Nasrin R. Mawji

Regression of Castrate-Recurrent Prostate Cancer by a Small-Molecule Inhibitor of the Amino-Terminus Domain of the Androgen Receptor

Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.

An androgen receptor N-terminal domain antagonist for treating prostate cancer

Hormone therapies for advanced prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD), but these ultimately fail and the disease progresses to lethal castration-resistant prostate cancer (CRPC). The mechanisms that drive CRPC are incompletely understood, but may involve constitutively active AR splice variants that lack the LBD. The AR N-terminal domain (NTD) is essential for AR activity, but targeting this domain with small-molecule inhibitors is complicated by its intrinsic disorder. Here we investigated EPI-001, a small-molecule antagonist of AR NTD that inhibits protein-protein interactions necessary for AR transcriptional activity. We found that EPI analogs covalently bound the NTD to block transcriptional activity of AR and its splice variants and reduced the growth of CRPC xenografts. These findings suggest that the development of small-molecule inhibitors that bind covalently to intrinsically disordered proteins is a promising strategy for development of specific and effective anticancer agents.

An orthotopic metastatic prostate cancer model in SCID mice via grafting of a transplantable human prostate tumor line

Metastasis is the major cause of prostate cancer deaths and there is a need for clinically relevant in vivo models allowing elucidation of molecular and cellular mechanisms underlying metastatic behavior. Here we describe the development of a new in vivo model system for metastatic prostate cancer. Pieces of prostate cancer tissue from a patient were grafted in testosterone-supplemented male NOD-SCID mice at the subrenal capsule graft site permitting high tumor take rates. After five serial transplantations, the tumor tissues were grafted into mouse prostates. Resulting tumors and suspected metastatic lesions were subjected to histopathological and immunohistochemical analysis. Samples of metastatic tissue were regrafted in mouse anterior prostates and their growth and spread examined, leading to isolation from lymph nodes of a metastatic subline, PCa1-met. Orthotopic grafting of PCa1-met tissue in 47 hosts led in all cases to metastases to multiple organs (lymph nodes, lung, liver, kidney, spleen and, notably, bone). Histopathological analysis showed strong similarity between orthotopic grafts and their metastases. The latter were of human origin as indicated by immunostaining using antibodies against human mitochondria, androgen receptor, prostate-specific antigen and Ki-67. Spectral karyotyping showed few chromosomal alterations in the PCa1-met subline. This study indicates that transplantable subrenal capsule xenografts of human prostate cancer tissue in NOD-SCID mice can, as distinct from primary cancer tissue, be successfully grown in the orthotopic site. Orthotopic xenografts of the transplantable tumor lines and metastatic sublines can be used for studying various aspects of metastatic prostate cancer, including metastasis to bone.

Sintokamides A to E, Chlorinated Peptides from the Sponge Dysidea sp. that Inhibit Transactivation of the N-Terminus of the Androgen Receptor in Prostate Cancer Cells

The new chlorinated peptides sintokamides A to E (1-5) have been isolated from specimens of the marine sponge Dysidea sp. collected in Indonesia. Their structures were elucidated by a combination of spectroscopic and single-crystal X-ray diffraction analyses. Sintokamide A (1) is an inhibitor of N-terminus transactivation of the androgen receptor in prostate cancer cells.

Androgen receptor decoy molecules block the growth of prostate cancer

The androgen receptor (AR) is activated by both ligand-dependent and -independent mechanisms. Current therapies for prostate cancer target the ligand-binding domain in the C terminus of the AR. However, ligand-independent activation of the AR occurs by the N-terminal domain (NTD), making the NTD a potential novel target for the treatment of hormone refractory prostate cancer. A possible therapeutic approach is to overexpress an AR NTD peptide to create decoy molecules that competitively bind the interacting proteins required for activation of the endogenous full-length AR. We provide evidence that in vivo expression of AR NTD decoys decreased tumor incidence and inhibited the growth of prostate cancer tumors. This growth inhibition was characterized by a 10-fold decrease in serum levels of prostate-specific antigen (PSA) (46.7 ng/ml ± 19.9 vs. 432.4 ng/ml ± 201.3; P = 0.0299) and a 4-fold decrease in tumor volume (92.2 mm3 ± 43.4 vs. 331.4 mm3 ± 85.5; P = 0.011). AR NTD decoy molecules also delayed hormonal progression, as determined by time to rising PSA levels after castration of the host. The tumors treated with AR NTD decoys contained more apoptotic cells and fewer proliferating cells, whereas no effect was seen on the viability of cells that did not depend on the AR. This work provides further evidence of the importance of the NTD of the AR in the progression of prostate cancer and presents a target for the development of antagonists of the AR in the clinical management of this disease.

