown of Snail prevents EGF-induced loss of E-cadherin expression. (*C*) Knockdown of Snail blocks EGF-induced EMT process. (*D*) Knockdown of Snail reduces EGF-driven cell migration measured by transwell assay. NS siRNA, nonspecific siRNA (control); \*\*, *P* < 0.01. For details see (Gan et

**6. Negative feedback loop between EGFR-directed ERK and Akt signaling** 

As described above, Ras/Raf/MEK/ERK and PI3K/Akt signaling pathways play central roles in many aspects related to tumorigenesis and cancer progression. Thus, inhibition of these signaling cascades could hold powerful therapeutic potentials. Given that many receptors utilize the common downstream pathways such as MEK/ERK and PI3K/Akt, targeting these kinases is expected to have greater therapeutic efficacy and broader applicability. For example, blockade of signaling through MEK offers the potential advantage of inhibiting both proliferation-promoting and anti-apoptotic signals originating from either activated receptors or mutation of RAS/Raf in breast cancer (Adeyinka et al., 2002). However, clinical studies of MEK inhibitors have only shown limited antitumor effects (Adjei et al., 2008; Rinehart et al.,

The molecular features of breast cancer cells that determine sensitivity to pharmacological inhibition of the Ras/Raf/MEK/ERK signaling pathway have been recently examined. Using a large set of human breast cancer cell lines as a model system, it was found that activation of PI3K/Akt pathway in response to MEK inhibition through a negative MEK-EGFR-PI3K feedback loop counteracts the efficacy of MEK inhibition on cell cycle and apoptosis induction (Mirzoeva et al., 2009). In concert with this finding, we uncovered that in prostate cancer cells, in contrast to inhibition of PI3K/Akt pathway, inhibition of MEK/ERK pathway rather enhanced EGF-directed cell motility, accompanied by enhanced EGF-induced Akt activation (Figure 10) (Gan et al., 2010). This phenomenon highly supports the notion that Akt is the key node in EGFR-mediated migratory pathways (see Section 5.3). It also raises a key question as to how ERK inactivation exerts its feedback effect to EGFinduced Akt activation. Based on our data, we believe that one mechanism could be through the feedback of ERK on EGFR phosphorylation (Figure 6). One can envision that inhibition

Fig. 8. Akt signaling contributes to EGF-driven EMT through the route of EGFR→Akt→GSK3β→Snail→E-cadherin. (*A*) LY294002 abolishes EGF-induced downregulation of E-cadherin and upregulation of vimentin. (*B*) LY294002 prevents EGFinduced phosphorylation (inactivation) of GSK3β via Akt inhibition. (*C*) LY294002 blocks EGF-induced upregulation of Snail. \*, *P* < 0.05; \*\*, *P* < 0.01; *NS*, not statistically significant. For details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

Snail is one of the several transcriptional factors that can suppress E-cadherin gene expression (Batlle et al., 2000; Cano et al., 2000) via binding to E-box sequences in the proximal E-cadherin promoter (Hemavathy et al., 2000). Snail is regulated by glycogen synthase kinase 3β (GSK3β, a downstream effector of Akt) by direct binding and phosphorylation, and inhibition of GSK3β results in upregulation of Snail and downregulation of E-cadherin (Zhou et al., 2004). This implies that Snail and GSK3β together, function as a molecular switch for many signaling pathways leading to EMT, and may provide a new connection of Akt to EMT. Along this line, we uncovered that in prostate cancer cells, EGF induced robust GSK3β phosphorylation (inactivation) and LY294002 markedly inhibited this phosphorylation, which correlated with the Akt activity. Consistent with Akt-mediated inactivation of GSK3β, Snail was upregulated upon EGF stimulation. Intriguingly, LY294002 pretreatment abolished such an EGF-induced upregulation of Snail, presumably by inactivating Akt and restoring GSK3β activity (Figure 8). As an alternative approach, we also demonstrated that knockdown of endogenous Snail in DU145 cells significantly prevented the EGF-induced loss of E-cadherin expression and concomitantly suppressed EGF-driven EMT, which correlated with a decrease in EGFdirected cell migration (Figure 9) (Gan et al., 2010). These results implicate Snail as a central effector of EMT and cell motility mediated by EGF/EGFR-activated Akt within prostate cancer cells. Collectively, our findings that EGF-mediated Akt signaling affects both phenotypic and molecular attributes, typical of EMT, provide new insights into the molecular mechanisms of EGFR-driven prostate cancer progression and metastasis.

Fig. 8. Akt signaling contributes to EGF-driven EMT through the route of EGFR→Akt→GSK3β→Snail→E-cadherin. (*A*) LY294002 abolishes EGF-induced

For details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

downregulation of E-cadherin and upregulation of vimentin. (*B*) LY294002 prevents EGFinduced phosphorylation (inactivation) of GSK3β via Akt inhibition. (*C*) LY294002 blocks EGF-induced upregulation of Snail. \*, *P* < 0.05; \*\*, *P* < 0.01; *NS*, not statistically significant.

Snail is one of the several transcriptional factors that can suppress E-cadherin gene expression (Batlle et al., 2000; Cano et al., 2000) via binding to E-box sequences in the proximal E-cadherin promoter (Hemavathy et al., 2000). Snail is regulated by glycogen synthase kinase 3β (GSK3β, a downstream effector of Akt) by direct binding and phosphorylation, and inhibition of GSK3β results in upregulation of Snail and downregulation of E-cadherin (Zhou et al., 2004). This implies that Snail and GSK3β together, function as a molecular switch for many signaling pathways leading to EMT, and may provide a new connection of Akt to EMT. Along this line, we uncovered that in prostate cancer cells, EGF induced robust GSK3β phosphorylation (inactivation) and LY294002 markedly inhibited this phosphorylation, which correlated with the Akt activity. Consistent with Akt-mediated inactivation of GSK3β, Snail was upregulated upon EGF stimulation. Intriguingly, LY294002 pretreatment abolished such an EGF-induced upregulation of Snail, presumably by inactivating Akt and restoring GSK3β activity (Figure 8). As an alternative approach, we also demonstrated that knockdown of endogenous Snail in DU145 cells significantly prevented the EGF-induced loss of E-cadherin expression and concomitantly suppressed EGF-driven EMT, which correlated with a decrease in EGFdirected cell migration (Figure 9) (Gan et al., 2010). These results implicate Snail as a central effector of EMT and cell motility mediated by EGF/EGFR-activated Akt within prostate cancer cells. Collectively, our findings that EGF-mediated Akt signaling affects both phenotypic and molecular attributes, typical of EMT, provide new insights into the

molecular mechanisms of EGFR-driven prostate cancer progression and metastasis.

Fig. 9. Knockdown of endogenous Snail prevents EGF-induced E-cadherin loss, EMT, and cell migration. (*A*) Knockdown of Snail in DU145 cells. (*B*) Knockdown of Snail prevents EGF-induced loss of E-cadherin expression. (*C*) Knockdown of Snail blocks EGF-induced EMT process. (*D*) Knockdown of Snail reduces EGF-driven cell migration measured by transwell assay. NS siRNA, nonspecific siRNA (control); \*\*, *P* < 0.01. For details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

## **6. Negative feedback loop between EGFR-directed ERK and Akt signaling**

As described above, Ras/Raf/MEK/ERK and PI3K/Akt signaling pathways play central roles in many aspects related to tumorigenesis and cancer progression. Thus, inhibition of these signaling cascades could hold powerful therapeutic potentials. Given that many receptors utilize the common downstream pathways such as MEK/ERK and PI3K/Akt, targeting these kinases is expected to have greater therapeutic efficacy and broader applicability. For example, blockade of signaling through MEK offers the potential advantage of inhibiting both proliferation-promoting and anti-apoptotic signals originating from either activated receptors or mutation of RAS/Raf in breast cancer (Adeyinka et al., 2002). However, clinical studies of MEK inhibitors have only shown limited antitumor effects (Adjei et al., 2008; Rinehart et al., 2004). The underlying mechanisms remain poorly understood.

The molecular features of breast cancer cells that determine sensitivity to pharmacological inhibition of the Ras/Raf/MEK/ERK signaling pathway have been recently examined. Using a large set of human breast cancer cell lines as a model system, it was found that activation of PI3K/Akt pathway in response to MEK inhibition through a negative MEK-EGFR-PI3K feedback loop counteracts the efficacy of MEK inhibition on cell cycle and apoptosis induction (Mirzoeva et al., 2009). In concert with this finding, we uncovered that in prostate cancer cells, in contrast to inhibition of PI3K/Akt pathway, inhibition of MEK/ERK pathway rather enhanced EGF-directed cell motility, accompanied by enhanced EGF-induced Akt activation (Figure 10) (Gan et al., 2010). This phenomenon highly supports the notion that Akt is the key node in EGFR-mediated migratory pathways (see Section 5.3). It also raises a key question as to how ERK inactivation exerts its feedback effect to EGFinduced Akt activation. Based on our data, we believe that one mechanism could be through the feedback of ERK on EGFR phosphorylation (Figure 6). One can envision that inhibition

Epidermal Growth Factor Receptor (EGFR)

**8. Acknowledgments** 

**9. References** 

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Phosphorylation, Signaling, and Trafficking in Prostate Cancer 161

activates the PI3K/Akt pathway through a negative MEK/ERK-EGFR-PI3K/Akt feedback loop. We believe that ERK-mediated threonine-669 phosphorylation is critically involved in such a negative feedback and thereby contributes to invasive migration (metastasis). Thus, inhibition of the MEK/ERK-EGFR-PI3K/Akt feedback loop is likely to result in therapeutic synergism. Future detailed studies along these lines and a deeper understanding of various mechanisms of cell signaling from EGFR and other ErbBs will undoubtedly generate new avenues for drug and biomarker development to combat cancers including prostate cancer.

