A cell permeable bimane-constrained PCNA-interacting peptide

The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 3₁₀-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143–151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with K(D) values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 3₁₀-helical binding conformation. This suggests the 3₁₀-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.Aimee J. Horsfall, Beth A. Vandborg, Zoya Kikhtyak, Denis B. Scanlon, Wayne D. Tilley, Theresa E. Hickey, John B. Bruning and Andrew D. Abel

High-throughput imaging assay for drug screening of 3D prostate cancer organoids

First Published June 11, 2021New treatments are required for advanced prostate cancer; however, there are fewer preclinical models of prostate cancer than other common tumor types to test candidate therapeutics. One opportunity to increase the scope of preclinical studies is to grow tissue from patient-derived xenografts (PDXs) as organoid cultures. Here we report a scalable pipeline for automated seeding, treatment and an analysis of the drug responses of prostate cancer organoids. We established organoid cultures from 5 PDXs with diverse phenotypes of prostate cancer, including castrate-sensitive and castrate-resistant disease, as well as adenocarcinoma and neuroendocrine pathology. We robotically embedded organoids in Matrigel in 384-well plates and monitored growth via brightfield microscopy before treatment with poly ADP-ribose polymerase inhibitors or a compound library. Independent readouts including metabolic activity and live-cell imaging-based features provided robust measures of organoid growth and complementary ways of assessing drug efficacy. Single organoid analyses enabled in-depth assessment of morphological differences between patients and within organoid populations and revealed that larger organoids had more striking changes in morphology and composition after drug treatment. By increasing the scale and scope of organoid experiments, this automated assay complements other patient-derived models and will expedite preclinical testing of new treatments for prostate cancer.Nicholas Choo, Susanne Ramm, Jennii Luu, Jean M. Winter, Luke A. Selth, Amy R. Dwyer … et al

A patient-derived explant (PDE) model of hormone-dependent cancer

Breast and prostate cancer research to date has largely been predicated on the use of cell lines in vitro or in vivo. These limitations have led to the development of more clinically relevant models, such as organoids or murine xenografts that utilize patient-derived material; however, issues related to low take rate, long duration of establishment, and the associated costs constrain use of these models. This study demonstrates that ex vivo culture of freshly resected breast and prostate tumor specimens obtained from surgery, termed patient-derived explants (PDEs), provides a high-throughput and cost-effective model that retains the native tissue architecture, microenvironment, cell viability, and key oncogenic drivers. The PDE model provides a unique approach for direct evaluation of drug responses on an individual patient's tumor, which is amenable to analysis using contemporary genomic technologies. The ability to rapidly evaluate drug efficacy in patient-derived material has high potential to facilitate implementation of personalized medicine approaches.Margaret M. Centenera, Theresa E. Hickey, Shalini Jindal, Natalie K. Ryan, Preethi Ravindranathan, Hisham Mohammed, Jessica L. Robinson, Matthew J. Schiewer, Shihong Ma, Payal Kapur, Peter D. Sutherland, Clive E. Hoffmann, Claus G. Roehrborn, Leonard G. Gomella, Jason S. Carroll, Stephen N. Birrell, Karen E. Knudsen, Ganesh V. Raj, Lisa M. Butler, Wayne D. Tille

Arming androgen receptors to oppose oncogenic estrogen receptor activity in breast cancer

Published online: 22 July 2021Most breast cancers are driven by oncogenic activity of the estrogen receptor alpha (ER). Resistance to ER target therapies is the major cause of breast cancer death. Recently, there has been renewed interest in targeting the androgen receptor (AR) to treat ER-driven breast cancers. Herein, we discuss evidence for an AR agonist, not antagonist, treatment strategy.Theresa E. Hickey, Amy R. Dwyer and Wayne D. Tille

