Seminar article
Oncogenic activation of androgen receptor

https://doi.org/10.1016/j.urolonc.2008.06.002Get rights and content

Abstract

Background

There is considerable evidence implicating the aberrant activation or “reactivation” of androgen receptor in the course of androgen-ablation therapy as a potential cause for the development of castration-resistant prostate cancer. Several non-mutually exclusive mechanisms including the inappropriate activation of androgen receptor (AR) by non-steroids have been postulated. The present work is aimed to understand the role of neuropeptides released by neuroendocrine transdifferentiated prostate cancer cells in the aberrant activation of AR.

Objectives

The study was designed to study how neuropeptides such as gastrin-releasing peptide activate AR and to define the crucial signal pathways involved, in the hope to identify therapeutic targets.

Methods and Materials

Androgen-dependent LNCaP cell line was used to study the effects of bombesin/gastrin-releasing peptide on the growth of the cell line and the transactivation of AR. The neuropeptide was either added to the media or introduced as a transgene in LNCaP cells to study its paracrine or autocrine effect on LNCaP growth under androgen-deprived conditions. The activation of AR was monitored by reporter assay, chromatin immunoprecipitation (ChIP) of AR, translocation into the nucleus and cDNA microarray of the AR response genes.

Results

Bombesin/gastrin releasing peptides induce androgen-independent growth of LNCaP in vitro and in vivo. It does so by activating AR, which is accompanied by the activation of Src tyrosine kinase and its target c-myc oncogene. The bombesin or Src-activated AR induces an overlapping set of AR response genes as androgen, but they also a unique set of genes. Intriguingly, the Src-activated and androgen-bound ARs differ in their binding specificity toward AR response elements, indicating the receptors activated by these 2 mechanisms are not conformationally identical. Finally, Src inhibitor was shown to effectively block the activation of AR and the growth effects induced by bombesin.

Conclusion

The results showed that AR can be activated by neuropeptide, a ligand for G-protein coupled receptor, in the absence of androgen. The activation goes through Src-tyrosine kinase pathway, and tyrosine kinase inhibitor is a potentially useful adjunctive therapy during androgen ablation.

Introduction

Prostate cancer (PCA) represents the most frequently diagnosed malignancy of men in the United States. PCA is a hormonally regulated malignancy and AR plays an important role in disease progression. One of the most troubling aspects of PCA progression is the conversion from an androgen-dependent to independent (AI) state, which at present defies any effective treatment. In the majority of end-stage, hormone-refractory (HR) tumors, AR continues to be expressed and appears to be activated by castration levels of androgen and adrenal androgens [1], [2], [3]. Thus, a required step towards solving the clinical problem of prostate cancer androgen independence, becomes one of understanding how AR is inappropriately activated and how to inhibit such aberrant signals.

Section snippets

AR and androgen-independence

AR plays a vital role in the development of male reproductive organs. Genetic defects in AR results in the failure to develop a prostate gland, a notion corroborated by recent AR knockout experiments in mice [4], [5]. In normal development, androgen is primarily required for differentiation functions. By contrast, during the development of PCA, androgen becomes a growth and survival factor for tumor cells. At the early stage of localized and metastatic PCA, proliferation depends on androgen,

Androgen-independent AR activation by non-steroids

AR mediates androgen action by being a transcriptional factor that binds specific DNA sequences and recruits RNA polymerase II and a basal transcriptional complex for efficient transcription of cellular genes. The transcriptional activity of AR is mediated by coregulators (coactivators and corepressors) [6], [7] which, in response to androgen's binding to AR and nuclear translocation, are assembled in a dynamic way at different response elements along the genome. The best recognized

Neuroendocrine differentiation and the development of hormone-refractory PCA

Our interest in neuropeptide and its possible role in HR PCA stems from the well documented observation that increased neuroendocrine cells accompany the development of HR PCA. We first demonstrated that IL-6, a progression factor of HR PCA, induced neuroendocrine differentiation of LNCaP [35], which is now supported by several studies reported in the literature [36], [37], [38], [39]. Others reported that androgen-deprivation and forskolin also induced neuroendocrine differentiation of LNCaP,

Inappropriate activation of AR by neuropeptides

A major effort of our laboratory was directed toward studying of the effect of neuropeptide bombesin (and its human homolog GRP) in the induction of androgen-resistance of prostate cancer cells. Our results [48] showed that GRP/bombesin induces LNCaP growth under androgen-free or low-androgen conditions. It activates Src/Etk/FAK tyrosine kinase complex, effectively translocates AR into the nucleus and activates AR transcription activity. Based on microarray analysis, bombesin was found to

Inappropriate activation of AR by other ligands engaging GPCR

Bombesin engages G-protein coupled receptor and channels its signal through Src/Etk/FAK tyrosine kinase complex [20], [48]. Since many of the neurokines released from neuroendocrine cells are ligands for GPCR, and GPCR activation is associated with HR PCA [56], we decided to study whether the above results can be extended to other GPCR ligands such as IL-8 [53] and Relaxin [57]. We found indeed both ligands are able to aberrantly activate AR and induce androgen-independent growth, via similar

In vivo neuropeptide model

To explore Src as a target, we hypothesized that NE cells are androgen-independent and secrete neuropeptides that further support androgen sensitive cell proliferation in the absence of androgens. We developed an in vitro and in vivo model by stable overexpression of the GRP in LNCaP cells (LNCaP-GRP) through transfection and selection. LNCaP-GRP cells demonstrated androgen- and anchorage-independent growth and enhanced cell motility via Src activation. LNCaP-GRP cells developed orthotopic

Clinical implications

Using a novel and specific Src kinase oral inhibitor AZD0530, we demonstrated inhibition of growth and metastases in vivo. The relationship of Src to FAK appears very important in that there was 100% inhibition of lymph node metastases in AZD0530 treated mice. The inhibition of FAK was through its complex to Src. In clinical samples, Src hyperactivation correlates with aberrant androgen receptor activation of high-grade prostate cancer cells and also androgen-independent disease. Furthermore,

Summary and discussion

In this article, we described the mechanism whereby neuropeptides such as bombesin or GRP activates androgen receptor in an androgen-independent fashion. Although our studies were carried out in charcoal-stripped conditions, our results are directly applicable to castration conditions where a low level of androgen is still present. We and others showed that the effects of neuropeptides and androgen on AR activation are synergistic. Perhaps the most provocative finding of this study is the

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