This work was supported by a St. Joseph's Foundation (SJF) Startup Fund, an American Heart Association (AHA) Beginning Grant-in-Aid Award, and a Science Foundation Arizona (SFAz) Competitive Advantage Award (to YH). The authors have nothing to disclose. Correspondence should be addressed to Dr. Yao Huang, 445 N 5th Street, Suite

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oncogenesis: signal diversification through combinatorial ligand-receptor

EGFR-induced cell migration is mediated predominantly by the JAK-STAT pathway in primary esophageal keratinocytes. *Am J Physiol Gastrointest Liver* 

Compartmentalized signal transduction by receptor tyrosine kinases. *Trends Cell* 

a multivalent ubiquitin-binding complex on early endosomes. *J Biol Chem*,

Lukacs, G.L.; Brech, A. & Stenmark, H. (2006). The ESCRT-III subunit hVps24 is

110, Phoenix, Arizona, 85004, USA. E-mail: yhuang@chw.edu.

of ERK activity eliminates EGFR threonine-669 phosphorylation, resulting in enhanced EGFR tyrosine phosphorylation (kinase activation), and subsequently augmented activation of the downstream PI3K/Akt pathway. The discovery of the negative feedback loop of MEK/ERK-EGFR-PI3K/Akt on several cellular aspects implies that targeting single MEK/ERK pathway in some cancers (e.g., breast and prostate carcinomas) may have undesirable outcomes, which deserves further investigation.

Fig. 10. Effects of ERK and Akt pathways on EGF-driven prostate cancer cell migration. (*A*) Inhibition of the ERK pathway by PD98059 augments EGF-induced Akt activation in both DU145 and PC3 cells, revealed by immunoblotting (IB) with anti-phospho-Akt antibody (*top panel*). (*B*) Transwell assay shows that blockade of the Akt pathway by LY294002 significantly inhibits EGF-driven cell migration. In contrast, blockade of the ERK pathway by PD98059 rather enhances EGF-induced migration. \*\*, *P* < 0.01. For details see (Gan et al., 2010). Reprinted with permission from *Oncogene*.

## **7. Concluding remarks**

Recent advances in the ErbB field have broadened our understanding of the important roles of EGFR/ErbB signaling in human cancer. However, the complexity of the ErbB signaling network, which involves numerous ligands, multiple dimerization partners, and a variety of downstream signaling components, makes it a real challenge to establish which pathways are activated or critical in the context of tumorigenesis and progression of specific cancer types. In this chapter, several aspects of EGFR/ErbB signaling and their potential roles in prostate cancer initiation and progression are discussed. In particular, we focus on the mechanisms of how Ras/Raf/MEK/ERK and PI3K/Akt pathways impact EGFR phosphorylation, trafficking, and cell motility. New insights into prostate cancer biology gained from our own work and the studies of other investigators highlight the importance of ERK activity-dependent threonine-669 phosphorylation of EGFR and its profound feedback on EGFR tyrosine phosphorylation/kinase activation, ubiquitination, and trafficking. Recent data from our group demonstrates that the Akt pathway plays a pivotal role in EGFR-driven prostate cancer cell migration by activating EMT. In particular, our results in prostate cancer (Gan et al., 2010) and data from a recent study in breast cancer (Mirzoeva et al., 2009) suggest that therapeutic targeting of ERK signaling may have undesirable outcomes. For example, inhibition of the MEK/ERK pathway conversely activates the PI3K/Akt pathway through a negative MEK/ERK-EGFR-PI3K/Akt feedback loop. We believe that ERK-mediated threonine-669 phosphorylation is critically involved in such a negative feedback and thereby contributes to invasive migration (metastasis). Thus, inhibition of the MEK/ERK-EGFR-PI3K/Akt feedback loop is likely to result in therapeutic synergism. Future detailed studies along these lines and a deeper understanding of various mechanisms of cell signaling from EGFR and other ErbBs will undoubtedly generate new avenues for drug and biomarker development to combat cancers including prostate cancer.

## **8. Acknowledgments**

160 Prostate Cancer – From Bench to Bedside

of ERK activity eliminates EGFR threonine-669 phosphorylation, resulting in enhanced EGFR tyrosine phosphorylation (kinase activation), and subsequently augmented activation of the downstream PI3K/Akt pathway. The discovery of the negative feedback loop of MEK/ERK-EGFR-PI3K/Akt on several cellular aspects implies that targeting single MEK/ERK pathway in some cancers (e.g., breast and prostate carcinomas) may have

Fig. 10. Effects of ERK and Akt pathways on EGF-driven prostate cancer cell migration. (*A*) Inhibition of the ERK pathway by PD98059 augments EGF-induced Akt activation in both DU145 and PC3 cells, revealed by immunoblotting (IB) with anti-phospho-Akt antibody (*top* 

significantly inhibits EGF-driven cell migration. In contrast, blockade of the ERK pathway by PD98059 rather enhances EGF-induced migration. \*\*, *P* < 0.01. For details see (Gan et al.,

Recent advances in the ErbB field have broadened our understanding of the important roles of EGFR/ErbB signaling in human cancer. However, the complexity of the ErbB signaling network, which involves numerous ligands, multiple dimerization partners, and a variety of downstream signaling components, makes it a real challenge to establish which pathways are activated or critical in the context of tumorigenesis and progression of specific cancer types. In this chapter, several aspects of EGFR/ErbB signaling and their potential roles in prostate cancer initiation and progression are discussed. In particular, we focus on the mechanisms of how Ras/Raf/MEK/ERK and PI3K/Akt pathways impact EGFR phosphorylation, trafficking, and cell motility. New insights into prostate cancer biology gained from our own work and the studies of other investigators highlight the importance of ERK activity-dependent threonine-669 phosphorylation of EGFR and its profound feedback on EGFR tyrosine phosphorylation/kinase activation, ubiquitination, and trafficking. Recent data from our group demonstrates that the Akt pathway plays a pivotal role in EGFR-driven prostate cancer cell migration by activating EMT. In particular, our results in prostate cancer (Gan et al., 2010) and data from a recent study in breast cancer (Mirzoeva et al., 2009) suggest that therapeutic targeting of ERK signaling may have undesirable outcomes. For example, inhibition of the MEK/ERK pathway conversely

*panel*). (*B*) Transwell assay shows that blockade of the Akt pathway by LY294002

2010). Reprinted with permission from *Oncogene*.

**7. Concluding remarks** 

undesirable outcomes, which deserves further investigation.

This work was supported by a St. Joseph's Foundation (SJF) Startup Fund, an American Heart Association (AHA) Beginning Grant-in-Aid Award, and a Science Foundation Arizona (SFAz) Competitive Advantage Award (to YH). The authors have nothing to disclose. Correspondence should be addressed to Dr. Yao Huang, 445 N 5th Street, Suite 110, Phoenix, Arizona, 85004, USA. E-mail: yhuang@chw.edu.

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**9** 

*1University of Campinas 2Federal Fluminense University* 

*3TopLab GmbH* 

*1,2Brazil 3Germany* 

**Prostate Cancer Dephosphorylation Atlas** 

Carmen Veríssima Ferreira1, Renato Milani1, Willian Fernando Zambuzzi2, Thomas Martin Halder3, Eduardo Galembeck1 and Hiroshi Aoyama1

The widespread nature of protein phosphorylation/dephosphorylation underscores its key role in cell metabolism. Phosphate moiety balance on proteins is regulated by protein kinases (PK) and protein phosphatases (PP), which are milestone players of eukaryotic signaling pathways. In general, signaling proteins involved in intracellular pathways are transiently active or inactive by phosphorylation and dephosphorylation mechanisms, covalently executed by PK and PP, respectively (Hooft et al. 2002; Tonks, 2005). It is accepted that the phosphorylation state of these proteins must be kept at a dynamic equilibrium in biological systems. Any deviation in this balance (generally associated with augmented PK signaling) can cause the intracellular accumulation of serine, threonine, tyrosine-phosphorylated proteins, which will cause abnormal cell proliferation and differentiation, thereby resulting in different kinds of diseases (Souza et al., 2009). Similar deviation from this equilibrium can be also induced by decreased activity of protein tyrosine phosphatases (PTP) resulting from gene mutation or gene deletion, leading to an increase in tyrosine phosphorylated proteins in cells. PPs are subdivided into two major families, with regard to their physiological substrates: protein tyrosine phosphatases and serine/threonine phosphatases. In particular, tyrosine phosphorylation of key proteins is a critical event in the regulation of intracellular signaling pathways (Aoyama et al., 2003; Gee and Mansuy, 2004; Souza et al., 2009). There is strong evidence pointing that low SHP-1 PTP activity is associated with a high proliferation rate and an increased risk of recurrence after radical prostatectomy for localized prostate cancer (Tassidis et al., 2010). Moreover, it has been proposed that specific PTPs may be related to determining the developmental stage and aggressiveness degree of prostate cancer (Chuang et al, 2010). Thus, it is reasonable to suggest that the chemical modulation of PTPs may, therefore, be a good spot for pharmacological intervention for overcoming prostate cancer, in combination with conventional cancer chemotherapeutic strategies. However, the critical bottleneck in deciphering the role of PTPs in prostate cancer biology is the identification of their physiological substrates and how their enzymatic activity is related to molecular changes in proliferation and cell death. In this chapter we shall focus on the contribution of the low molecular weight protein tyrosine phosphatase (LMWPTP), Src homology 2 (SH2) domaincontaining PTP (SHP-1), cell division cycle 25 (Cdc25), acid phosphatase, phosphatase and tensin homolog (PTEN) and dual-specificity phosphatase (DUSP) for prostate carcinogenesis and describe their participation in the molecular events that lead to tumor survival and

**1. Introduction** 


## **Prostate Cancer Dephosphorylation Atlas**

Carmen Veríssima Ferreira1, Renato Milani1, Willian Fernando Zambuzzi2, Thomas Martin Halder3, Eduardo Galembeck1 and Hiroshi Aoyama1

> *1University of Campinas 2Federal Fluminense University 3TopLab GmbH 1,2Brazil 3Germany*

