Upregulation of epidermal growth factor receptor (EGFR) and subsequent increases in extracellular-regulated kinase (ERK) and Akt signaling are implicated in prostate cancer progression. Impaired endocytic downregulation of EGFR also contributes to oncogenic phenotypes such as metastasis. Thus, understanding the roles of divergent signaling pathways in the regulation of EGFR trafficking and EGFRdriven invasive migration may enable the development of more effective therapies. In this study, we use the human prostate cancer cell lines, DU145 and PC3, to investigate the effects of both the ERK and Akt pathways on epidermal growth factor (EGF)-mediated EGFR signaling, trafficking and cell motility. We show that DU145 and PC3 cells overexpress EGFR and migrate in a ligand (EGF)-dependent manner. Next, we show that pharmacological inhibition of ERK (but not Akt) signaling enhances EGFinduced EGFR activation, ubiquitination and downregulation, and may lead to enhanced receptor turnover. These findings negatively correlate with ERK-mediated threonine phosphorylation of EGFR, implicating it as a possible mechanism. Further, we uncover that EGF promotes disassembly of cell-cell junctions, downregulation of E-cadherin and upregulation of the transcriptional repressor, Snail, typical characteristics of epithelial-mesenchymal transition (EMT). These effects are dependent on activation of Akt, as inhibition of Akt signaling abolishes EGF/EGFR-driven cell migration and EMT. Knockdown of endogenous Snail also prevents EGFR-mediated downregulation of E-cadherin, EMT and cell migration. Surprisingly, inhibition of the ERK pathway augments EGFR-dependent motility, occurring concomitantly with elevation of EGF-induced Akt activity. Collectively, our results suggest that EGF-triggered ERK activation has profound feedback on EGFR signaling and trafficking by EGFR threonine phosphorylation, and Akt has a pivotal role in EGFR-mediated cell migration by activating EMT. More important, our results also suggest that therapeutic targeting of ERK signaling may have undesirable outcomes (for example, augmenting EGFR-driven motility).

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Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair, and occurs during pathologic processes of tumor invasiveness, metastasis, and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. Although it is well documented that transforming growth factor (TGF)-β plays a central role in the induction of EMT, the targets of TGF-β signaling are poorly defined. We have shown earlier that Na,K-ATPase β 1 -subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-β 1 -mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-β 1 , the surface expression of the β 1 -subunit of Na,K-ATPase is reduced, before well-characterized EMT markers, and is associated with the acquisition of a mesenchymal phenotype. RNAi-mediated knockdown confirmed the specific involvement of the Na,K-ATPase β 1 -subunit in the loss of the epithelial phenotype and exogenous overexpression of the Na,K-ATPase β 1 -subunit attenuated TGF-β 1 -mediated EMT. We further show that both Na,K-ATPase α-and β-subunit levels are highly reduced in renal fibrotic tissues. These findings reveal for the first time that Na,K-ATPase is a target of TGF-β 1 -mediated EMT and is associated with the progression of EMT in cancer and fibrosis. Mol Cancer Ther; 9(6); 1515-24. ©2010 AACR.

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Lung cancer is the leading cause of cancer deaths worldwide; approximately 85% of these cancers are non-small cell lung cancer (NSCLC). Patients with NSCLC frequently have tumors harboring somatic mutations in the epidermal growth factor receptor (EGFR) gene that cause constitutive receptor activation. These patients have the best clinical response to EGFR tyrosine kinase inhibitors (TKIs). Herein, we show that fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is frequently overexpressed in NSCLC tumors, and Fn14 levels correlate with p-EGFR expression. We also report that NSCLC cell lines that contain EGFR-activating mutations show high levels of Fn14 protein expression. EGFR TKI treatment of EGFR-mutant HCC827 cells decreased Fn14 protein levels, whereas EGF stimulation of EGFR wild-type A549 cells transiently increased Fn14 expression. Furthermore, Fn14 is highly expressed in EGFR-mutant H1975 cells that also contain an EGFR TKI-resistance mutation, and high TKI doses are necessary to reduce Fn14 levels. Constructs encoding EGFRs with activating mutations induced Fn14 expression when expressed in rat lung epithelial cells. We also report that short hairpin RNA-mediated Fn14 knockdown reduced NSCLC cell migration and invasion in vitro. Finally, Fn14 overexpression enhanced NSCLC cell migration and invasion in vitro and increased experimental lung metastases in vivo. Thus, Fn14 may be a novel therapeutic target for patients with NSCLC, in particular for those with EGFR-driven tumors who have either primary or acquired resistance to EGFR TKIs.

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Glioblastoma multiforme (GBM) is the most common brain malignancies in adults. Most GBM patients succumb to the disease less than 1 year after diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radioresistant and chemoresistant properties to the tumor cells; therefore, there is a critical need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat signaling pathway. Here, it is reported that EGFRvIII activates Stat5 and GBM invasion by inducing the expression of a previously established mediator of glioma cell invasion and survival: fibroblast growth factor-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is Stat5 dependent and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon Src-MEK/ERK-Stat3 activation. Notably, treatment of EGFRvIII-expressing GBM cells with the FDA-approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration and survival. Because EGFR inhibitors display limited therapeutic efficacy in GBM patients, the EGFRvIII-Stat5-Fn14 signaling pathway represents a node of vulnerability in the invasive GBM cell populations. Targeting critical effectors in the EGFRvIII-Stat5-Fn14 pathway may limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival. .

sEC, MMP-9, TIMP-1, and CCL15 levels correlate with response to combination therapy with erlotinib and celecoxib in patients with NSCLC. A randomized phase II trial is planned comparing erlotinib and celecoxib with erlotinib plus placebo in advanced NSCLC. This study will prospectively assess these and other biomarkers in serum and tumor tissue.