O. Harris Ford

The Androgen Axis in Recurrent Prostate Cancer

Purpose. Prostate cancer that recurs during androgen deprivation therapy is referred to as androgen-independent. High levels of expression of androgen receptor and androgen receptor-regulated genes in recurrent prostate cancer suggest a role for androgen receptor and its ligands in prostate cancer recurrence. Experimental Design. Recurrent prostate cancer specimens from 22 men whose prostate cancer recurred locally during androgen deprivation therapy and benign prostate specimens from 48 men who had received no prior treatment were studied. Androgen receptor expression was measured using monoclonal antibody and automated digital video image analysis. Tissue androgens were measured using radioimmunoassay. Results. Epithelial nuclei androgen receptor immunostaining in recurrent prostate cancer (mean optical density, 0.284 ± SD 0.115 and percentage positive nuclei, 83.7 ± 11.6) was similar to benign prostate (mean optical density, 0.315 ± 0.044 and percentage positive nuclei, 77.3 ± 13.0). Tissue levels of testosterone were similar in recurrent prostate cancer (2.78 ± 2.34 pmol/g tissue) and benign prostate (3.26 ± 2.66 pmol/g tissue). Tissue levels of dihydrotestosterone, dehydroepiandrosterone, and androstenedione were lower (Wilcoxon, P = 0.0000068, 0.00093, and 0.0089, respectively) in recurrent prostate cancer than in benign prostate, and mean dihydrotestosterone levels, although reduced, remained 1.45 nm. Androgen receptor activation in recurrent prostate cancer was suggested by the androgen-regulated gene product, prostate-specific antigen, at 8.80 ± 10.80 nmol/g tissue. Conclusions. Testosterone and dihydrotestosterone occur in recurrent prostate cancer tissue at levels sufficient to activate androgen receptor. Novel therapies for recurrent prostate cancer should target androgen receptor directly and prevent the formation of androgens within prostate cancer tissue.

Steroid 5α-Reductase Isozymes I and II in Recurrent Prostate Cancer

Purpose: Prostate cancer recurs during androgen deprivation therapy despite reduced circulating androgens. We showed that recurrent prostate cancer tissue has testosterone levels similar to androgen-stimulated benign prostate, whereas dihydrotestosterone levels were reduced 82% to 1.45 nmol/L, sufficient for androgen receptor activation. The altered testosterone/dihydrotestosterone ratio in recurrent prostate cancer suggests loss of 5α-reducing capability. The aim of this study was to characterize steroid 5α-reductase isozymes I (S5αRI) and II (S5αRII) in prostate tissues. Experimental Design: A tissue microarray was constructed from 22 recurrent prostate cancer specimens and matched pairs of androgen-stimulated benign prostate and androgen-stimulated prostate cancer from 23 radical prostatectomy specimens. Immunoblots were constructed from eight recurrent prostate cancers, eight androgen-stimulated benign prostate, and eight androgen-stimulated prostate cancer specimens. Isozyme expression was examined in microarray sections and immunoblots using S5αRI and S5αRII polyclonal antibodies. Isozyme activities were measured in 12 recurrent prostate cancer, 12 androgen-stimulated benign prostate, and 12 androgen-stimulated prostate cancer specimens. Results: Nuclear immunostaining exhibited higher S5αRI expression than S5αRII in recurrent prostate cancer, androgen-stimulated benign prostate, and androgen-stimulated prostate cancers (P < 0.0001); mean expression was 125, 150, and 115 for S5αRI versus 10, 29, and 37 for S5αRII, respectively. Cytoplasmic immunostaining was moderate and similar for both isozymes in the three tissue types (P > 0.05). Immunoblots confirmed immunohistochemistry; S5αRI was expressed in recurrent prostate cancer specimens and S5αRII was not detected. The activity of S5αRI (114.4 pmol/mg epithelial protein/minute) was 3.7-fold higher than S5αRII (30.7 pmol/mg epithelial protein/minute) in recurrent prostate cancer specimens. Conclusions: Expression levels and isozyme activity shifts from S5αRII toward S5αRI in recurrent prostate cancer. Dual inhibition of S5αRI and S5αRII should reduce dihydrotestosterone biosynthesis and may prevent or delay growth of recurrent prostate cancer.

