Publications by Year: 1999

The role of androgen receptor (AR) mutations in androgen-independent prostate cancer (PCa) was determined by examining AR transcripts and genes from a large series of bone marrow metastases. Mutations were found in 5 of 16 patients who received combined androgen blockade with the AR antagonist flutamide, and these mutant ARs were strongly stimulated by flutamide. In contrast, the single mutant AR found among 17 patients treated with androgen ablation monotherapy was not flutamide stimulated. Patients with flutamide-stimulated AR mutations responded to subsequent treatment with bicalutamide, an AR antagonist that blocks the mutant ARs. These findings demonstrate that AR mutations occur in response to strong selective pressure from flutamide treatment.

BACKGROUND: Functional inactivation of the tsg101 gene in mouse fibroblasts leads to cell transformation and the ability to form metastatic tumors in nude mice. Abnormal TSG101 transcripts with highly-specific deletions in the protein-coding region have been identified in human tumor samples and cancer cell lines, including prostate and breast carcinomas, and have been attributed to alternative splicing of TSG101 mRNA. The function of the TSG101 protein is not known, although its predicted sequence has suggested that it may function as a transcription factor.

METHODS: Human TSG101 N-terminal (encoding amino acids 10-240) and C-terminal (encoding amino acids 230-391) fragments were cloned and used in both transient transfection and protein binding experiments. The transient transfections were carried in CV-1 cells. Protein-protein interactions were determined by both glutathione-S-transferase fusion protein binding and co-immunoprecipitation.

RESULTS: The N-terminal region of TSG101, when fused to the GAL4 DNA binding domain, can activate transcription; whereas the C-terminal region mediates transcriptional repression. Full-length TSG101 or its separated regions repressed ligand-dependent transcriptional activation by nuclear receptors, including androgen receptor and estrogen receptor, which play central roles in prostate carcinoma and breast carcinoma, respectively. In addition, a direct association between TSG101 and the transcriptional co-factor p300 was demonstrated in vitro and in vivo.

CONCLUSIONS: These results indicate that TSG101 can function as a transcription modulator to affect nuclear receptor-mediated transcriptional activation, which raises the possibility that the tumor suppression by TSG101 observed previously may be mediated at least in part by its effects on nuclear receptor function.

An important biological feature of prostate cancer (PCa) is its marked preference for bone marrow as a metastatic site. To identify factors that may support the growth of PCa in bone marrow, expression of receptor and nonreceptor tyrosine kinases by androgen-independent PCa bone marrow metastases was assessed. Bone marrow biopsies largely replaced by PCa were analyzed using reverse transcriptase-polymerase chain reaction amplification with degenerate primers that amplified the conserved kinase domain. Sequence analyses of the cloned products demonstrated expression of multiple kinases. Expression of the receptor and nonreceptor tyrosine kinases, alpha platelet-derived growth factor receptor and Jak 1, respectively, was confirmed by immunohistochemistry. In contrast, the type 1 insulin-like growth factor receptor, thought to play a role in PCa development, was lost in metastatic PCa. These results implicate several specific growth factors and signaling pathways in metastatic androgen-independent PCa and indicate that loss of the type 1 insulin-like growth factor receptor contributes to PCa progression.