Purpose: Prostate cancer metastasizes to the skeleton to form osteoblastic lesions. Androgen ablation is the current treatment for metastatic prostate cancer. This therapy is palliative, and the disease will return in an androgenindependent form that is preceded by a rising titer of prostate-specific antigen (PSA). Here, we investigated the possibility that human osteoblasts might secrete factors that contribute to the emergence of androgen-independent prostate cancer.Experimental Design: Primary cultures of human osteoblasts were used as a source of conditioned medium (OCM). Proliferation, expression of androgen-regulated genes, and transactivation of the androgen receptor (AR) were monitored in LNCaP human prostate cancer cells in response to OCM using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Northern blot analysis, and reporter gene constructs. Levels of interleukin-6 (IL-6) present in OCM were measured, and its contribution to proliferation and expression of PSA were investigated by neutralization studies with anti IL-6 antibodies.Results: OCM increased the proliferation and expression of PSA at both the protein and RNA levels in LNCaP cells. Synergistic increases in the activities of PSA (6.1 kb)-and pARR 3 -tk-luciferase reporters were measured in cells cotreated with both OCM and androgen. OCM targeted the NH 2 -terminal domain of the AR. The effect of OCM on transcriptional activity of the AR was inhibited by an antiandrogen. Neutralizing antibodies to IL-6 blocked proliferation and expression of PSA by OCM.Conclusion: Osteoblasts secrete factors, such as IL-6, that cause androgen-independent induction of PSA gene expression and proliferation of prostate cancer cells by a mechanism that partially relies on the AR. Identifying such molecular mechanisms may lead to improved clinical management of metastatic prostate cancer.
Context:Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene.Objective:The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls.Design:Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus.Setting:The study was conducted at a university hospital endocrine research laboratory.Patients:GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46).Intervention(s):There were no interventions.Main Outcome Measure(s):DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured.Results:The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling.Conclusions:AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high clinical relevance.
A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5’ untranslated region (5’-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5′UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5′UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.
BackgroundTo better understand the molecular programs of normal and abnormal genital development, clear-cut definition of androgen-dependent gene expression patterns, without the influence of genotype (46, XX vs. 46, XY), is warranted. Previously, we have identified global gene expression profiles in genital-derived fibroblasts that differ between 46, XY males and 46, XY females with complete androgen insensitivity syndrome (CAIS) due to inactivating mutations of the androgen receptor (AR). While these differences could be due to cell autonomous changes in gene expression induced by androgen programming, recent work suggests they could also be influenced by the location from which the fibroblasts were harvested (topology). To minimize the influence of topology, we compared gene expression patterns of fibroblasts derived from identical urogenital anlagen: the scrotum in normally virilized 46, XY males and the labia majora from completely feminized 46, XY individuals with CAIS.Results612 transcripts representing 440 unique genes differed significantly in expression levels between scrotum and CAIS labia majora, suggesting the effects of androgen programming. While some genes coincided with those we had identified previously (TBX3, IGFBP5, EGFR, CSPG2), a significant number did not, implying that topology had influenced gene expression in our previous experiments. Supervised clustering of gene expression data derived from a large set of fibroblast cultures from individuals with partial AIS revealed that the new, topology controlled data set better classified the specimens.ConclusionInactivating mutations of the AR, in themselves, appear to induce lasting changes in gene expression in cultured fibroblasts, independent of topology and genotype. Genes identified are likely to be relevant candidates to decipher androgen-dependent normal and abnormal genital development.
Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symptoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. We report a G -a' T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46,XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein -5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.
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