#### **1. Introduction**

172 Prostate Cancer – From Bench to Bedside

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The widespread nature of protein phosphorylation/dephosphorylation underscores its key role in cell metabolism. Phosphate moiety balance on proteins is regulated by protein kinases (PK) and protein phosphatases (PP), which are milestone players of eukaryotic signaling pathways. In general, signaling proteins involved in intracellular pathways are transiently active or inactive by phosphorylation and dephosphorylation mechanisms, covalently executed by PK and PP, respectively (Hooft et al. 2002; Tonks, 2005). It is accepted that the phosphorylation state of these proteins must be kept at a dynamic equilibrium in biological systems. Any deviation in this balance (generally associated with augmented PK signaling) can cause the intracellular accumulation of serine, threonine, tyrosine-phosphorylated proteins, which will cause abnormal cell proliferation and differentiation, thereby resulting in different kinds of diseases (Souza et al., 2009). Similar deviation from this equilibrium can be also induced by decreased activity of protein tyrosine phosphatases (PTP) resulting from gene mutation or gene deletion, leading to an increase in tyrosine phosphorylated proteins in cells. PPs are subdivided into two major families, with regard to their physiological substrates: protein tyrosine phosphatases and serine/threonine phosphatases. In particular, tyrosine phosphorylation of key proteins is a critical event in the regulation of intracellular signaling pathways (Aoyama et al., 2003; Gee and Mansuy, 2004; Souza et al., 2009). There is strong evidence pointing that low SHP-1 PTP activity is associated with a high proliferation rate and an increased risk of recurrence after radical prostatectomy for localized prostate cancer (Tassidis et al., 2010). Moreover, it has been proposed that specific PTPs may be related to determining the developmental stage and aggressiveness degree of prostate cancer (Chuang et al, 2010). Thus, it is reasonable to suggest that the chemical modulation of PTPs may, therefore, be a good spot for pharmacological intervention for overcoming prostate cancer, in combination with conventional cancer chemotherapeutic strategies. However, the critical bottleneck in deciphering the role of PTPs in prostate cancer biology is the identification of their physiological substrates and how their enzymatic activity is related to molecular changes in proliferation and cell death. In this chapter we shall focus on the contribution of the low molecular weight protein tyrosine phosphatase (LMWPTP), Src homology 2 (SH2) domaincontaining PTP (SHP-1), cell division cycle 25 (Cdc25), acid phosphatase, phosphatase and tensin homolog (PTEN) and dual-specificity phosphatase (DUSP) for prostate carcinogenesis and describe their participation in the molecular events that lead to tumor survival and

Prostate Cancer Dephosphorylation Atlas 175

 Class III cysteine-based PTPs are tyrosine/threonine specific phosphatases and probably evolved from a bacterial rhodanese-like enzyme. In humans, this class is represented by the group of Cdc 25 phosphatases: Cdc25A, Cdc25B and Cdc25C. These three cell cycle regulators act by dephosphorylation of Cdks at their inhibitory N-terminal phosphor-Thr/Tyr motifs, a reaction that is required for the activation of these kinases to drive progression of the cell cycle (Hoffman et al., 2004; Kristjansdottir and Rudolph, 2004). The fourth class of PTPs is represented by aspartate-based PTPs, which use a different catalytic mechanism with a key aspartic acid and dependence on a cation (Rayapureddi

> CD45, RPTP, RPTP, RPTP, RPTP, RPTP, RPTP, RPTP, RPTP, RPTP, RPTP, RPTP, DEP1, SAP1, GLEPP, PTPS31,

> PTP1B, TCPTP, PTP-MEG2, HePTP, STEP, LYP, PTP-PEST,

PAC-1, MKP1, MKP2, MKP3, MKP4, VH3, VH5, PYST2,

VHR, PIR1, BEDP, TMDP, MKP6, DSP20, SKRP, DSP21, MOSP, MGC1136, VHZ, FMDSP, VHX, VHY, HYVH1, VHP,

MTM1, MTMR1, MTMR2, MTMR3, MTMR4, MTMR5, MTMR6, MTMR7, MTMR8, MTMR9, MTMR10, MTMR11,

involved in pivotal processes in cell physiology (Malentacchi et al., 2005).

PTP family **Members** 

Nonreceptor PTP

Atypical DSPs

MPKs

PRLs

CDC14s

Slingshots

Myotubularins

PTENs

Class III cys-based CDC25A, CDC25B and CDC25C Class IV asp-based EyA1, EyA2, EyA3 and EyA4

Class II cys-based LMWPTP

Alonso et al., 2004; Souza et al., 2009.

the catalytic domains

PCPTP, STEP, IA2 and IA2

MKP5, MKP7 and MK-STYX

Laforin, RNGTT and STYX

CDC14A, CDC14B, KAP and PTP9Q22

PTEN, TPIP, TPTE, tensin and C-1-TEN

MTMR12, MTMR13 and MTMR14

Table 1. Classification of protein tyrosine phosphatases based on amino acid sequences of

PRL1, PRL2 and PRL3,

SSH1, SSH2 and SSH3

PTP-HSCF, Typ-PTP and HD-PTP

Receptor PTP

et al., 2003).

Class I cys-based

dephosphorylate tyrosine kinases and their substrates but its biological functions remain unclear. The correlation between expression and activity of variants of this PTP with some human diseases, including cancer, indicates that this phosphatase may be

osteomimetic properties, highlighting perspectives and directions for future research that improve current knowledge on these critical signaling molecules.

## **2. Protein phosphatases**

The ubiquitous nature of protein phosphorylation/dephosphorylation underscores its key role in cell signaling metabolism, growth and differentiation. In fact, cells respond to internal and external stimuli through integrated networks of intracellular signaling pathways that act via cascades of sequential phosphorylation or dephosphorylation reactions which are governed by the action of PK and PPs, respectively (Hooft van Huijsduijnen et al., 2002; Tonks, 2005).

PPs have been classified by structure and substrate specificity into protein serine/threonine phosphatases (PSTPs) and protein tyrosine phosphatases (PTPs) (Aoyama et al., 2003; Gee and Mansuy, 2005).

In general, PTPs control fundamental physiological processes such as cell growth and differentiation, cell cycle, metabolism, immune response and cytoskeletal function. Furthermore, interfering with the delicate balance between counteracting PTKs and PTPs is involved in the development of numerous inherited and acquired human diseases such as autoimmunity, diabetes and cancer (Alonso et al., 2004; Andersen et al., 2004; Ferreira et al., 2006; Souza et al., 2009; Zambuzzi et al., 2010; Zambuzzi et al., 2011).

#### **2.1 PTPs classification**

Up to now, 107 genes encoding PTPs have been discovered in the human genome, whereas 81 of them have been predicted to be active PTPs (Alonso et al., 2004). Classically, PTPs were divided into four classes: receptor type PTPs, non-receptor PTPs, dual specificity PTPs and low molecular weight PTPs. However, some authors have proposed an alternative way to classify this enzyme family based on the amino acid residues of their catalytic domains (Alonso et al., 2004; Bialy and Waldmann et al., 2005). In fact, comparison of the crystal structure of the PTPs that have been solved to date demonstrates that the PTPs domains are conserved in both sequence and structure. Additionally the sequences (domains) outside the catalytic domain are diverse and may regulate PTP activity and/or function (Table 1).


osteomimetic properties, highlighting perspectives and directions for future research that

The ubiquitous nature of protein phosphorylation/dephosphorylation underscores its key role in cell signaling metabolism, growth and differentiation. In fact, cells respond to internal and external stimuli through integrated networks of intracellular signaling pathways that act via cascades of sequential phosphorylation or dephosphorylation reactions which are governed by the action of PK and PPs, respectively (Hooft van Huijsduijnen et al., 2002; Tonks, 2005). PPs have been classified by structure and substrate specificity into protein serine/threonine phosphatases (PSTPs) and protein tyrosine phosphatases (PTPs) (Aoyama et al., 2003; Gee and

In general, PTPs control fundamental physiological processes such as cell growth and differentiation, cell cycle, metabolism, immune response and cytoskeletal function. Furthermore, interfering with the delicate balance between counteracting PTKs and PTPs is involved in the development of numerous inherited and acquired human diseases such as autoimmunity, diabetes and cancer (Alonso et al., 2004; Andersen et al., 2004; Ferreira et al.,

Up to now, 107 genes encoding PTPs have been discovered in the human genome, whereas 81 of them have been predicted to be active PTPs (Alonso et al., 2004). Classically, PTPs were divided into four classes: receptor type PTPs, non-receptor PTPs, dual specificity PTPs and low molecular weight PTPs. However, some authors have proposed an alternative way to classify this enzyme family based on the amino acid residues of their catalytic domains (Alonso et al., 2004; Bialy and Waldmann et al., 2005). In fact, comparison of the crystal structure of the PTPs that have been solved to date demonstrates that the PTPs domains are conserved in both sequence and structure. Additionally the sequences (domains) outside the catalytic domain are diverse and may regulate PTP activity and/or function (Table 1). Class I cysteine-based PTPs catalyze the enzymatic reaction in which an active-site cysteine group plays a central role and renders the PTP susceptible to oxidant agents that can lead to oxidation of the key cysteine and inhibition of PTP activity. This class contains the "classical" PTPs and the "dual specificity" protein phosphatases (DSPs), both evolved from a common ancestor. The "classical PTPs" members are strictly tyrosine-specific and according to their subcellular localization can be further divided into intracellular PTPs (PTP1B and SHP) and receptor-like PTPs (CD45, PTP and PTP), both containing one or two catalytic domain(s) of approximately 240 amino acids. The DSPs (VH1-like enzymes) are the most diverse group in terms of substrate specificity and can be distinguished by their ability to hydrolyze pSer/pThr as well as pTyr residues and non-protein substrates, such as inositol phospholipids. The DSP family contains, amongst others, highly specialized types of phosphatases. For instance, members of this family include mitogenactivated protein kinases phosphatases (MKPs), members of the myotubularin family, RNA triphosphatases, and PTEN (phosphatase and tensin homologue deleted on

chromosome 10) type phosphatase (Alonso et al., 2004; Wishart and Dixon, 2006). Class II cysteine-based PTPs are especially common in bacteria and enzymes of this class appear to be more ancient than class I PTPs. In humans this class is represented by an 18 kDa tyrosine-specific low Mr phosphatase (LMPTP). LMPTP is able to

improve current knowledge on these critical signaling molecules.

2006; Souza et al., 2009; Zambuzzi et al., 2010; Zambuzzi et al., 2011).

**2. Protein phosphatases** 

Mansuy, 2005).