Bringing androgens up a NOTCH in breast cancer

While it has been known for decades that androgen hormones influence normal breast development and breast carcinogenesis, the underlying mechanisms have only been recently elucidated. To date, most studies have focused on androgen action in breast cancer cell lines, yet these studies represent artificial systems that often do not faithfully replicate/recapitulate the cellular, molecular and hormonal environments of breast tumours in vivo. It is critical to have a better understanding of how androgens act in the normal mammary gland as well as in in vivo systems that maintain a relevant tumour microenvironment to gain insights into the role of androgens in the modulation of breast cancer development. This in turn will facilitate application of androgen-modulation therapy in breast cancer. This is particularly relevant as current clinical trials focus on inhibiting androgen action as breast cancer therapy but, depending on the steroid receptor profile of the tumour, certain individuals may be better served by selectively stimulating androgen action. Androgen receptor (AR) protein is primarily expressed by the hormone-sensing compartment of normal breast epithelium, commonly referred to as oestrogen receptor alpha (ERa (ESR1))-positive breast epithelial cells, which also express progesterone receptors (PRs) and prolactin receptors and exert powerful developmental influences on adjacent breast epithelial cells. Recent lineage-tracing studies, particularly those focussed on NOTCH signalling, and genetic analysis of cancer risk in the normal breast highlight how signalling via the hormone-sensing compartment can influence normal breast development and breast cancer susceptibility. This provides an impetus to focus on the relationship between androgens, AR and NOTCH signalling and the crosstalk between ERa and PR signalling in the hormone-sensing component of breast epithelium in order to unravel the mechanisms behind the ability of androgens to modulate breast cancer initiation and growth.Gerard A Tarulli, Lisa M Butler, Wayne D Tilley and Theresa E Hicke

Interplay between the androgen receptor signaling axis and microRNAs in prostate cancer

The androgen receptor (AR) is a ligand activated transcription factor that drives prostate cancer. Since therapies that target the AR are the mainstay treatment for men with metastatic disease, it is essential to understand the molecular mechanisms underlying oncogenic AR signaling in the prostate. MicroRNAs (miRNAs) are small, non-coding regulators of gene expression that play a key role in prostate cancer, and are increasingly recognized as targets or modulators of the AR signaling axis. In this review, we examine the regulation of AR signalling by miRNAs and vice versa, and discuss how this interplay influences prostate cancer growth, metastasis and resistance to therapy. Finally, we explore the potential clinical applications of miRNAs implicated in the regulation of AR signaling in this prevalent hormone-driven disease.Rayzel C Fernandes, Theresa E Hickey, Wayne D Tilley and Luke A Selt

Unraveling an identity for the androgen receptor-expressing mammary epithelial cell

Abstracts of the Endocrine Society of Australia Annual Scientific Meeting 2015, 23–26 August 2015Gerard A Tarulli, Geraldine Laven-Law, Wayne D Tilley, and Theresa E Hicke

ANTI-proliferative transcriptional effects of medroxyprogesterone acetate in Estrogen receptor positive breast cancer cells are predominantly mediated by ThE progesterone receptor.

Medroxyprogesterone acetate (MPA) is a first generation progestin that has been in clinical use for various hormonal conditions in women since the 1960s. Although developed as a progesterone receptor (PR) agonist, MPA also has strong binding affinity for other steroid receptors. This promiscuity confounds the mechanistic action of MPA in target cells that express multiple steroid receptors. This study is the first to assess the relative contribution of progesterone, androgen and glucocorticoid receptors in mediating the transcriptional activity of MPA on endogenous targets in breast cancer cells that endogenously express all three receptors at comparable levels. Gene expression profiling in estrogen receptor positive (ER+) ZR-75-1 breast cancer cells demonstrated that although the MPA-regulated transcriptome strongly overlapped with that of Progesterone (PROG), 5α-dihydrotestosterone (DHT) and Dexamethasone (DEX), it clustered most strongly with that of PROG, suggesting that MPA predominantly acts via the progesterone receptor (PR) rather than androgen receptor (AR) or glucocorticoid receptor (GR). Subsequent experiments manipulating levels of these receptors, either through specific culture conditions or with lentiviral shRNAs targeting individual receptors, also revealed a stronger contribution of PR compared to AR and GR on the expression of endogenous target genes that are either commonly regulated by all ligands or specifically regulated only by MPA. A predominant contribution of PR to MPA action in ER + T-47D breast cancer cells was also observed, although a stronger role for AR was evident in T-47D compared to that observed in ZR-75-1 cells. Network analysis of ligand-specific and commonly regulated genes demonstrated that MPA utilises different transcription factors and signalling pathways to inhibit proliferation compared with PROG. This study reaffirms the importance of PR in mediating MPA action in an endogenous breast cancer context where multiple steroid receptors are co-expressed and has potential implications for PR-targeting therapeutic strategies in ER + breast cancer.Nicole L.Moore, Adrienne R.Hanson, Esmaeil Ebrahimie, Theresa E.Hickey, Wayne D.Tille