Protein kinase C ɛ interacts with Bax and promotes survival of human prostate cancer cells

Prostatic glandular epithelial cells express protein kinase Cɛ (PKCɛ), an oncoprotein that coordinately disrupts the reactivation of the tumor suppressor Rb, derepressess transcriptional elongation of the c-myc oncogene, and propagates survival signals in LNCaP cells. Since the activation of such a program may contribute to the progression of human prostate cancer, a proteomic analysis was performed to gain a more global perspective on the signaling network that PKCɛ might be capable of engaging in prostate cancer cells. Using CWR22 xenografts, we identified at least 18 different structural, signaling, and stress-related proteins that associated with PKCɛ, including an interaction with the proapoptotic protein Bax that was novel to recurrent CWR22 tumors. An investigation into the biological significance of the PKCɛ association with Bax provided the first evidence of an inverse relationship between endogenous levels of PKCɛ and susceptibility of prostate cancer cells to the apoptotic effects of phorbol esters. Western blot and antisense experiments demonstrated that CWR-R1 cells expressed moderate levels of PKCɛ and relied on this protein to survive in the presence of phorbol esters, while the apoptosis normally induced by phorbol esters in PKCɛ-deficient LNCaP cells was dependent on the presence of Bax. Forced expression of PKCɛ in LNCaP cells was sufficient to confer a significant resistance to phorbol esters and this resistance was associated with an inhibition of phorbol ester-induced Bax conformational rearrangements that are important for Bax oligomerization, mitochondrial integration, and cytochrome c release. Considered in their entirety, our data suggest that an association of PKCɛ with Bax may neutralize apoptotic signals propagated through a mitochondrial death-signaling pathway.

14-3-3η Amplifies Androgen Receptor Actions in Prostate Cancer

Purpose: Androgen receptor abundance and androgen receptor–regulated gene expression in castration-recurrent prostate cancer are indicative of androgen receptor activation in the absence of testicular androgen. Androgen receptor transactivation of target genes in castration-recurrent prostate cancer occurs in part through mitogen signaling that amplifies the actions of androgen receptor and its coregulators. Herein we report on the role of 14-3-3η in androgen receptor action. Experimental Design and Results: Androgen receptor and 14-3-3η colocalized in COS cell nuclei with and without androgen, and 14-3-3η promoted androgen receptor nuclear localization in the absence of androgen. 14-3-3η interacted with androgen receptor in cell-free binding and coimmunoprecipitation assays. In the recurrent human prostate cancer cell line, CWR-R1, native endogenous androgen receptor transcriptional activation was stimulated by 14-3-3η at low dihydrotestosterone concentrations and was increased by epidermal growth factor. Moreover, the dihydrotestosterone- and epidermal growth factor–dependent increase in androgen receptor transactivation was inhibited by a dominant negative 14-3-3η. In the CWR22 prostate cancer xenograft model, 14-3-3η expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3η and androgen receptor actions. 14-3-3η mRNA and protein decreased following castration of tumor-bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3η levels similar to the androgen-stimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3η localized with androgen receptor in nuclei, and the similar amounts expressed in castration-recurrent prostate cancer, androgen-stimulated prostate cancer, and benign prostatic hyperplasia were consistent with androgen receptor activation in recurrent prostate cancer. Conclusion: 14-3-3η enhances androgen- and mitogen-induced androgen receptor transcriptional activity in castration-recurrent prostate cancer. (Clin Cancer Res 2009;15(24):7571–81)

Mechanism of androgen receptor corepression by CKβBP2/CRIF1, a multifunctional transcription factor coregulator expressed in prostate cancer

The transcription factor coregulator Casein kinase IIβ-binding protein 2 or CR6-interacting factor 1 (CKβBP2/CRIF1) binds the androgen receptor (AR) in prostate cancer cells and in response to dihydrotestosterone localizes with AR on the prostate-specific antigen gene enhancer, but does not bind DNA suggesting CKβBP2/CRIF1 localization in chromatin is determined by AR. In this study we show also that CKβBP2/CRIF1 inhibits wild-type AR and AR N-terminal transcriptional activity, binds to the AR C-terminal region, inhibits interaction of the AR N- and C-terminal domains (N/C interaction) and competes with p160 coactivator binding to the AR C-terminal domain, suggesting CKβBP2/CRIF1 interferes with AR activation functions 1 and 2. CKβBP2/CRIF1 is expressed mainly in stromal cells of benign prostatic hyperplasia and in stroma and epithelium of prostate cancer. CKβBP2/CRIF1 protein is increased in epithelium of androgen-dependent prostate cancer compared to benign prostatic hyperplasia and decreased slightly in castration recurrent epithelium compared to androgen-dependent prostate cancer. The multifunctional CKβBP2/CRIF1 is a STAT3 interacting protein and reported to be a coactivator of STAT3. CKβBP2/CRIF1 is expressed with STAT3 in prostate cancer where STAT3 may help to offset the AR repressor effect of CKβBP2/CRIF1 and allow AR regulation of prostate cancer growth.

Comparison of ACINUS, caspase-3, and TUNEL as apoptotic markers in determination of tumor growth rates of clinically localized prostate cancer using image analysis.

The balance between apoptotic and proliferative processes determines the enlargement of a tumor. Accurate measurement of apoptotic and proliferative rates from diagnostic prostate biopsies would allow calculation of tumor growth rates in a population‐based prostate cancer (CaP) study. Automated image analysis may be used if proliferation and apoptotic biomarkers provide clearly resolved immunostained images.