Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence

Despite earlier detection and recent advances in surgery and radiation, prostate cancer is second only to lung cancer in male cancer deaths in the United States. Hormone therapy in the form of medical or surgical castration remains the mainstay of systemic treatment in prostate cancer. Over the last 15 years with the clinical use of prostate specific antigen (PSA), there has been a shift to using hormone therapy earlier in the disease course and for longer duration. Despite initial favorable response to hormone therapy, over a period of time these tumors will develop androgen‐independence that results in death. The androgen receptor (AR) is central to the initiation and growth of prostate cancer and to its response to hormone therapy. Analyses have shown that AR continues to be expressed in androgen‐independent tumors and AR signaling remains intact as demonstrated by the expression of the AR regulated gene, PSA. Androgen‐independent prostate cancers have demonstrated a variety of AR alterations that are either not found in hormone naïve tumors or found at lower frequency. These changes include AR amplification, AR point mutation, and changes in expression of AR co‐regulatory proteins. These AR changes result in a “super AR” that can respond to lower concentrations of androgens or to a wider variety of agonistic ligands. There is also mounting evidence that AR can be activated in a ligand independent fashion by compounds such as growth factors or cytokines working independently or in combination. These growth factors working through receptor tyrosine kinase pathways may promote AR activation and growth in low androgen environments. The clinical significance of these AR alterations in the development and progression of androgen‐independent prostate cancer remains to be determined. Understanding the changes in AR signaling in the evolution of androgen‐independent prostate cancer will be key to the development of more effective hormone therapy. © 2003 Wiley‐Liss, Inc.     

Hormone status selects for spontaneous somatic androgen receptor variants that demonstrate specific ligand and cofactor dependent activities in autochthonous prostate cancer

We have used the autochthonous transgenic adenocarcinoma of mouse prostate (TRAMP) model to investigate the relationship between somatic mutation in the androgen receptor (AR) and the emergence of androgen-independent prostate cancer. Here we report the identification, isolation, and characterization of distinct classes of AR variants from spontaneous prostate tumors in the TRAMP model. Using cDNA cloning, single stranded conformation polymorphism and sequencing strategies, 15 unique somatic mutations in the AR were identified in prostate tumors obtained from eight TRAMP mice between 24 and 29 weeks of age. At least one mutation was isolated from each mouse. All mutations were single base substitutions, 10 were missense and 5 were silent. Nine mutations in the AR were identified in tumors of four mice that were castrated at 12 weeks of age. Interestingly, the majority of mutations (seven out of nine, 78%) identified in the androgen-independent tumors colocalized in the AR transactivation domain. The remaining mutations colocalized in the AR ligand binding domain. In general, the AR variants demonstrated promoter-, cell-, and cofactor-specific activities in response to various hormones. All AR variants isolated in this study maintained strong sensitivity for androgens, and four AR variants isolated from castrated mice demonstrated increased activities in the absence of ligand. The K638M and F677S variants demonstrated increased activities in response to androgen, and K638M also demonstrated increased response to estradiol. In the presence of AR coactivator ARA70 the E231G variant demonstrated increased activity in response to both androgen and estradiol. However, in the presence of AR coactivator ARA160 the E231G variant was selectively responsive to androgen. Collectively these analyses not only indicate that somatic mutations in the AR gene occur spontaneously in TRAMP tumors but also how changes in the hormonal environment may drive the selection of spontaneous somatic mutations that provide a growth advantage.     

Androgen receptor action in hormone-dependent and recurrent prostate cancer

The importance of androgens and androgen receptors (AR) in primary prostate cancer is well established. Metastatic disease is usually treated with some form of androgen ablation, which is effective for a limited amount of time. The role of AR in prostate cancers that recur despite androgen ablation therapy is less certain. Most of these tumors express prostate specific antigen (PSA), an androgen-regulated gene; moreover, AR is generally highly expressed in recurrent prostate cancer. We propose that AR continues to play a role in many of these tumors and that it is not only the levels of AR, ligands, and co-regulators, but also the changes in cell signaling that induce AR action in recurrent prostate cancer. These pathways are, therefore, potential therapeutic targets.     