**2.1 PTPs classification** 

dephosphorylate tyrosine kinases and their substrates but its biological functions remain unclear. The correlation between expression and activity of variants of this PTP with some human diseases, including cancer, indicates that this phosphatase may be involved in pivotal processes in cell physiology (Malentacchi et al., 2005).



Alonso et al., 2004; Souza et al., 2009.

Table 1. Classification of protein tyrosine phosphatases based on amino acid sequences of the catalytic domains

Prostate Cancer Dephosphorylation Atlas 177

isolated from the Chilean tree *Persea nubigena* and from the stem bark of *Podocarpus andina* (Podocarpaceae), modulates both expression and activity of LMWPTP in prostate cancer cells (PC3) which was important for diminishing the proliferation ratio of these cells (Bispo de Jesus et al. 2008). More recently, we observed that prostate cancer cells that had LMWPTP silenced showed considerable reduction in invasiveness (unpublished data). Thus, this enzyme is attracting great interest as a drug target. Zabell et al. (2004) described that specific inhibitors could be rationally designed according to each of the two isoform structures of this class of enzymes. Taddei et al. (2006) observed that, at least in part, the antitumoral activity of Aplidin could be due to the direct oxidation and inactivation of LMWPTP. Marzocchini et al. (2008) reported that the treatment of rats with 1,2 dimethylhydrazine provoked a significant increase in LMWPTP expression in adenocarcinomas, suggesting that this phenomenon is associated with the onset of

Among all members of PTPs, SHP-1 has been suggested as a key signaling protein to control cell growth. Specifically, SHP-1 (an SH2 domain-containing cytosolic PTP) is an important modulator of intracellular phosphotyrosine level in eukaryotic cells, controlling different cell fates, such as proliferation, migration and differentiation through regulating signaling of cytokines such as IL-3R, PDGF- and EGF receptors, and other tyrosine kinase receptors (Tomic et al., 1995; Keilhack et al., 1998). Disruption on SHP-1 regulation can cause abnormal cell growth and induce different kinds of cancers such as leukemia, lymphoma, breast and prostate cancers as well. In order to validate this hypothesis, some authors have inserted the SHP-1 gene into different cancer cell lines and they reported a diminishment on growth of those cells (Zapata et al., 2002). Altogether, these data reinforce that SHP-1 acts as a tumor

suppressor protein, regulating cell signaling responsible to growth of eukaryotic cells.

In men, it is known that androgen deprivation leads to development of a negative growthregulating loop involving antiproliferative molecules like somatostatin (SST) in prostate adenocarcinoma. Physiologically, SST presents an antiproliferative effect, impairing mitogenic signals upon growth factors signaling (Patel, 1999). The SST signaling starts upon activation of a family of transmembrane receptors (SSTRs), sharing common signaling pathways such as the inhibition of adenylate cyclase, activation of PTP, and modulation of mitogen-activated protein kinase (MAPK). A number of publications support an involvement of SHP- 1 on negative regulation of cellular proliferation by SST (Lahlou et al., 2003). The expression of SHP-1 in rat prostate (Valencia et al., 1997) and in human prostate was shown as well (Tassidis et al., 2010). Despite the limitation of cell culture, some authors have defined SHP-1 as a decisive protein on determining cancer cell phenotype *in vitro* by using two classical prostate cancer cell lines: PC3 and LNCap. They determined an inverse relationship between cell proliferation and secreted somatostain amount. Briefly, SST was able to inhibit both PC-3 and LNCap cell proliferation by an autocrine/paracrine manner, suggesting its participation on blocking cell cycle signaling. Moreover, when SST secretion was blocked, the expression and activity levels of SHP-1 protein were reduced, and PC-3 cell proliferation was increased (Zapata et al., 2002). These authors suggest that SHP-1 could

malignancy.

**3.2 SHP-1** 

**3.2.1 Signaling features** 

**3.2.2 Role in prostate cancer** 

#### **2.2 Mechanisms of PTP catalysis**

Different experimental approaches, such as X-ray crystallography, directed site mutagenesis and circular dichroism, have contributed to our understanding of catalysis and substrate recognition by PTPs. Although PTPs have conserved catalytic domains and share a common mechanism of action, substrate specificity of individual PTPs may display substantial specificity, thus resulting in these enzymes to regulate highly specialized and often fundamentally important processes.

The PTP family shares a strictly conserved active site comprising the "P-loop" residues (H/V)**C**(X)5**R**(S/T) and a conserved acidic residue (Denu et al., 1996; Fauman et al., 1996; Zhang 2003; Aoyama et al., 2003). In all structurally characterized PTPs to date, the threedimensional structure of active-site components is also highly conserved suggesting a common catalytic mechanism. In general, the catalytic site is located in a groove at the protein surface. Its size is responsible for explaining the higher substrate selectivity of classical PTPs (Alonso et al., 2004).

*In vitro* studies based on model substrates, such as phenyl phosphate or *p*-nitrophenyl phosphate, have provided much of the information on the mechanistic aspects of catalysis. In particular, it is well established that the enzyme completes its action in two major steps. In the first step, the phosphoryl group from the substrate is transferred to the nucleophilic cysteine, forming a phosphoenzyme intermediate. In the second step, this intermediate is hydrolyzed, leading to the regeneration of the enzyme and the release of an inorganic phosphate (Aoyama et al., 2003; Zhang, 1997). Although this two-step mechanism is well-established, some mechanistic aspects still need to be clarified, such as regulatory and inhibitory mechanisms.

## **3. Protein tyrosine phosphatases and prostate cancer**

## **3.1 LMWPTP**

#### **3.1.1 Signaling features**

Chernoff and Li (1985) purified a PTP from bovine heart whose characteristics were similar to those described for the low molecular weight acid phosphatase (See item 3.4.1). This low molecular weight (about 18 kDa) protein tyrosine phosphatase (LMWPTP) shares very low sequence homology in relation to the other protein tyrosine phosphatase families, except for the consensus active site motif CX5R, that contains the essential nucleophilic cysteinyl residue, and an identical catalytic mechanism (Tonks, 2006; Tabernero et al, 2008). All PTPs hydrolyze p-nitrophenylphosphate and show inhibition by vanadate, insensitivity to okadaic acid and lack of metal ion requirement for catalysis. LMWPTP contains two conserved adjacent tyrosines, Tyr131 and Tyr132, which are preferential sites for phosphorylation by protein tyrosine kinases and important for the regulation of its activity (Tailor et al, 1997; Buccciantini et al, 1999). This enzyme class is very important in cell signaling processes such as proliferation, adhesion and migration. It can associate with and dephosphorylate many growth factors and receptors, such as platelet-derived growth factor (PDGFR), fibroblast growth factor (FGFR), insulin receptor (IR) and ephrin receptor (Eph), causing downregulation of tyrosine kinase receptor functions and leading to cell division (Souza et al, 2009).

#### **3.1.2 Role in prostate cancer**

LMWPTP has been recognized as a positive regulator of tumor growth (Chiarugi et al., 2004). Our research group has a long-standing interest in the possible beneficial prostate cancer biological effects of LMWPTP. In this scenario, we demonstrated that a compound isolated from the Chilean tree *Persea nubigena* and from the stem bark of *Podocarpus andina* (Podocarpaceae), modulates both expression and activity of LMWPTP in prostate cancer cells (PC3) which was important for diminishing the proliferation ratio of these cells (Bispo de Jesus et al. 2008). More recently, we observed that prostate cancer cells that had LMWPTP silenced showed considerable reduction in invasiveness (unpublished data).

Thus, this enzyme is attracting great interest as a drug target. Zabell et al. (2004) described that specific inhibitors could be rationally designed according to each of the two isoform structures of this class of enzymes. Taddei et al. (2006) observed that, at least in part, the antitumoral activity of Aplidin could be due to the direct oxidation and inactivation of LMWPTP. Marzocchini et al. (2008) reported that the treatment of rats with 1,2 dimethylhydrazine provoked a significant increase in LMWPTP expression in adenocarcinomas, suggesting that this phenomenon is associated with the onset of malignancy.

## **3.2 SHP-1**

176 Prostate Cancer – From Bench to Bedside

Different experimental approaches, such as X-ray crystallography, directed site mutagenesis and circular dichroism, have contributed to our understanding of catalysis and substrate recognition by PTPs. Although PTPs have conserved catalytic domains and share a common mechanism of action, substrate specificity of individual PTPs may display substantial specificity, thus resulting in these enzymes to regulate highly specialized and often

The PTP family shares a strictly conserved active site comprising the "P-loop" residues (H/V)**C**(X)5**R**(S/T) and a conserved acidic residue (Denu et al., 1996; Fauman et al., 1996; Zhang 2003; Aoyama et al., 2003). In all structurally characterized PTPs to date, the threedimensional structure of active-site components is also highly conserved suggesting a common catalytic mechanism. In general, the catalytic site is located in a groove at the protein surface. Its size is responsible for explaining the higher substrate selectivity of classical PTPs

*In vitro* studies based on model substrates, such as phenyl phosphate or *p*-nitrophenyl phosphate, have provided much of the information on the mechanistic aspects of catalysis. In particular, it is well established that the enzyme completes its action in two major steps. In the first step, the phosphoryl group from the substrate is transferred to the nucleophilic cysteine, forming a phosphoenzyme intermediate. In the second step, this intermediate is hydrolyzed, leading to the regeneration of the enzyme and the release of an inorganic phosphate (Aoyama et al., 2003; Zhang, 1997). Although this two-step mechanism is well-established, some mechanistic aspects still need to be clarified, such as regulatory and inhibitory mechanisms.

Chernoff and Li (1985) purified a PTP from bovine heart whose characteristics were similar to those described for the low molecular weight acid phosphatase (See item 3.4.1). This low molecular weight (about 18 kDa) protein tyrosine phosphatase (LMWPTP) shares very low sequence homology in relation to the other protein tyrosine phosphatase families, except for the consensus active site motif CX5R, that contains the essential nucleophilic cysteinyl residue, and an identical catalytic mechanism (Tonks, 2006; Tabernero et al, 2008). All PTPs hydrolyze p-nitrophenylphosphate and show inhibition by vanadate, insensitivity to okadaic acid and lack of metal ion requirement for catalysis. LMWPTP contains two conserved adjacent tyrosines, Tyr131 and Tyr132, which are preferential sites for phosphorylation by protein tyrosine kinases and important for the regulation of its activity (Tailor et al, 1997; Buccciantini et al, 1999). This enzyme class is very important in cell signaling processes such as proliferation, adhesion and migration. It can associate with and dephosphorylate many growth factors and receptors, such as platelet-derived growth factor (PDGFR), fibroblast growth factor (FGFR), insulin receptor (IR) and ephrin receptor (Eph), causing downregulation

of tyrosine kinase receptor functions and leading to cell division (Souza et al, 2009).