Complexities of androgen receptor signalling in breast cancer

While the clinical benefit of androgen-based therapeutics in breast cancer has been known since the 1940s, we have only recently begun to fully understand the mechanisms of androgen action in breast cancer. Androgen signalling pathways can have either beneficial or deleterious effects in breast cancer depending on the breast cancer subtype and intracellular context. This review discusses our current knowledge of androgen signalling in breast cancer, including the relationship between serum androgens and breast cancer risk, the prognostic significance of androgen receptor (AR) expression in different breast cancer subtypes and the downstream molecular pathways mediating androgen action in breast cancer cells. Intracrine androgen metabolism has also been discussed and proposed as a potential mechanism that may explain some of the reported differences regarding dichotomous androgen actions in breast cancers. A better understanding of AR signalling in this disease is critical given the current resurgence in interest in utilising contemporary AR-directed therapies for breast cancer and the need for biomarkers that will accurately predict clinical response.Keely M McNamara, Nicole L Moore, Theresa E Hickey, Hironobu Sasano and Wayne D Tille

The combined actions of DHT and MPA lead to altered AR signaling in normal and malignant post-menopausal breast epithelial cells

Abstract Consistent with several observational studies examining combined hormone replacement therapy (cHRT: conjugated equine estrogen in conjunction with the synthetic progestin medroxyprogesterone acetate, MPA) in postmenopausal women, a re-analysis of the placebo-controlled randomized Women's Health Initiative clinical trial demonstrated a markedly increased breast cancer risk in newly menopausal women following ≥ 5 years of cHRT (Hazard Ratio, 3.05; 95% Confidence Interval, 1.62-5.70) [1]. We have previously demonstrated that androgen receptor (AR)-mediated effects of MPA impede 5α-dihydrotestosterone (DHT)-induced AR signaling in normal and malignant breast epithelial cells (AACR abstract 2010). The current study aimed to further investigate the biological actions of DHT and/or MPA on steroid receptor expression and cancer-related intracellular signaling pathways. Immunohistochemical analysis of estrogen receptor alpha (ERα), progesterone receptor (PR) and AR expression was performed on histologically normal human post-menopausal breast tissues and measured by image analysis in tissues cultured ex vivo with vehicle (0.1% ethanol control), DHT (1nM), MPA (1nM) or the AR antagonist, bicalutamide (Bic;1uM) for 48 hr, either alone or in combination. Microarray analysis and qRT-PCR validation were performed using the ERα positive breast cancer cell line, ZR-75-1 to determine changes in gene expression in key intracellular signaling pathways. The microarray data was analyzed with Ingenuity Gene Pathway Analysis and Gene Ontology software. Statistical tests included both Wilcoxon matched pairs test and one-way ANOVA (p<0.05). DHT treatment increased AR expression in cultured breast tissues compared to vehicle control (p<0.05), and co-treatment with either MPA or Bic impeded this effect. No change in ERα or PR protein levels was induced by the hormone treatments. Microarray studies revealed that DHT or MPA treatment for 6 hr altered the expression of 439 and 858 genes, respectively, whereas co-treatment altered 1494 genes (p<0.05). Only 114 genes were uniquely regulated by DHT, and the expression of 32% (41% induced and 27% repressed) of these genes was abrogated by MPA. Similarly, the expression of 38% (51% induced and 24% repressed) of the 690 genes uniquely regulated by co-treatment with DHT and MPA was altered compared to DHT alone (p<0.05). Examples of genes that were regulated by DHT (p<0.05) and this effect of DHT was antagonised by co-treatment with DHT and MPA (p<0.05) are FGFR2, OLR1 and C1ORF116. Co-treatment with DHT and MPA altered the expression of genes involved in cell growth, cell cycle, cell death, cancer and intracellular signaling pathways compared to individual treatments (p<0.05). Collectively, these findings suggest an AR-mediated mechanism for the action of MPA in breast cancer. 1.Prentice, R.L., et al. Am J Epidemiol, 2009 170(1): 12-23. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 274. doi:1538-7445.AM2012-274Aleksandra M. Ochnik, Nicole L. Moore, Stephen N. Birrell, Lisa M. Butler, Shalini Jindal, Luke Selth, Wayne D. Tilley and Theresa E. Hicke