Mutation of Androgen Receptor N-Terminal Phosphorylation Site Tyr-267 Leads to Inhibition of Nuclear Translocation and DNA Binding

Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.     

Alterations of androgen receptor in prostate cancer

The significance of androgens in the development of prostate cancer has been known for more than half century. During the last decade, a lot of effort has been put to study the significance of the specific nuclear receptor of the hormone, androgen receptor (AR). It has been suggested that polymorphisms, especially the length of CAG repeat in exon 1 of the gene, are associated with the risk of prostate cancer. However, not all studies have confirmed the association. Most surprisingly, it has now become clear that prostate carcinomas emerging during the androgen withdrawal therapy (i.e. hormone-refractory tumors) are capable of reactivating the AR-mediated signalling despite of the low levels of androgens. In addition, it has been shown that AR gene itself is genetically targeted. One-third of the hormone-refractory prostate carcinomas contains amplification of the gene. In addition, 10-30% of prostate carcinomas treated by antiandrogens acquire point mutation in the AR gene. The genetic alterations in AR indicate that receptor should be considered as putative treatment target. Evidently, the currently available antiandrogens are not capable to abolish the AR-mediated signalling efficiently enough.     

Amino-terminus domain of the androgen receptor as a molecular target to prevent the hormonal progression of prostate cancer

Prostate cancer has a propensity to metastasize to the bone. Currently the only effective systemic treatment for these patients is androgen ablation therapy. However, the tumor will invariably progress to an androgen-independent stage and the patient will succumb to his disease within approximately 2 years. The earliest indication of hormonal progression is the rising titer of serum prostate specific antigen. Current evidence implicates the androgen receptor (AR) as a key factor in maintaining the growth of prostate cancer cells in an androgen-depleted state. Under normal conditions, binding of ligand activates the receptor, allowing it to effectively bind to its respective DNA element. However, AR is also transformed in the absence of androgen (ligand-independent activation) in prostate cells via multiple protein kinase pathways and the interleukin-6 (IL-6) pathway that converge upon the N-terminal domain of the AR. This domain is the main region for phosphorylation and is also critical for normal coregulator recruitment. Here we discuss evidence supporting the role of the AR, IL-6 and other protein kinase pathways in the hormonal progression of prostate cancer to androgen independence and the mechanisms involved in activation of the AR by these pathways. Receptor-targeted therapy, especially potential drugs targeting the N-terminal domain, may effectively prevent or delay the hormonal progression of AR-dependent prostate cancer.     

Loss of Androgen Receptor-Dependent Growth Suppression by Prostate Cancer Cells Can Occur Independently from Acquiring Oncogenic Addiction to Androgen Receptor Signaling

The conversion of androgen receptor (AR) signaling as a mechanism of growth suppression of normal prostate epithelial cells to that of growth stimulation in prostate cancer cells is often associated with AR mutation, amplification and over-expression. Thus, down-regulation of AR signaling is commonly therapeutic for prostate cancer. The E006AA cell line was established from a hormone naïve, localized prostate cancer. E006AA cells are genetically aneuploid and grow equally well when xenografted into either intact or castrated male NOG but not nude mice. These cells exhibit: 1) X chromosome duplication and AR gene amplification, although paradoxically not coupled with increased AR expression, and 2) somatic, dominant-negative Serine-599-Glycine loss-of-function mutation within the dimerization surface of the DNA binding domain of the AR gene. No effect on the growth of E006AA cells is observed using targeted knockdown of endogenous mutant AR, ectopic expression of wild-type AR, or treatment with androgens or anti-androgens. E006AA cells represent a prototype for a newly identified subtype of prostate cancer cells that exhibit a dominant-negative AR loss-of-function in a hormonally naïve patient. Such loss-of-function eliminates AR-mediated growth suppression normally induced by normal physiological levels of androgens, thus producing a selective growth advantage for these malignant cells in hormonally naïve patients. These data highlight that loss of AR-mediated growth suppression is an independent process, and that, without additional changes, is insufficient for acquiring oncogene addiction to AR signaling. Thus, patients with prostate cancer cells harboring such AR loss-of-function mutations will not benefit from aggressive hormone or anti-AR therapies even though they express AR protein.     

