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The cells of the prostate gland are dependent on cell signaling pathways to regulate their growth, maintenance and function. However, perturbations in key signaling pathways, resulting in neoplastic transformation of cells in the prostate epithelium, are likely to generate subtypes of prostate cancer which may subsequently require different treatment regimes. Accumulating evidence supports multiple sources of stem cells in the prostate epithelium with distinct cellular origins for prostate tumorigenesis documented in animal models, while human prostate cancer stem-like cells (PCSCs) are typically enriched by cell culture, surface marker expression and functional activity assays. As future therapies will require a deeper understanding of its cellular origins as well as the pathways that drive PCSC maintenance and tumorigenesis, we review the molecular and functional evidence supporting dysregulation of PI3K/AKT, RAS/MAPK and STAT3 signaling in PCSCs, the development of castration resistance, and as a novel treatment approach for individual men with prostate cancer.

SOX2 is an essential transcription factor for stem cells and plays a role in tumorigenesis, however its role in prostate cancer stem cells (PCSCs) remains unclear. We report here a significant upregulation of SOX2 at both mRNA and protein levels in DU145 PCSCs propagated as suspension spheres in vitro. The expression of SOX2 in DU145 PCSCs is positively regulated by epidermal growth factor receptor (EGFR) signaling. Activation of EGFR signaling, following the addition of epidermal growth factor (EGF) or ectopic expression of a constitutively-active EGFR mutant (EGFRvIII), increased SOX2 expression and the self-renewal of DU145 PCSCs. Conversely, a small molecule EGFR inhibitor (AG1478) blocked EGFR activation, reduced SOX2 expression and inhibited PCSC self-renewal activity, implicating SOX2 in mediating EGFR-dependent self-renewal of PCSCs. In line with this notion, ectopic SOX2 expression enhanced EGF-induced self-renewal of DU145 PCSCs, while SOX2 knockdown reduced PCSC self-renewal with EGF treatment no longer capable of enhancing their propagation. Furthermore, SOX2 knockdown reduced the capacity of DU145 PCSCs to grow under anchorage-independent conditions. Finally, DU145 PCSCs generated xenograft tumors more aggressively with elevated levels of SOX2 expression compared to xenograft tumors derived from non-PCSCs. Collectively, we provide evidence that SOX2 plays a critical role in EGFR-mediated self-renewal of DU145 PCSCs., (© 2013.)

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.

While accumulating evidence demonstrates the existence of prostate cancer stem cells (PCSCs), PCSCs have not been isolated and thoroughly characterized. We report here the enrichment and characterization of sphere-propagating cells with stem-like properties from DU145 PC cells in a defined serum-free medium (SFM). Approximately 1.25% of monolayer DU145 cells formed spheres in SFM and 26% of sphere cells formed secondary spheres. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins (34βE12 and CK18) and for cancer stem cell markers, including CD44, CD24, and integrin α2β1. Upon culturing spheres under differentiating media conditions in the presence of 10% serum, cells positive for CD44 and CD24 were substantially reduced. Furthermore, spheres could be generated from the sphere-derived adherent cell cultures and xenograft tumors, demonstrating the stemness of DU145 spheres. We have maintained spheres for more than 30 passages within 1.5years without noticeable loss of their "stemness". Sphere cells possess self-renewal capacity, display significant increases in proliferation potential, and initiate xenograft tumors with enhanced capacity compared to monolayer DU145 cells. While EGF promoted the generation and maintenance of these stem-like cells, bFGF inhibited these events. Sphere cells proliferate slowly with a significant reduction in the activation of the PI3K-AKT pathway compared to monolayer DU145 cells. While knockdown of PTEN enhanced AKT activation, this did not affect the generation of primary spheres and the propagation of secondary spheres. Consistent with this observation, we were able to demonstrate the generation and propagation of spheres without the addition of external growth factors. This article is part of a Special Issue entitled: 11th European Symposium on Calcium., (2011 Elsevier B.V. All rights reserved.)