leupeptins and bicalutamide

The androgen receptor (AR) acts as a ligand-dependent transcription factor, whereas mutant AR lacking the C-terminal ligand-binding domain functions in a ligand-independent manner. In the present study we report that the C-terminal truncated AR, which we named AR-NH1 (the N-terminal fragment of AR cleaved in the neighborhood of helix 1 of the ligand-binding domain), is produced in LNCaP prostatic carcinoma cells. The AR-NH1 of ~90 kDa was observed in an androgen-independent LNCaP subline and was further accumulated by the proteasome inhibitor MG132. MG132 treatment caused the accumulation of AR-NH1 even in parent LNCaP cells. AR-NH1 was produced in the absence of ligand or in the presence of the AR antagonist bicalutamide, whereas AR agonists suppressed its production. AR-NH1 was detected with different AR antibodies recognizing amino acid residues 1-20 and 300-316 and was also generated from exogenous AR. Both siRNA-mediated AR knockdown and treatment with a serine protease inhibitor (4-(2-aminoethyl)-benzenesulfonyl fluoride) reduced AR-NH1 levels. According to the predicted cleavage site (between amino acid residues 660-685) and its nuclear localization, it is assumed that AR-NH1 functions as a constitutively active transcription factor. These data suggest that AR-NH1 is produced under hormone therapy and contributes to the development of castration-resistant prostate cancer due to its ligand-independent transcriptional activity.

Topics: Androgen Receptor Antagonists; Androgens; Anilides; Antineoplastic Agents; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Male; Nitriles; Prostatic Neoplasms; Receptors, Androgen; RNA Interference; RNA, Small Interfering; Serine Proteinase Inhibitors; Sulfones; Tosyl Compounds

A stem cell model of prostate cancer tumourigenesis explains progression to castration resistant prostate cancer (CRPC) and offers novel perspectives in targeting this cancer in its more advanced forms. Androgen receptor (AR) regulated pathways are central mechanisms in progression to CRPC. However, AR was thought to be lacking in prostate stem cell enriched fractions. Potential low levels of AR expression in stem cell enriched cells were investigated and potential direct effects of androgen were examined.. Human prostate stem cell enriched populations, based on high α(2)β(1) integrin expression (α(2)β(1)(hi)), were selected from primary human prostate tissue in men undergoing transurethral prostatectomy or cystoprostatectomy. Effects on differentiation were assayed with flow cytometry using differentiation-specific markers.. Low levels of AR were demonstrable in α(2)β(1)(hi) cells following inhibition of the proteasome using MG132. Furthermore, a direct effect of androgen was shown in stabilising/inducing AR expression. Androgen treatment of α(2)β(1)(hi) cells was associated with the induction of differentiation using a number of differentiation-specific markers (prostatic acid phosphatase, cytokeratin 18 and AR) with increases ranging from 49% to 67% (p<0.05). These effects were blocked with the AR-specific inhibitor bicalutamide (p<0.05). These data support a role of direct androgen activity on stem cell enriched cells in the prostate and the implications of these findings are discussed.

Topics: Aged; Aged, 80 and over; Androgen Antagonists; Androgen Receptor Antagonists; Androgens; Anilides; Antigens, Differentiation; Biomarkers, Tumor; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Epithelial Cells; Humans; Leupeptins; Male; Metribolone; Middle Aged; Neoplastic Stem Cells; Nitriles; Prostatic Neoplasms; Receptors, Androgen; Testosterone Congeners; Tosyl Compounds

Abnormal differentiation in epithelial stem cells or their immediate proliferative progeny, the transiently amplifying population (TAP), may explain malignant pathogenesis in the human prostate. These models are of particular importance as differing sensitivities to androgen among epithelial cell subpopulations during differentiation are recognised and may account for progression to androgen independent prostate cancer. Androgens are crucial in driving terminal differentiation and their indirect effects via growth factors from adjacent androgen responsive stroma are becoming better characterised. However, direct effects of androgen on immature cells in the context of a prostate stem cell model have not been investigated in detail and are studied in this work. In alpha2beta1hi stem cell enriched basal cells, androgen analogue R1881 directly promoted differentiation by the induction of differentiation-specific markers CK18, androgen receptor (AR), PSA and PAP. Furthermore, treatment with androgen down-regulated alpha2beta1 integrin expression, which is implicated in the maintenance of the immature basal cell phenotype. The alpha2beta1hi cells were previously demonstrated to lack AR expression and the direct effects of androgen were confirmed by inhibition using the anti-androgen bicalutamide. AR protein expression in alpha2beta1hi cells became detectable when its degradation was repressed by the proteosomal inhibitor MG132. Stratifying the alpha2beta1hi cells into stem (CD133(+)) and transient amplifying population (TAP) (CD133(-)) subpopulations, AR mRNA expression was found to be restricted to the CD133(-) (TAP) cells. The presence of a functional AR in the TAP, an androgen independent subpopulation for survival, may have particular clinical significance in hormone resistant prostate cancer, where both the selection of immature cells and functioning AR regulated pathways are involved.

Topics: AC133 Antigen; Acid Phosphatase; Aged; Aged, 80 and over; Androgen Antagonists; Anilides; Antigens, CD; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Epithelial Cells; Fibroblast Growth Factor 7; Glycoproteins; Humans; Integrin alpha2beta1; Keratin-18; Leupeptins; Male; Metribolone; Middle Aged; Neoplastic Stem Cells; Nitriles; Peptides; Phenotype; Prostate-Specific Antigen; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Tyrosine Phosphatases; Receptors, Androgen; RNA, Messenger; Signal Transduction; Testosterone Congeners; Tosyl Compounds