Osteoblast-Derived Factors Induce Androgen-Independent Proliferation and Expression of Prostate-Specific Antigen in Human Prostate Cancer Cells

Purpose: Prostate cancer metastasizes to the skeleton to form osteoblastic lesions. Androgen ablation is the current treatment for metastatic prostate cancer. This therapy is palliative, and the disease will return in an androgen-independent form that is preceded by a rising titer of prostate-specific antigen (PSA). Here, we investigated the possibility that human osteoblasts might secrete factors that contribute to the emergence of androgen-independent prostate cancer. Experimental Design: Primary cultures of human osteoblasts were used as a source of conditioned medium (OCM). Proliferation, expression of androgen-regulated genes, and transactivation of the androgen receptor (AR) were monitored in LNCaP human prostate cancer cells in response to OCM using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Northern blot analysis, and reporter gene constructs. Levels of interleukin-6 (IL-6) present in OCM were measured, and its contribution to proliferation and expression of PSA were investigated by neutralization studies with anti IL-6 antibodies. Results: OCM increased the proliferation and expression of PSA at both the protein and RNA levels in LNCaP cells. Synergistic increases in the activities of PSA (6.1 kb)- and pARR3-tk-luciferase reporters were measured in cells cotreated with both OCM and androgen. OCM targeted the NH2-terminal domain of the AR. The effect of OCM on transcriptional activity of the AR was inhibited by an antiandrogen. Neutralizing antibodies to IL-6 blocked proliferation and expression of PSA by OCM. Conclusion: Osteoblasts secrete factors, such as IL-6, that cause androgen-independent induction of PSA gene expression and proliferation of prostate cancer cells by a mechanism that partially relies on the AR. Identifying such molecular mechanisms may lead to improved clinical management of metastatic prostate cancer.

Niphatenones, glycerol ethers from the sponge Niphates digitalis block androgen receptor transcriptional activity in prostate cancer cells: structure elucidation, synthesis, and biological activity.

Extracts of the marine sponge Niphates digitalis collected in Dominica showed strong activity in a cell-based assay designed to detect antagonists of the androgen receptor (AR) that could act as lead compounds for the development of a new class of drugs to treat castration recurrent prostate cancer (CRPC). Assay-guided fractionation showed that niphatenones A (3) and B (4), two new glycerol ether lipids, were the active components of the extracts. The structures of 3 and 4 were elucidated by analysis of NMR and MS data and confimed via total synthesis. Biological evaluation of synthetic analogues of the niphatenones has shown that the enantiomers 7 and 8 are more potent than the natural products in the screening assay and defined preliminary SAR for the new AR antagonist pharmacophore, including the finding that the Michael acceptor enone functionality is not required for activity. Niphatenone B (4) and its enantiomer 8 blocked androgen-induced proliferation of LNCaP prostate cancer cells but had no effect on the proliferation of PC3 prostate cancer cells that do not express functional AR, consistent with activity as AR antagonists. Use of the propargyl ether 44 and Click chemistry showed that niphatenone B binds covalently to the activation function-1 (AF1) region of the AR N-terminus domain (NTD).

Targeting Androgen Receptor Activation Function-1 with EPI to Overcome Resistance Mechanisms in Castration-Resistant Prostate Cancer.

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

Co-targeting Androgen Receptor Splice Variants and mTOR signaling pathway for the Treatment of Castration-Resistant Prostate Cancer

Purpose: The PI3K/Akt/mTOR pathway is activated in most castration-resistant prostate cancers (CRPC). Transcriptionally active androgen receptor (AR) plays a role in the majority of CRPCs. Therefore, cotargeting full-length (FL) AR and PI3K/Akt/mTOR signaling has been proposed as a possible, more effective therapeutic approach for CRPC. However, truncated AR-splice variants (AR-V) that are constitutively active and dominant over FL-AR are associated with tumor progression and resistance mechanisms in CRPC. It is currently unknown how blocking the PI3K/Akt/mTOR pathway impacts prostate cancer driven by AR-Vs. Here, we evaluated the efficacy and mechanism of combination therapy to block mTOR activity together with EPI-002, an AR N-terminal domain (NTD) antagonist that blocks the transcriptional activities of FL-AR and AR-Vs in models of CRPC. Experimental Design: To determine the functional roles of FL-AR, AR-Vs, and PI3K/Akt/mTOR pathways, we employed EPI-002 or enzalutamide and BEZ235 (low dose) or everolimus in human prostate cancer cells that express FL-AR or FL-AR and AR-Vs (LNCaP95). Gene expression and efficacy were examined in vitro and in vivo. Results: EPI-002 had antitumor activity in enzalutamide-resistant LNCaP95 cells that was associated with decreased expression of AR-V target genes (e.g., UBE2C). Inhibition of mTOR provided additional blockade of UBE2C expression. A combination of EPI-002 and BEZ235 decreased the growth of LNCaP95 cells in vitro and in vivo. Conclusions: Cotargeting mTOR and AR-NTD to block transcriptional activities of FL-AR and AR-Vs provided maximum antitumor efficacy in PTEN-null, enzalutamide-resistant CRPC. Clin Cancer Res; 22(11); 2744–54. ©2015 AACR.

Characterization of Niphatenones that Inhibit Androgen Receptor N-Terminal Domain

Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC). The androgen receptor (AR) remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD). Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD). Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S)-niphatenone had significantly better activity against the AR NTD compared to (R)-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR) activity and covalently bound to GR activation function-1 (AF-1) region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.