LMWPTP has been recognized as a positive regulator of tumor growth (Chiarugi et al., 2004). Our research group has a long-standing interest in the possible beneficial prostate cancer biological effects of LMWPTP. In this scenario, we demonstrated that a compound

**3. Protein tyrosine phosphatases and prostate cancer** 

**2.2 Mechanisms of PTP catalysis** 

fundamentally important processes.

(Alonso et al., 2004).

**3.1 LMWPTP** 

**3.1.1 Signaling features** 

**3.1.2 Role in prostate cancer** 

#### **3.2.1 Signaling features**

Among all members of PTPs, SHP-1 has been suggested as a key signaling protein to control cell growth. Specifically, SHP-1 (an SH2 domain-containing cytosolic PTP) is an important modulator of intracellular phosphotyrosine level in eukaryotic cells, controlling different cell fates, such as proliferation, migration and differentiation through regulating signaling of cytokines such as IL-3R, PDGF- and EGF receptors, and other tyrosine kinase receptors (Tomic et al., 1995; Keilhack et al., 1998). Disruption on SHP-1 regulation can cause abnormal cell growth and induce different kinds of cancers such as leukemia, lymphoma, breast and prostate cancers as well. In order to validate this hypothesis, some authors have inserted the SHP-1 gene into different cancer cell lines and they reported a diminishment on growth of those cells (Zapata et al., 2002). Altogether, these data reinforce that SHP-1 acts as a tumor suppressor protein, regulating cell signaling responsible to growth of eukaryotic cells.

#### **3.2.2 Role in prostate cancer**

In men, it is known that androgen deprivation leads to development of a negative growthregulating loop involving antiproliferative molecules like somatostatin (SST) in prostate adenocarcinoma. Physiologically, SST presents an antiproliferative effect, impairing mitogenic signals upon growth factors signaling (Patel, 1999). The SST signaling starts upon activation of a family of transmembrane receptors (SSTRs), sharing common signaling pathways such as the inhibition of adenylate cyclase, activation of PTP, and modulation of mitogen-activated protein kinase (MAPK). A number of publications support an involvement of SHP- 1 on negative regulation of cellular proliferation by SST (Lahlou et al., 2003). The expression of SHP-1 in rat prostate (Valencia et al., 1997) and in human prostate was shown as well (Tassidis et al., 2010). Despite the limitation of cell culture, some authors have defined SHP-1 as a decisive protein on determining cancer cell phenotype *in vitro* by using two classical prostate cancer cell lines: PC3 and LNCap. They determined an inverse relationship between cell proliferation and secreted somatostain amount. Briefly, SST was able to inhibit both PC-3 and LNCap cell proliferation by an autocrine/paracrine manner, suggesting its participation on blocking cell cycle signaling. Moreover, when SST secretion was blocked, the expression and activity levels of SHP-1 protein were reduced, and PC-3 cell proliferation was increased (Zapata et al., 2002). These authors suggest that SHP-1 could

Prostate Cancer Dephosphorylation Atlas 179

CDC25A in PC-3 and LNCap cells and CDC25A inhibitors induced both extracellular signalregulated kinase (Erk) activation and augmented Raf-1 tyrosine phosphorylation. These results indicate that CDC25A phosphatase regulates Raf-1/MEK/Erk kinase activation in human prostate cancer cells. Indeed, CDC25A controls proliferation and survival signaling,

Moreover, to determine whether CDC25C activity is altered in prostate cancer, Ozen and Ittman (2005) have examined the expression of CDC25C and an alternatively spliced variant in human prostate cancer samples and cell lines. Interestingly, they showed that an active dephosphorylated form of CDC25C was up-regulated in prostate cancer in comparison with normal prostate tissue. In addition, they showed that at the transcriptional level, CDC25C and alternatively spliced variants were both overexpressed in prostate cancer. Finally, their findings suggest that expression of the spliced variants is

Regarding CDC25B, Ngan et al. (2003) described that its overexpression is associated with the stage of prostate cancer, transiting from a hormone-dependent to a hormoneindependent state and contributing to prostate cancer development and progression.

Acid phosphatases (EC 3.1.3.2), enzymes that catalyze the hydrolysis of a wide range of orthophosphate monoesters, are largely distributed in nature and have been studied in numerous organisms and tissues (Granjeiro et al, 1997; Ferreira et al, 1998a, 1998b; Granjeiro et al, 1999; Fernandes et al, 2003; Jonsson et al, 2007). The enzyme found in mammalian tissues occurs in multiple forms that differ in regard to molecular mass, substrate specificity and sensitivity to inhibitors (Granjeiro et al, 1997). Low relative molecular mass (Mr) enzymes (Mr < 20.0 kDa) are insensitive to tartrate and fluoride and strongly inhibited by SH-reacting compounds. High Mr acid phosphatases (Mr > 100.0 kDa) are inhibited by tartrate and intermediary Mr enzymes (30.0 kDa < Mr<60.0 kDa) by fluoride. In contrast to high Mr acid phosphatases, low Mr enzymes present more restricted substrate specificity, preferentially hydrolyzing p-nitrophenylphosphate, flavin mononucleotide and tyrosine-

In 1985, Chernoff and Li reported several similarities between the low molecular weight acid phosphatase and one class of protein tyrosine phosphatase (PTP), the low molecular

The phosphatidic acid phosphatase (PAP) is a key enzyme in both glycerolipid biosynthesis and cellular signal transduction. It was observed that the plasma membrane-bound type 2 PAP, now known as lipid phosphate phosphatase, participates in germ cell migration, epithelial differentiation and other signaling processes (Kanoh et al, 1997; Brindley and

Altered acid phosphatase activities can be related to several pathological processes, such as those involving infectious, inflammatory or tumoral processes. For instance, high and intermediary molecular weight acid phosphatases levels are increased in the serum of patients with prostate carcinoma (Hudson et al. 1955), of patients suffering from spleen disorders (Kumar and Gupta, 1971), of patients with endothelial reticulum leukemia

culminating on modulation of prostate cancer progression and aggressiveness.

correlated with biochemical recurrence.

**3.4 Acid phosphatase 3.4.1 Signaling features** 

phosphorylated proteins.

**3.4.2 Role in prostate cancer** 

(Ketcham et al, 1985), etc.

Pilquil, 2009).

weight protein tyrosine phosphatase (LMWPTP).

play a key role in controlling prostatic cell proliferation, which also indicates that SHP-1 expression might be a therapeutic target for treatment of prostate cancer (Zapata et al., 2002). On the other hand, by using human prostate biopsies, Cariaga-Martinez et al. (2009) observed a decrease of SHP-1 and somatostatin in prostate cancer cells, and they demonstrated that this is consistent with aggressiveness of the tumor. In addition, Wu et al. (2003) proposed the diminished or abolished SHP-1 expression could be due to mutation of the SHP-1 gene, methylation of the promoter region or post-transcriptional regulation of SHP-1 protein synthesis. It might also be explained by the action of specific families of miRNAs.

#### **3.3 CDC25**

#### **3.3.1 Signaling features**

Unbalance on either expression or activity of proteins related to control of cell cycle progression provokes a wide variety of malignant diseases, including prostate cancer. Biochemically, cell cycle progression is a well orchestrated event regulated by well-defined sequential activities of cyclin-dependent kinases (CDKs), cyclins, and other proteins (Karlsson-Rosenthal and Millar, 2006). During mitosis, Cdc2/Cyclin B complexes can be dephosphorylated by the CDC25 phosphatase (a dual-specificity protein tyrosine phosphatase). CDC25 phosphatases play a critical role in regulating cell cycle progression by dephosphorylating CDKs at inhibitory residues and, therefore, have been shown to possess oncogenic potential (Karlsson-Rosenthal and Millar, 2006). In human, CDC25 proteins are encoded by a multigene family: CDC25A, CDC25B, and CDC25C (Turowski et al., 2003). It has been suggested that phosphorylation of CDC25C at Ser216 (activated Chk kinases) negatively regulate the activity of this phosphatase by an immediate cytoplasmic sequestration (Peng et al., 1997). Despite its potential role in prostate cancer, its exact involvement remains unclear.

#### **3.3.2 Role in prostate cancer**

Due to its hormone-dependent nature, prostate cancer at the metastatic stage is usually treated with hormone ablation therapy. Androgen receptor (AR) is a ligand-dependent transcription factor and its activity is regulated by numerous AR coregulators. Inadequate incidence of these AR coregulators contributes for the development of prostate cancer. Current studies have shown that AR activity is modulated by phosphorylation at specific sites performed by mitogen-activated protein kinases, Akt/PKB, and cAMP-activated protein kinase A, which control AR transcriptional activity. Guo et al. (2006) reported that AR was tyrosine-phosphorylated in prostate cancer cell lines and that an elevated level of phosphorylation was detected in hormone refractory prostate tumor xenografts, demonstrating that such AR modification may contribute to androgen-independent activation of AR. Chiu et al. (2009) demonstrated for the first time that CDC25A could interact with AR and inhibit its transcriptional activity. Since CDC25A overexpression is implicated in cancer development, their findings may provide an insight into the pathological role of CDC25A and AR in the development of prostate cancer.

