Oncogene (2010) 29, 4947–4958 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Differential roles of ERK and Akt pathways in regulation of EGFR-mediated signaling and motility in prostate cancer cells YGan1, C Shi1,3, L Inge2, M Hibner1, J Balducci1 and Y Huang1 1Department of Obstetrics and Gynecology, St Joseph’s Hospital and Medical Center, Phoenix, AZ, USA; 2Heart and Lung Institute, St Joseph’s Hospital and Medical Center, Phoenix, AZ, USA and 3Department of Pathogen Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China Upregulation of epidermal growth factor receptor (EGFR) Oncogene (2010) 29, 4947–4958; doi:10.1038/onc.2010.240; and subsequent increases in extracellular-regulated kinase published online 21 June 2010 (ERK) and Akt signaling are implicated in prostate cancer progression. Impaired endocytic downregulation of EGFR Keywords: EGFR; ERK; Akt; phosphorylation; signal- also contributes to oncogenic phenotypes such as metastasis. ing; cell migration Thus, understanding the roles of divergent signaling path- ways in the regulation of EGFR trafficking and EGFR- driven invasive migration may enable the development of Introduction more effective therapies. In this study, we use the human prostate cancer cell lines, DU145 and PC3, to investigate The molecular mechanisms of prostate cancer are still the effects of both the ERK and Akt pathways on epidermal poorly understood, despite the threat that prostate growth factor (EGF)-mediated EGFR signaling, trafficking cancer poses to the health of men worldwide. As and cell motility. We show that DU145 and PC3 cells prostate tumors are initially dependent on androgens overexpress EGFR and migrate in a ligand (EGF)- for growth and survival, androgen deprivation therapy dependent manner. Next, we show that pharmacological is commonly the first-line treatment for prostate cancer inhibition of ERK (but not Akt) signaling enhances EGF- patients. However, prostate cancer cells can develop a induced EGFR activation, ubiquitination and downregula- hormonal-refractory (androgen independent) state, ren- tion, and may lead to enhanced receptor turnover. These dering principal treatment options palliative because of findings negatively correlate with ERK-mediated threonine the acquisition of invasive and metastatic capacities phosphorylation of EGFR, implicating it as a possible associated with androgen independence. To date, no mechanism. Further, we uncover that EGF promotes effective therapy allows the abrogation of prostate disassembly of cell–cell junctions, downregulation of cancer’s progression to advanced, invasive forms. E-cadherin and upregulation of the transcriptional Recent evidence suggests that acquisition of androgen repressor, Snail, typical characteristics of epithelial–me- independence may be due to upregulation of growth senchymal transition (EMT). These effects are dependent factor/receptor signaling pathways, principally the on activation of Akt, as inhibition of Akt signaling abolishes epidermal growth factor receptor (EGFR) (Kambham- EGF/EGFR-driven cell migration and EMT. Knockdown pati et al., 2005; Traish and Morgentaler, 2009), making of endogenous Snail also prevents EGFR-mediated down- EGFR an attractive target for therapeutic intervention. regulation of E-cadherin, EMT and cell migration. However, the exact contribution that EGFR makes to Surprisingly, inhibition of the ERK pathway augments prostate cancer progression remains unclear. EGFR-dependent motility, occurring concomitantly with EGFR regulates cell growth, differentiation, motility, elevation of EGF-induced Akt activity. Collectively, our adhesion and tumorigenesis through interaction with its results suggest that EGF-triggered ERK activation has cognate ligand, epidermal growth factor (EGF). EGFR profound feedback on EGFR signaling and trafficking by is the prototype of the ErbB family, which also includes EGFR threonine phosphorylation, and Akt has a pivotal ErbB-2, 3 and 4, and is expressed in nearly all epithelial role in EGFR-mediated cell migration by activating EMT. tissues. EGF engagement activates EGFR’s intrinsic More important, our results also suggest that therapeutic kinase and leads to activation of several downstream targeting of ERK signaling may have undesirable outcomes intracellular signaling pathways, including rat sarcoma– (for example, augmenting EGFR-driven motility). MAPK kinase (MEK)–extracellular-related kinase (ERK) and phosphoinositide 3-kinase (PI3K)–Akt pathways, responsible for a variety of mitogenic, metastatic and other tumor-promoting cellular activities Correspondence: Dr Y Huang, Department of Obstetrics and (Wells, 1999; Grant et al., 2002). On EGF binding, Gynecology, St Joseph’s Hospital and Medical