Molecular defects of the androgen receptor

Defects of the androgen receptor cause a wide spectrum of abnormalities of phenotypic male development, ranging from individuals with mild defects of virilization to those with complete female phenotypes. In parallel with this phenotypic spectrum, a large number of different mutations have been identified that alter the synthesis or functional activity of the receptor protein. In many instances, the genetic mutations identified lead to an absence of the intact, full-length receptor protein. Such defects (splicing defects, termination codons, partial or complete gene deletions) invariably result in the phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, single amino acid substitutions in the androgen receptor protein can result in the entire phenotypic spectrum of androgen resistant phenotypes and provide far more information on the functional organization of the receptor protein. Amino acid substitutions in different segments of the AR open-reading frame disturb AR function by distinct mechanisms. Substitutions in the DNA binding domain of the receptor appear to comprise a relatively homogeneous group. These substitutions impair the capacity of the receptor to bind to specific DNA sequence elements and to modulate the function of responsive genes. Amino acid substitutions in the hormone-binding domain of the receptor have a more varied effect on receptor function. In some instances, the resulting defect is obvious and causes an inability of the receptor to bind hormone. In other instances, the effect is subtler, and may result in the production of a receptor protein that displays qualitative abnormalities of hormone binding or from which hormone dissociates more rapidly. Often it is not possible to correlate the type of binding defect with the phenotype that is observed. Instead, it is necessary to measure the capacity of the receptor that is synthesized in functional assays in order to discern any type of correlation with phenotype. Finally, two types of androgen receptor mutation do not fit such a categorization. The first of these—the glutamine repeat expansion that is observed in spinal and bulbar muscular atrophy—leads to a reduction of receptor function that can be measured in heterologous cells or in fibroblasts established from such patients. The expression of ARs containing such expanded repeats in men is associated with a degeneration of motor neurons in the spinal cords of affected patients. Likewise, the alterations of androgen receptor structure that have been detected in advanced forms of prostate cancer also behave as gain-of-function mutations. In this latter type of mutation, the exquisite specificity of the normal androgen receptor is relaxed and the mutant receptors can be activated by a variety of steroidal and non-steroidal ligands.     

雄激素受体信号转导途径及其在前列腺癌发展中的作用

前列腺癌是西方男性发病率最高的癌症之一,在采用雄激素阻断疗法后,大部分患者的病情可得到控制,但经过一段时间又会转变为雄激素非依赖型前列腺癌。雄激素受体(AR)在前列腺细胞中扮演重要的角色,它可调节大量基因的表达。在前列腺癌由雄激素依赖型向雄激素非依赖型的转变过程中,AR及其信号途径通过多种方式发挥作用,AR基因的扩增、AR的突变,以及与共激活子之间作用的改变都可能使细胞获得雄激素非依赖型的生长能力。此外,AR还受到多肽生长因子和细胞因子等的调节,表现激素非依赖型的转录激活活性。AR在前列腺癌中作用的阐明对前列腺癌的诊断与治疗有着重大的意义。     

Androgen receptor gene mutations in human prostate cancer

To investigate the structural abnormality of the androgen receptor (AR) in human prostate cancers, exons B-H encoding DNA- and hormone-binding domains were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products using originally designed oligoprimers. Tissues from 7 cases of untreated stage B prostate cancer surgically removed and from 8 cases of endocrine therapy-resistant cancers obtained at autopsy were used in the study. Two different mutations were identified in exons D and H in the different cancer foci of the same cancer death patient. One mutation in exon D (at codon 701, Leu to His) was detected in the prostate, and the other in exon H (at codon 877, Thr to Ala) was found in metastatic tissues. In untreated cancer tissues and the other autopsy samples, no mutations were detected. The mutation in exon H was identical to that reported in LNCaP cells. These results indicate that AR gene mutations occur in relation to endocrine therapy-resistance, although the mutation was found in 1 out of 8 resistant cases (12.5%) at autopsy.