In addition, CDC25A phosphatase has been implicated in the regulation of Raf-1 and the MAPK pathway. Raf-1 controls the mitogen activated protein kinase (MAPK) pathway, which has been associated with the progression of prostate cancer to the more advanced and androgen-independent disease. Nemoto et al. (2004) showed that Raf-1 interacts with

play a key role in controlling prostatic cell proliferation, which also indicates that SHP-1 expression might be a therapeutic target for treatment of prostate cancer (Zapata et al., 2002). On the other hand, by using human prostate biopsies, Cariaga-Martinez et al. (2009) observed a decrease of SHP-1 and somatostatin in prostate cancer cells, and they demonstrated that this is consistent with aggressiveness of the tumor. In addition, Wu et al. (2003) proposed the diminished or abolished SHP-1 expression could be due to mutation of the SHP-1 gene, methylation of the promoter region or post-transcriptional regulation of SHP-1 protein synthesis. It might also be explained by the action of specific

Unbalance on either expression or activity of proteins related to control of cell cycle progression provokes a wide variety of malignant diseases, including prostate cancer. Biochemically, cell cycle progression is a well orchestrated event regulated by well-defined sequential activities of cyclin-dependent kinases (CDKs), cyclins, and other proteins (Karlsson-Rosenthal and Millar, 2006). During mitosis, Cdc2/Cyclin B complexes can be dephosphorylated by the CDC25 phosphatase (a dual-specificity protein tyrosine phosphatase). CDC25 phosphatases play a critical role in regulating cell cycle progression by dephosphorylating CDKs at inhibitory residues and, therefore, have been shown to possess oncogenic potential (Karlsson-Rosenthal and Millar, 2006). In human, CDC25 proteins are encoded by a multigene family: CDC25A, CDC25B, and CDC25C (Turowski et al., 2003). It has been suggested that phosphorylation of CDC25C at Ser216 (activated Chk kinases) negatively regulate the activity of this phosphatase by an immediate cytoplasmic sequestration (Peng et al., 1997). Despite its potential role in prostate cancer, its exact

Due to its hormone-dependent nature, prostate cancer at the metastatic stage is usually treated with hormone ablation therapy. Androgen receptor (AR) is a ligand-dependent transcription factor and its activity is regulated by numerous AR coregulators. Inadequate incidence of these AR coregulators contributes for the development of prostate cancer. Current studies have shown that AR activity is modulated by phosphorylation at specific sites performed by mitogen-activated protein kinases, Akt/PKB, and cAMP-activated protein kinase A, which control AR transcriptional activity. Guo et al. (2006) reported that AR was tyrosine-phosphorylated in prostate cancer cell lines and that an elevated level of phosphorylation was detected in hormone refractory prostate tumor xenografts, demonstrating that such AR modification may contribute to androgen-independent activation of AR. Chiu et al. (2009) demonstrated for the first time that CDC25A could interact with AR and inhibit its transcriptional activity. Since CDC25A overexpression is implicated in cancer development, their findings may provide an insight into the

In addition, CDC25A phosphatase has been implicated in the regulation of Raf-1 and the MAPK pathway. Raf-1 controls the mitogen activated protein kinase (MAPK) pathway, which has been associated with the progression of prostate cancer to the more advanced and androgen-independent disease. Nemoto et al. (2004) showed that Raf-1 interacts with

pathological role of CDC25A and AR in the development of prostate cancer.

families of miRNAs.

**3.3.1 Signaling features** 

involvement remains unclear.

**3.3.2 Role in prostate cancer** 

**3.3 CDC25** 

CDC25A in PC-3 and LNCap cells and CDC25A inhibitors induced both extracellular signalregulated kinase (Erk) activation and augmented Raf-1 tyrosine phosphorylation. These results indicate that CDC25A phosphatase regulates Raf-1/MEK/Erk kinase activation in human prostate cancer cells. Indeed, CDC25A controls proliferation and survival signaling, culminating on modulation of prostate cancer progression and aggressiveness.

Moreover, to determine whether CDC25C activity is altered in prostate cancer, Ozen and Ittman (2005) have examined the expression of CDC25C and an alternatively spliced variant in human prostate cancer samples and cell lines. Interestingly, they showed that an active dephosphorylated form of CDC25C was up-regulated in prostate cancer in comparison with normal prostate tissue. In addition, they showed that at the transcriptional level, CDC25C and alternatively spliced variants were both overexpressed in prostate cancer. Finally, their findings suggest that expression of the spliced variants is correlated with biochemical recurrence.

Regarding CDC25B, Ngan et al. (2003) described that its overexpression is associated with the stage of prostate cancer, transiting from a hormone-dependent to a hormoneindependent state and contributing to prostate cancer development and progression.

#### **3.4 Acid phosphatase**

#### **3.4.1 Signaling features**

Acid phosphatases (EC 3.1.3.2), enzymes that catalyze the hydrolysis of a wide range of orthophosphate monoesters, are largely distributed in nature and have been studied in numerous organisms and tissues (Granjeiro et al, 1997; Ferreira et al, 1998a, 1998b; Granjeiro et al, 1999; Fernandes et al, 2003; Jonsson et al, 2007). The enzyme found in mammalian tissues occurs in multiple forms that differ in regard to molecular mass, substrate specificity and sensitivity to inhibitors (Granjeiro et al, 1997). Low relative molecular mass (Mr) enzymes (Mr < 20.0 kDa) are insensitive to tartrate and fluoride and strongly inhibited by SH-reacting compounds. High Mr acid phosphatases (Mr > 100.0 kDa) are inhibited by tartrate and intermediary Mr enzymes (30.0 kDa < Mr<60.0 kDa) by fluoride. In contrast to high Mr acid phosphatases, low Mr enzymes present more restricted substrate specificity, preferentially hydrolyzing p-nitrophenylphosphate, flavin mononucleotide and tyrosinephosphorylated proteins.

In 1985, Chernoff and Li reported several similarities between the low molecular weight acid phosphatase and one class of protein tyrosine phosphatase (PTP), the low molecular weight protein tyrosine phosphatase (LMWPTP).

The phosphatidic acid phosphatase (PAP) is a key enzyme in both glycerolipid biosynthesis and cellular signal transduction. It was observed that the plasma membrane-bound type 2 PAP, now known as lipid phosphate phosphatase, participates in germ cell migration, epithelial differentiation and other signaling processes (Kanoh et al, 1997; Brindley and Pilquil, 2009).

#### **3.4.2 Role in prostate cancer**

Altered acid phosphatase activities can be related to several pathological processes, such as those involving infectious, inflammatory or tumoral processes. For instance, high and intermediary molecular weight acid phosphatases levels are increased in the serum of patients with prostate carcinoma (Hudson et al. 1955), of patients suffering from spleen disorders (Kumar and Gupta, 1971), of patients with endothelial reticulum leukemia (Ketcham et al, 1985), etc.

Prostate Cancer Dephosphorylation Atlas 181

PTEN loss effects also extend to the androgen receptor (AR) activity, associated to androgen-independence. AR is shown to be inhibited by PTEN through blockage of the Akt pathway (Shen & Abate-Shen, 2007; Nan *et al.*, 2003). However, a recent study points to the opposite activities of AR and PI3K signaling pathways and their cross-regulation, with inhibition of one activating the other, maintaining cancer cell survival by distinct means. Through combined pharmacological inhibition of both pathways, the authors could achieve near-complete prostate cancer regressions in a PTEN-deficient murine model and in human

Recent studies have also implicated PTEN loss in chemokine receptor 4 (CXCR4)- mediated prostate cancer progression and metastasis, as well as showing that reactive oxygen species (ROS) can increase this outcome through direct inactivation of PTEN by active site oxidation

Dual-specificity phosphatases (DUSPs) are enzymes able to dephosphorylate both tyrosine and serine/threonine residues within their substrate (Patterson *et al.*, 2009). There are 49 gene products characterized as human DUSPs in the Gene Ontology database (The Gene Ontology Consortium, 2000). These are divided into subgroups, according to their substrate specificity. MKPs, one of the best-characterized subgroups, are able to dephosphorylate mitogen-associated protein kinases (MAPKs), which are in turn increasingly implicated in the development and progression of several cancers, including prostate cancer. Another subgroup is named atypical DUSPs. Some of them also show a preference for MAPKs as substrates, but, unlike MKPs, they are mostly of low-molecular mass and lack the N-

In spite of some clear links between some DUSPs, their substrates and specific cancer types, there is still variability in respect to their role in distinct tissue environments. The existing reports regarding prostate cancer are diverse, sometimes even antagonistic. Thus, the precise role of DUSPs in carcinogenesis remains to be clarified (Arnoldussen &

DUSP1 is a member of the MKP group. It is able to dephosphorylate all members of the MAPK family, although displaying preference for p38 and JNK substrates (Magi-Galluzzi *et* 

DUSP3 is an atypical dual-specificity phosphatase that has controversial substrate specificity. ERK 1/2 and JNK were identified as direct substrates for DUSP3 (Todd *et al.*, 1999; Todd *et al.*, 2002), although a later report points to ERK2 as an unlikely substrate for DUSP3 (Zhou *et al.*, 2002). STAT5 was also identified as a substrate for DUSP3 (Hoyt *et al.*, 2007). DUSP10 is a MKP with preference for p38 and JNK rather than ERK as substrates. It has been implicated in the regulation of innate and adaptive immune responses (Zhang *et al*., 2004) and also has been shown to have a potent anti-inflammatory activity in prostate cells

DUSP18 is a member of the atypical subgroup of dual-specificity phosphatases whose mRNA expression was identified in several cancer tissues and cell lines, including prostate, among others (Patterson *et al.*, 2009; Wu *et al.*, 2006). It presents phosphatase activity against ERK, JNK and p38 synthetic substrates, with a preference for ERK and

terminal CH2 (Cdc25 homology 2) domain (Patterson *et al.*, 2009).

*al.*, 1997; Sun *et al.*, 1993; Franklin & Kraft, 1997).

xenografts (Carver *et al.*, 2011).

(Chetram *et al.*, 2011).

Saatcioglu, 2009).

(Nonn *et al*., 2007).

JNK (Hood *et al.*, 2002).

**3.6.1 Signaling features** 

**3.6 DUSP** 

Human prostatic acid phosphatase has been used as a valuable marker for prostate cancer, before the evaluation by the prostate-specific antigen (PSA). Increased prostatic acid phosphatase serum levels are well correlated with metastatic prostate cancer (Ahmann and Schifman, 1987). In normal human prostate epithelial cells, human prostatic acid phosphatase expression is very high and guarantees the slow proliferation rate of those cells (Goldfarb et al., 1986; Veeramani et al, 2005). On the other hand, decreased activity of this phosphatase correlates with the poor differentiation of high-grade prostate cancer. One possible mechanism by which this phosphatase regulates the proliferation of prostate cancer is due to the dephosphorylation of the receptor HER-2. Uncontrolled phosphorylation of HER-2 leads to increased hormone-refractory growth of prostate cancer cells (Chuang et al, 2010).

