Propagation of Human Prostate Cancer Stem-Like Cells Occurs through EGFR-Mediated ERK Activation

Adrian P Rybak,Damu Tang,Alistair J Ingram,Dean G Tang

doi:10.1371/journal.pone.0061716

Abstract

Prostate cancer stem-like cells (PCSCs) are being intensely investigated largely owing to their contributions towards prostate tumorigenesis, however, our understanding of PCSC biology, including their critical pathways, remains incompletely understood. While epidermal growth factor (EGF) is widely used in maintaining PCSC cells in vitro, the importance of EGF-dependent signaling and its downstream pathways in PCSC self-renewal are not well characterized. By investigating DU145 sphere cells, a population of prostate cancer cells with stem-like properties, we report here that epidermal growth factor receptor (EGFR) signaling plays a critical role in the propagation of DU145 PCSCs. Activation of EGFR signaling via addition of EGF and ectopic expression of a constitutively-active EGFR mutant (EGFRvIII) increased sphere formation. Conversely, inhibition of EGFR signaling by using EGFR inhibitors (AG1478 and PD168393) and knockdown of EGFR significantly inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signaling, activation of the MEK-ERK pathway contributed to EGFR-facilitated PCSC propagation. Modulation of EGFR signaling affected extracellular signal-related kinase (ERK) activation. Inhibition of ERK activation through multiple approaches, including treatment with the MEK inhibitor U0126, ectopic expression of dominant-negative MEK1(K97M), and knockdown of either ERK1 or ERK2 resulted in a robust reduction in PCSC propagation. Collectively, the present study provides evidence that EGFR signaling promotes PCSC self-renewal, in part, by activating the MEK-ERK pathway.

Highlights

Prostate cancer is the most common male malignancy and the second leading cause of cancer-related deaths in males in Western countries [1,2]
We have shown that epidermal growth factor (EGF) plays a critical role in long-term propagation of DU145 prostate cancer stem-like cells (PCSCs), and that these stem-like cells were capable of initiating tumors with a significantly enhanced ability in non-obese, diabetic/severe combined immunodeficient (NOD/ SCID) mice [11]
We have previously reported that while EGF supplementation significantly promoted the generation and propagation of DU145 spheres, the formation of spheres did not require the addition of exogenous EGF [11] suggesting that epidermal growth factor receptor (EGFR) signaling may be activated in the absence of exogenous EGF

Summary

Introduction

Prostate cancer is the most common male malignancy and the second leading cause of cancer-related deaths in males in Western countries [1,2]. Despite the mounting evidence suggesting the existence of PCSCs, identification of human PCSCs in vivo has appeared to be a challenging task This challenge is largely due to the heterogeneous nature of prostate cancer and the limited samples available from clinical sources. To advance our knowledge of PCSCs, several research groups, including ours, have enriched for PCSCs from human prostate cancer cell lines This is largely attributable to the demonstration that cancer stem cells (CSCs) can be studied using the sphere culture assay under serum-free (SF) media conditions. This assay has been used to derive and propagate CSCs from brain [8], breast [9], colon [10] and prostate cancer cells [11,12,13,14,15,16] in vitro. The sphere culture approach has allowed the propagation of prostate cancer stem-like cells that display CSC properties of self-renewal and the ability to initiate tumor formation in vivo [11,12,15,17]

Methods

Results

Conclusion