title: Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome.
abstract: Most prostate cancers (PCs) become resistant to combined androgen blockade therapy with surgical or medical castration and antiandrogens after several years. Some of these refractory PCs regress after discontinuation of antiandrogen administration [antiandrogen withdrawal syndrome (AWS)]. Although the molecular mechanisms of the AWS are not fully understood because of the lack of suitable experimental models, one hypothesis of the mechanism is mutation of androgen receptor (AR). However, bicalutamide, which has become the most prevalent pure antiandrogen, does not work as an agonist for any mutant AR detected thus far in PC. To elucidate the mechanisms of the AWS, we established and characterized novel LNCaP cell sublines, LNCaP-cxDs, which were generated in vitro by culturing androgen-dependent LNCaP-FGC human PC cells in androgen-depleted medium with bicalutamide to mimic the combined androgen blockade therapy. LNCaP-FGC cells did not grow at first, but they started to grow after 6-13 weeks of culture. Bicalutamide stimulated LNCaP-cxD cell growth and increased prostate-specific antigen secretion from LNCaP-cxD cells both in vitro and in vivo. Sequencing of AR transcripts revealed that the AR in LNCaP-cxD cells harbors a novel mutation in codon 741, TGG (tryptophan) to TGT (cysteine; W741C), or in codon 741, TGG to TTG (leucine; W741L), in the ligand-binding domain. Transactivation assays showed that bicalutamide worked as an agonist for both W741C and W741L mutant ARs. Importantly, another antiandrogen, hydroxyflutamide, worked as an antagonist for these mutant ARs. In summary, we demonstrate for the first time that within only 6-13 weeks of in vitro exposure to bicalutamide, LNCaP-FGC cells, whose growth had initially been suppressed, came to use bicalutamide as an AR agonist via W741 AR mutation to survive. Our data strongly support the hypothesis that AR mutation is one possible mechanism of the AWS and suggest that flutamide might be effective as a second-line therapy for refractory PC previously treated with bicalutamide.
authors.affiliation: Pharmaceutical Research Laboratories, Takeda Chemical Industries, Ltd., Osaka 532-8686, Japan. Hara_Takahito@takedo.co.jp
authors.forename: Takahito
authors.initials: T
authors.lastname: Hara
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Miyazaki
authors.forename: Hideo
authors.initials: H
authors.lastname: Araki
authors.forename: Masuo
authors.initials: M
authors.lastname: Yamaoka
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Kanzaki
authors.forename: Masami
authors.initials: M
authors.lastname: Kusaka
authors.forename: Masaomi
authors.initials: M
authors.lastname: Miyamoto
mesh_terms: D019943:Amino Acid Substitution; D000726:Androgen Antagonists / Q000009:adverse effects*; D000813:Anilides / Q000009:adverse effects*; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004195:Disease Models, Animal; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009570:Nitriles; D017430:Prostate-Specific Antigen / Q000032:analysis; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D011944:Receptors, Androgen / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013375:Substance Withdrawal Syndrome / Q000503:physiopathology* / Q000517:prevention & control; D014105:Tosyl Compounds; D014158:Transcription, Genetic; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
chemical_list: D000726:Androgen Antagonists; D000813:Anilides; D017931:DNA Primers; D018076:DNA, Complementary; D009570:Nitriles; D011944:Receptors, Androgen; D014105:Tosyl Compounds; C053541:bicalutamide; D017430:Prostate-Specific Antigen