#### **3.5 PTEN**

#### **3.5.1 Signaling features**

PTEN is a tumor suppressor protein, acting as a dual-specificity protein phosphatase. It is one of several enzymes with the ability to dephosphorylate tyrosine-, serine- and threoninephosphorylated residues (Pulido & van Huijsduijnen, 2008). It also presents lipid phosphatase activity, mainly towards phosphatidylinositol-3,4,5-triphosphate (Maehama & Dixon, 1998). This is crucial to its tumor suppressor function, since it opposes the survival and proliferative actions of many growth factors (Uzoh *et al.*, 2008).

PTEN was first described in 1997. Mutations in its encoding gene were detected, at the time, in several human cancer tissues and cell lines, including prostate cancer (Li *et al.*, 1997). Its loss has been associated mainly with activation of the PI3K/Akt/mTOR pathway, leading to proliferation and survival of cancer cells (Hollander *et al.*, 2011).

#### **3.5.2 Role in prostate cancer**

The lack of PTEN has been implicated in the resistance of prostate cancer cells to conventional chemo- and radiotherapy, as well as androgen-independence (Uzoh *et al.*, 2008; Huang *et al*., 2001; Priulla *et al.*, 2007; Anai *et al.*, 2006; Shen & Abate-Shen, 2007). In a mouse model of prostate cancer, PTEN inactivation was shown to induce growth arrest through the p53-dependent cellular senescence pathway both *in vitro* and *in vivo* (Chen *et al.*, 2005).

Chemo- and radiotherapy resistance is linked to overexpression of Bcl-2, an anti-apoptotic protein that blocks PTEN-mediated apoptosis. Huang *et al.* showed this overexpression to be related to PTEN-loss, as well as establishing an association between PTEN-induced chemosensitivity and inhibition of Bcl-2 expression (Huang *et al.*, 2001).

mTOR inhibition has also been shown to sensitize Pten-null prostate cancer cells to chemoand radiotherapy (Grunwald *et al.*, 2002; Cao *et al.*, 2006), pointing to PTEN's role in resistance. Interestingly, Cao and colleagues used an mTOR inhibitor other than rapamycin (RAD001 - everolimus) to enhance the cytotoxic effects of radiotherapy on two prostate cancer cell lines (PC-3 and DU145). They found that the increased susceptibility to radiation presented by both cell lines was due to autophagy, instead of apoptosis. They also showed that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA leads to the same effects (Cao *et al.*, 2006). TORC1/TORC2 inhibition in association with docetaxel and cisplatin also led to promising results in mice with chemoresistant prostate cancer (Gravina *et al.*, 2011).

PTEN loss effects also extend to the androgen receptor (AR) activity, associated to androgen-independence. AR is shown to be inhibited by PTEN through blockage of the Akt pathway (Shen & Abate-Shen, 2007; Nan *et al.*, 2003). However, a recent study points to the opposite activities of AR and PI3K signaling pathways and their cross-regulation, with inhibition of one activating the other, maintaining cancer cell survival by distinct means. Through combined pharmacological inhibition of both pathways, the authors could achieve near-complete prostate cancer regressions in a PTEN-deficient murine model and in human xenografts (Carver *et al.*, 2011).

Recent studies have also implicated PTEN loss in chemokine receptor 4 (CXCR4)- mediated prostate cancer progression and metastasis, as well as showing that reactive oxygen species (ROS) can increase this outcome through direct inactivation of PTEN by active site oxidation (Chetram *et al.*, 2011).

#### **3.6 DUSP**

180 Prostate Cancer – From Bench to Bedside

Human prostatic acid phosphatase has been used as a valuable marker for prostate cancer, before the evaluation by the prostate-specific antigen (PSA). Increased prostatic acid phosphatase serum levels are well correlated with metastatic prostate cancer (Ahmann and Schifman, 1987). In normal human prostate epithelial cells, human prostatic acid phosphatase expression is very high and guarantees the slow proliferation rate of those cells (Goldfarb et al., 1986; Veeramani et al, 2005). On the other hand, decreased activity of this phosphatase correlates with the poor differentiation of high-grade prostate cancer. One possible mechanism by which this phosphatase regulates the proliferation of prostate cancer is due to the dephosphorylation of the receptor HER-2. Uncontrolled phosphorylation of HER-2 leads to increased hormone-refractory growth of prostate

PTEN is a tumor suppressor protein, acting as a dual-specificity protein phosphatase. It is one of several enzymes with the ability to dephosphorylate tyrosine-, serine- and threoninephosphorylated residues (Pulido & van Huijsduijnen, 2008). It also presents lipid phosphatase activity, mainly towards phosphatidylinositol-3,4,5-triphosphate (Maehama & Dixon, 1998). This is crucial to its tumor suppressor function, since it opposes the survival

PTEN was first described in 1997. Mutations in its encoding gene were detected, at the time, in several human cancer tissues and cell lines, including prostate cancer (Li *et al.*, 1997). Its loss has been associated mainly with activation of the PI3K/Akt/mTOR pathway, leading to

The lack of PTEN has been implicated in the resistance of prostate cancer cells to conventional chemo- and radiotherapy, as well as androgen-independence (Uzoh *et al.*, 2008; Huang *et al*., 2001; Priulla *et al.*, 2007; Anai *et al.*, 2006; Shen & Abate-Shen, 2007). In a mouse model of prostate cancer, PTEN inactivation was shown to induce growth arrest through the p53-dependent cellular senescence pathway both *in vitro* and *in vivo* (Chen *et* 

Chemo- and radiotherapy resistance is linked to overexpression of Bcl-2, an anti-apoptotic protein that blocks PTEN-mediated apoptosis. Huang *et al.* showed this overexpression to be related to PTEN-loss, as well as establishing an association between PTEN-induced

mTOR inhibition has also been shown to sensitize Pten-null prostate cancer cells to chemoand radiotherapy (Grunwald *et al.*, 2002; Cao *et al.*, 2006), pointing to PTEN's role in resistance. Interestingly, Cao and colleagues used an mTOR inhibitor other than rapamycin (RAD001 - everolimus) to enhance the cytotoxic effects of radiotherapy on two prostate cancer cell lines (PC-3 and DU145). They found that the increased susceptibility to radiation presented by both cell lines was due to autophagy, instead of apoptosis. They also showed that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA leads to the same effects (Cao *et al.*, 2006). TORC1/TORC2 inhibition in association with docetaxel and cisplatin also led to promising results in mice with chemoresistant prostate cancer

and proliferative actions of many growth factors (Uzoh *et al.*, 2008).

proliferation and survival of cancer cells (Hollander *et al.*, 2011).

chemosensitivity and inhibition of Bcl-2 expression (Huang *et al.*, 2001).

cancer cells (Chuang et al, 2010).

**3.5.1 Signaling features** 

**3.5.2 Role in prostate cancer** 

**3.5 PTEN** 

*al.*, 2005).

(Gravina *et al.*, 2011).

#### **3.6.1 Signaling features**

Dual-specificity phosphatases (DUSPs) are enzymes able to dephosphorylate both tyrosine and serine/threonine residues within their substrate (Patterson *et al.*, 2009). There are 49 gene products characterized as human DUSPs in the Gene Ontology database (The Gene Ontology Consortium, 2000). These are divided into subgroups, according to their substrate specificity. MKPs, one of the best-characterized subgroups, are able to dephosphorylate mitogen-associated protein kinases (MAPKs), which are in turn increasingly implicated in the development and progression of several cancers, including prostate cancer. Another subgroup is named atypical DUSPs. Some of them also show a preference for MAPKs as substrates, but, unlike MKPs, they are mostly of low-molecular mass and lack the Nterminal CH2 (Cdc25 homology 2) domain (Patterson *et al.*, 2009).

In spite of some clear links between some DUSPs, their substrates and specific cancer types, there is still variability in respect to their role in distinct tissue environments. The existing reports regarding prostate cancer are diverse, sometimes even antagonistic. Thus, the precise role of DUSPs in carcinogenesis remains to be clarified (Arnoldussen & Saatcioglu, 2009).

DUSP1 is a member of the MKP group. It is able to dephosphorylate all members of the MAPK family, although displaying preference for p38 and JNK substrates (Magi-Galluzzi *et al.*, 1997; Sun *et al.*, 1993; Franklin & Kraft, 1997).

DUSP3 is an atypical dual-specificity phosphatase that has controversial substrate specificity. ERK 1/2 and JNK were identified as direct substrates for DUSP3 (Todd *et al.*, 1999; Todd *et al.*, 2002), although a later report points to ERK2 as an unlikely substrate for DUSP3 (Zhou *et al.*, 2002). STAT5 was also identified as a substrate for DUSP3 (Hoyt *et al.*, 2007).

DUSP10 is a MKP with preference for p38 and JNK rather than ERK as substrates. It has been implicated in the regulation of innate and adaptive immune responses (Zhang *et al*., 2004) and also has been shown to have a potent anti-inflammatory activity in prostate cells (Nonn *et al*., 2007).

DUSP18 is a member of the atypical subgroup of dual-specificity phosphatases whose mRNA expression was identified in several cancer tissues and cell lines, including prostate, among others (Patterson *et al.*, 2009; Wu *et al.*, 2006). It presents phosphatase activity against ERK, JNK and p38 synthetic substrates, with a preference for ERK and JNK (Hood *et al.*, 2002).

Prostate Cancer Dephosphorylation Atlas 183

CDC25A nucleus cancer suppressor Androgen receptor (AR)

Our research on this field is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Ahmann, F.R. and Schifman, R.B. (1987) Prospective comparison between serum

Alonso, A., Sasin, J., Bottini, N., Friedberg, I., Friedberg, I., Osterman, A., Godzik, A.,

Anai, S *et al.* (2006) Combination of PTEN gene therapy and radiation inhibits the growth of

Andersen, J.N., Jansen, P.G., Echwald, S.M., Mortensen, O.H., Fukada, T., Del Vecchio, R.,

Arnoldussen, Y.J., Lorenzo, P.I., Pretorius, M.E., Waehre, H., Risberg, B., Maelandsmo, G.M.,

Arnoldussen, YJ & Saatcioglu, F (2009) Dual specificity phosphatases in prostate cancer. *Mol* 

Bialy, L., Waldmann, H. Inhibitors of protein tyrosine phosphatases: next-generation drugs?

