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PDF File - 72K, Reporter gene assay and growth assay of LNCaP95 cells.

Androgen receptor is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiologic ligands for androgen receptor ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit androgen receptor transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semisynthetic analogues (T2 and T3) revealed that these diterpenoids have antiandrogen properties that include inhibition of both androgen-dependent proliferation and androgen receptor transcriptional activity by a mechanism that involved competing with androgen for androgen receptor LBD and blocking essential N/C interactions required for androgen-induced androgen receptor transcriptional activity. Structure–activity relationship analyses revealed some chemical features of T1 that are associated with activity and yielded T3 as the most potent analogue. In vivo, T3 significantly reduced the weight of seminal vesicles, which are an androgen-dependent tissue, thereby confirming the on-target activity of T3. The ability to create analogues of diterpenoids that have varying antiandrogen activity represents a novel class of chemical compounds for the analysis of androgen receptor ligand-binding properties and therapeutic development. Mol Cancer Ther; 12(5); 621–31. ©2013 AACR.

PDF File - 81K, Growth assay for LNCaP, VCaP, and PC3 cells.

PDF - 121K, Methods and Supplemental Figure Legends and References

PDF File - 72K, Levels of AR protein in cell lines.

PDF File - 58K, PC3 cell proliferation data.

Immunohistochemistry of representative sections of LNCaP95 xenografts stained for cleaved caspase-3. Scale bars (red) indicate 20 µm. % of caspase-3 positive cells were counted in xenograft sections of each treatment. At least 1000 cells per xenograft were counted. Total number of cells counted: 3287 (Control), 4238 (EPI-002), and 4262 (ENZA). Bar graphs are mean {plus minus} SEM with n = at least 3 different xenograft sections. One-way ANOVA was used for statistical analyses; *p < 0.05; **p < 0.01; ***p

Purpose: Persistent androgen receptor (AR) transcriptional activity is clinically evident in castration-resistant prostate cancer (CRPC). Therefore, AR remains as a viable therapeutic target for CRPC. All current hormonal therapies target the C-terminus ligand-binding domain (LBD) of AR. By using EPI to target AR activation function-1 (AF-1), in the N-terminal domain that is essential for AR transactivation, we evaluate the ability of EPI to overcome several clinically relevant AR-related mechanisms of resistance.Experimental Design: To study the effect of EPI on AR transcriptional activity against overexpressed coactivators, such as SRC1-3 and p300, luciferase reporter assays were performed using LNCaP cells. AR-negative COS-1 cells were employed for reporter assays to examine whether the length of polyglutamine tract affects inhibition by EPI. The effect of EPI on constitutively active AR splice variants was studied in LNCaP95 cells, which express AR-V7 variant. To evaluate the effect of EPI on the proliferation of LNCaP95 cells, we performed in vitro BrdUrd incorporation assay and in vivo studies using xenografts in mice.Results: EPI effectively overcame several molecular alterations underlying aberrant AR activity, including overexpressed coactivators, AR gain-of-function mutations, and constitutively active AR-V7. EPI inhibited AR transcriptional activity regardless of the length of polyglutamine tract. Importantly, EPI significantly inhibited the in vitro and in vivo proliferation of LNCaP95 prostate cancer cells, which are androgen independent and enzalutamide resistant.Conclusions: These findings support EPI as a promising therapeutic agent to treat CRPC, particularly against tumors driven by constitutively active AR splice variants that are resistant to LBD-targeting drugs. Clin Cancer Res; 22(17); 4466–77. ©2016 AACR.See related commentary by Sharp et al., p. 4280

The Supplementary Materials and Methods contain information for the reagents and plasmids and procedures for experiments in the manuscript.