Center, 445 North EGFR undergoes a process of internalization, ubiqui- 5th Street, Suite 110, Phoenix, AZ 85004, USA. E-mail: [email protected] tination and destruction (known as EGFR endo- Received 21 January 2010; revised 3 May 2010; accepted 22 May 2010; cytosis and trafficking), resulting in temporary EGFR published online 21 June 2010 downregulation (Wiley, 2003; Citri and Yarden, 2006). ERK and Akt in EGFR signaling and cell motility Y Gan et al 4948 Impaired endocytic downregulation of EGFR is fre- may have undesirable outcomes (for example, enhancing quently associated with cancer, as it can lead to EGFR-driven prostate cancer cell migration). uncontrolled signaling and thus to oncogenic pheno- types (Grandal and Madshus, 2008; Roepstorff et al., 2008). Further data suggest that signaling can also emanate from the EGFR in the process of postendocytic Results trafficking (Wiley, 2003; Sebastian et al., 2006). Indeed, DU145 and PC3 cells highly express EGFR, but not we have previously shown that phosphorylation of ErbB-2, and are responsive to EGF EGFR at threonine-669 by ERK can influence receptor signaling and trafficking (Huang et al., 2003, 2004, 2006; The well-characterized human prostate cancer cell lines, Li et al., 2008). However, the effects that ERK- DU145 and PC3, are both androgen insensitive (van et al. dependent EGFR phosphorylation has on the quanti- Bokhoven , 2003), making them excellent models to tative and qualitative output from EGFR and cancerous study the consequences of EGF-mediated signaling in behaviors, such as invasive migration, remain poorly hormone-refractory prostate cancer. Compared with an understood. Clinically, upregulation of EGFR and/or androgen-responsive human prostate cancer cell line, et al. ErbB-2 signaling is associated with more aggressive LnCap (van Bokhoven , 2003), EGFR proteins behavior in a broad spectrum of human cancers and were overexpressed in DU145 and PC3, whereas ErbB-2 correlates with poor prognosis (Yarden and Sliwkowski, was mainly expressed in LnCap cells (Supplementary 2001; Mendelsohn and Baselga, 2003). Thus, it is Figure 1a), indicating that the two androgen-indepen- of particular importance to understand the roles of dent cell lines (DU145 and PC3) predominantly divergent downstream pathways in the regulation of expressed EGFR, but not ErbB-2. Phosphorylation of 1068Y is known to correlate with EGFR kinase EGFR trafficking and EGFR-mediated cellular pro- residue activation (Rojas et al., 1996). As shown in Supplemen- cesses, as it will enable the development of more effective and selective therapies. tary Figure 1b, EGF induced phosphorylation (activa- tion) of EGFR and activated ERK and Akt in both Acquisition of migratory properties is a prerequisite DU145 and PC3 cells. Notably, Akt was basally for cancer progression and for invasive migration of tumor cells into surrounding tissue. Within carcinoma activated in PC3 (Supplementary Figure 1b, lane 3), consistent with a previous report that this cell line (cancer of epithelial origin) cells, acquisition of inva- harbors PTEN deletion (Davies et al., 2002). Further, siveness requires a dramatic morphological alteration, EGF markedly promoted prostate cancer cell migration termed epithelial–mesenchymal transition (EMT), as measured by wound closure assays (Supplementary wherein carcinoma cells lose their epithelial character- Figure 1c). Collectively, overexpression of EGFR and istics of cell polarity and cell–cell adhesion and switch to robust activation of ERK and Akt in response to EGF a motile mesenchymal phenotype (Thiery, 2002; Thiery and Sleeman, 2006). Disruption of cell–cell adherens in DU145 and PC3 cells make these cells appealing systems for evaluating the roles of ERK and Akt junctions mediated by E-cadherin (one of the epithelial pathways in EGFR-mediated actions. markers) is considered a crucial step in EMT and downregulation of E-cadherin is common in metastatic carcinomas (Cavallaro and Christofori, 2004). Reduced Inhibition of the ERK pathway enhances EGF-induced E-cadherin expression has been found in high-grade EGFR activation and downregulation prostate cancers and is associated with poor prognosis To understand the functional impact of ERK and Akt (Umbas et al., 1992, 1994), reflective of its critical role in inhibition on EGFR activation and trafficking in tumor progression. It is widely believed that down- prostate cancer cells, we used two pharmacological regulation of E-cadherin occurs through transcriptional inhibitors, PD98059 and LY294002, to specifically repression mediated by the protein, Snail (Cano et al., block the MEK–ERK and PI3K–Akt pathway, respec- 2000; Peinado et al., 2007; Moreno-Bueno et al., 2008). tively (Figure