Bispo de Jesus, M., Zambuzzi, W.F., Ruela de Sousa, R.R., Areche, c., Souza, A.C.S., Aoyama,

human prostate cancer xenografts. *Hum Gene Ther* 17, 975-984.

and is overexpressed in prostate cancer. *Cancer Res* 68, 9255-9264.

monoclonal prostate specific antigen and acid phosphatase measurements in

Hunter, T., Dixon, J. and Mustelin, T. (2004) Protein tyrosine phosphatases in the

Tonks, N.K., Moller, N.P. (2004) A genomic perspective on protein tyrosine phosphatases: gene structure, pseudogenes, and genetic disease linkage, *FASEB J.*

Danielsen, H.E., Saatcioglu, F. (2008) The mitogen-activated protein kinase phosphatase vaccinia H1-related protein inhibits apoptosis in prostate cancer cells

H., Schmeda-Hirschmann, G., Rodrigues, J.A., Brito, A.R.M.S., Peppelenbosch, M.P., den Hertog, J., de Paula, E. and Ferreira, C.V. (2008) Ferruginol suppresses

DUSP nucleus cancer promoter JNK, p38 and ERK

LMWPTP cytosol cancer promoter unknown

CDC25C nucleus cancer promoter unknown

Acid phosphatase cytosol cancer suppressor Her-2

SHP-1 cytosol cancer suppressor

metastatic prostatic cancer. *J. Urol.* 137, 431-434.

(2005) *Angew. Chem. Int. Ed. Engl.* 44, 3814-3839.

human genome. *Cell* 117, 699-711

Table 2. Prostate cancer protein tyrosine phosphatases

cell localization Main action Main targets in prostate

nucleus cancer suppressor phosphatidylinositol-

cancer

IL-3R, PDGF- and EGF

3,4,5-triphosphate

receptors

Phosphatase Prostate cancer

PTEN cytosol and

**5. Acknowledgment** 

18, 8-30.

*Cell Endocrinol* 309, 1-7.

**6. References** 

CDC25B and

#### **3.6.2 Role in prostate cancer**

DUSP1 mRNA was found to be overexpressed in the early phases of prostate cancer, but this did not prevent high ERK-1 expression (Loda *et al.*, 1996). Overall, ERK appears to increase DUSP1 expression, decreasing JNK activity and inhibiting apoptosis. In a 2008 study it was shown that coordinate inhibition of AKT/mTOR and ERK-1/MAPK pathways leads to reduced cell growth and proliferation, as well as upregulation of the apoptotic regulator Bcl-2-interacting mediator of cell death (Bim) in a preclinical mouse model of hormone-refractory prostate cancer (Kinkade *et al.*, 2008). Accordingly, a later study showed DUSP1 mRNA expression to be lower in hormone-refractory prostate carcinomas than in benign prostate hyperplasia (BPH) or untreated prostate carcinomas. Higher DUSP1 protein levels were found in BPH, normal prostate and high-grade prostate intraepithelial neoplasia (Rauhala *et al.*, 2005). Consistent with the low levels of DUSP1 in response to androgen ablation, DUSP1 mRNA was found to be upregulated upon androgen treatment of LNCaP cells (Arnoldussen *et al.*, 2008) The androgen receptor (AR) has been identified as responsible for increased expression of DUSP1 (and several other DUSPs) upon interaction with testosterone. However, DUSP1 implication in prostate cancer is not yet fully resolved, since there have been reports showing that both high and low levels of DUSP1 may have an antiapoptotic effect, depending on which MAP kinase DUSP1 is targeting (Rauhala *et al.*, 2005).

Similarly, androgens protect LNCaP cells from 12-O-tetradecanoylphorbol-13-acetate- and thapsigargin-induced apoptosis via down-regulation of JNK activity through an increase in DUSP3 expression. This effect was not observed in androgen-independent DU145 cells (Arnoldussen *et al.*, 2008). Expression analysis in human prostate cancer specimens also show that DUSP3 is increased in prostate cancer compared with normal prostate, evidencing a direct DUSP3 role in prostate cancer progression through JNK-mediated apoptosis inhibition (Arnoldussen *et al*, 2008).

Another DUSP that has been related to prostate cancer is DUSP10. DUSP10 presents antiinflammatory activity and its expression is increased after treatment with calcitriol, the hormonally active form of vitamin D. This results in the subsequent inhibition of p38 stress kinase signaling and the attenuation of the production of pro-inflammatory cytokines (Nonn, *et al.*, 2006; Krishnan & Feldman, 2010).

## **4. Concluding remarks**

Deciphering the molecular networks that distinguish progressive from non-progressive prostate cancer will bring light on the biology of this tumor, as well as lead to the identification of biomarkers that will aid to the selection of better-suited treatments for each patient. For this, it is crucial to characterize and integrate the molecular mediators involved in prostate cancer biology. In this chapter we pointed out some evidences of the contribution of protein tyrosine phosphatases for prostate cancer pathogenesis. PTPs, as a large enzyme family, can act as prostate cancer suppressors or promoters, depending on their target protein (Table 2). However, studies quantifying PTPs on gene and protein levels in prostate cancer have been limited. New efforts to raise this kind of combinatory data might reveal a spectacular relationship between genotype and PTP activity levels and lead to an understanding of the fundamental role of this enzyme family in controlling malignant cell transformation. This, in turn, may open new avenues to treat prostate cancer based on PTP activity modulation.

DUSP1 mRNA was found to be overexpressed in the early phases of prostate cancer, but this did not prevent high ERK-1 expression (Loda *et al.*, 1996). Overall, ERK appears to increase DUSP1 expression, decreasing JNK activity and inhibiting apoptosis. In a 2008 study it was shown that coordinate inhibition of AKT/mTOR and ERK-1/MAPK pathways leads to reduced cell growth and proliferation, as well as upregulation of the apoptotic regulator Bcl-2-interacting mediator of cell death (Bim) in a preclinical mouse model of hormone-refractory prostate cancer (Kinkade *et al.*, 2008). Accordingly, a later study showed DUSP1 mRNA expression to be lower in hormone-refractory prostate carcinomas than in benign prostate hyperplasia (BPH) or untreated prostate carcinomas. Higher DUSP1 protein levels were found in BPH, normal prostate and high-grade prostate intraepithelial neoplasia (Rauhala *et al.*, 2005). Consistent with the low levels of DUSP1 in response to androgen ablation, DUSP1 mRNA was found to be upregulated upon androgen treatment of LNCaP cells (Arnoldussen *et al.*, 2008) The androgen receptor (AR) has been identified as responsible for increased expression of DUSP1 (and several other DUSPs) upon interaction with testosterone. However, DUSP1 implication in prostate cancer is not yet fully resolved, since there have been reports showing that both high and low levels of DUSP1 may have an antiapoptotic effect, depending on which MAP kinase

Similarly, androgens protect LNCaP cells from 12-O-tetradecanoylphorbol-13-acetate- and thapsigargin-induced apoptosis via down-regulation of JNK activity through an increase in DUSP3 expression. This effect was not observed in androgen-independent DU145 cells (Arnoldussen *et al.*, 2008). Expression analysis in human prostate cancer specimens also show that DUSP3 is increased in prostate cancer compared with normal prostate, evidencing a direct DUSP3 role in prostate cancer progression through JNK-mediated apoptosis

Another DUSP that has been related to prostate cancer is DUSP10. DUSP10 presents antiinflammatory activity and its expression is increased after treatment with calcitriol, the hormonally active form of vitamin D. This results in the subsequent inhibition of p38 stress kinase signaling and the attenuation of the production of pro-inflammatory cytokines

Deciphering the molecular networks that distinguish progressive from non-progressive prostate cancer will bring light on the biology of this tumor, as well as lead to the identification of biomarkers that will aid to the selection of better-suited treatments for each patient. For this, it is crucial to characterize and integrate the molecular mediators involved in prostate cancer biology. In this chapter we pointed out some evidences of the contribution of protein tyrosine phosphatases for prostate cancer pathogenesis. PTPs, as a large enzyme family, can act as prostate cancer suppressors or promoters, depending on their target protein (Table 2). However, studies quantifying PTPs on gene and protein levels in prostate cancer have been limited. New efforts to raise this kind of combinatory data might reveal a spectacular relationship between genotype and PTP activity levels and lead to an understanding of the fundamental role of this enzyme family in controlling malignant cell transformation. This, in turn, may open new avenues to treat prostate cancer based on PTP

**3.6.2 Role in prostate cancer** 

DUSP1 is targeting (Rauhala *et al.*, 2005).

inhibition (Arnoldussen *et al*, 2008).

**4. Concluding remarks** 

activity modulation.

(Nonn, *et al.*, 2006; Krishnan & Feldman, 2010).


Table 2. Prostate cancer protein tyrosine phosphatases

#### **5. Acknowledgment**

Our research on this field is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

#### **6. References**


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**10** 

*USA* 

Glenn Tisman

*Cancer Research Building, Whittier, CA* 

**Modulation of One-Carbon Metabolism by** 

**and Progression of Prostate Cancer** 

**B Vitamins: Implications for Transformation** 

Extensive laboratory, epidemiological and clinical investigations suggest that prostate cancer might be affected by enhanced folate or B12 ingestion and or other perturbations of one-carbon (CH3—) metabolism. Over the last decade, largely due to government mandated dietary fortification with folic acid (FA), our clinic patients experienced a 4-6-fold increase in the median level of serum folate (5 ng/ml 24 ng/ml). The National Health and Nutrition Examination Surveys (NHANES) confirm similar elevated levels Figure 1 (Dietrich et al,

Fig. 1. Median serum/plasma blood folate levels NHANES and GT data.

**1. Introduction** 

2005; McDowell et al, 2008; Yang et al, 2010).

