Adrienne R. Hanson

Evidence for Efficacy of New Hsp90 Inhibitors Revealed by Ex Vivo Culture of Human Prostate Tumors

Purpose: Targeting Hsp90 has significant potential as a treatment for prostate cancer, but prototypical agents such as 17-allylamino-17 demethoxygeldanamycin (17-AAG) have been ineffective in clinical trials. Recently, a phase I study aimed at defining a biologically active dose reported the first response to an Hsp90 inhibitor in a patient with prostate cancer, which supports the development of new generation compounds for this disease. Experimental Design: The biological actions of two new synthetic Hsp90 inhibitors, NVP-AUY922 and NVP-HSP990, were evaluated in the prostate cancer cell lines PC-3, LNCaP, and VCaP and in an ex vivo culture model of human prostate cancer. Results: In cell lines, both NVP-AUY922 and NVP-HSP990 showed greater potency than 17-AAG with regard to modulation of Hsp90 client proteins, inhibition of proliferation, and induction of apoptotic cell death. In prostate tumors obtained from radical prostatectomy that were cultured ex vivo, treatment with 500 nmol/L of NVP-AUY922, NVP-HSP990, or 17-AAG caused equivalent target modulation, determined by the pharmacodynamic marker Hsp70, but only NVP-AUY922 and NVP-HSP990 showed antiproliferative and proapoptotic activity. Conclusions: This study provides some of the first evidence that new generation Hsp90 inhibitors are capable of achieving biologic responses in human prostate tumors, with both NVP-AUY922 and NVP-HSP990 showing potent on-target efficacy. Importantly, the ex vivo culture technique has provided information on Hsp90 inhibitor action not previously observed in cell lines or animal models. This approach, therefore, has the potential to enable more rational selection of therapeutic agents and biomarkers of response for clinical trials. Clin Cancer Res; 18(13); 3562–70. ©2012 AACR.

A ZEB1-miR-375-YAP1 pathway regulates epithelial plasticity in prostate cancer

MicroRNA-375 (miR-375) is frequently elevated in prostate tumors and cell-free fractions of patient blood, but its role in genesis and progression of prostate cancer is poorly understood. In this study, we demonstrated that miR-375 is inversely correlated with epithelial–mesenchymal transition signatures (EMT) in clinical samples and can drive mesenchymal–epithelial transition (MET) in model systems. Indeed, miR-375 potently inhibited invasion and migration of multiple prostate cancer lines. The transcription factor YAP1 was found to be a direct target of miR-375 in prostate cancer. Knockdown of YAP1 phenocopied miR-375 overexpression, and overexpression of YAP1 rescued anti-invasive effects mediated by miR-375. Furthermore, transcription of the miR-375 gene was shown to be directly repressed by the EMT transcription factor, ZEB1. Analysis of multiple patient cohorts provided evidence for this ZEB1-miR-375-YAP1 regulatory circuit in clinical samples. Despite its anti-invasive and anti-EMT capacities, plasma miR-375 was found to be correlated with circulating tumor cells in men with metastatic disease. Collectively, this study provides new insight into the function of miR-375 in prostate cancer, and more broadly identifies a novel pathway controlling epithelial plasticity and tumor cell invasion in this disease.

Expression of androgen receptor splice variants in clinical breast cancers

The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer.

Electrophoretic sperm isolation: optimization of electrophoresis conditions and impact on oxidative stress

BACKGROUND The purpose of this study was to optimize the electrophoretic conditions that should be used for the effective isolation of functional human spermatozoa and to determine whether this method of isolating cells was associated with oxidative stress and DNA damage. METHODS Human spermatozoa were prepared by repeated centrifugation, discontinuous density gradient centrifugation and electrophoresis followed by assessments of sperm quality. RESULTS Systematic analysis of optimal electrophoresis conditions demonstrated that field strength was positively correlated with sperm recovery rates but negatively correlated with sperm movement, irrespective of whether the current or the voltage was held constant. This loss of functionality observed at high power settings was not associated with a major increase in superoxide generation or the induction of oxidative DNA damage. In contrast, discontinuous Percoll gradient centrifugation was shown to produce a significant rise in oxidative DNA base adduct expression in live cells (P < 0.05). As a result of these analyses, optimized electrophoretic conditions were defined that permitted sperm recovery rates of around 20%. These electrophoretically isolated cells were not only free of oxidative stress but exhibited significantly enhanced motility (P < 0.01) and vitality (P < 0.001) compared with the original samples. CONCLUSIONS We conclude that while field strength is positively correlated with sperm recovery rates; it is negatively associated with sperm motility. Optimized conditions are described that represent a balance between these opposing forces and permit the isolation of highly motile, vital sperm populations, free from the oxidative DNA damage associated with conventional density gradient centrifugation technologies.

MicroRNA-194 promotes prostate cancer metastasis by inhibiting SOCS2

Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial-mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194-driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194-stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194-driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer. Cancer Res; 77(4); 1021-34. ©2016 AACR.

Novel Androgen Receptor Coregulator GRHL2 Exerts Both Oncogenic and Antimetastatic Functions in Prostate Cancer

Alteration to the expression and activity of androgen receptor (AR) coregulators in prostate cancer is an important mechanism driving disease progression and therapy resistance. Using a novel proteomic technique, we identified a new AR coregulator, the transcription factor Grainyhead-like 2 (GRHL2), and demonstrated its essential role in the oncogenic AR signaling axis. GRHL2 colocalized with AR in prostate tumors and was frequently amplified and upregulated in prostate cancer. Importantly, GRHL2 maintained AR expression in multiple prostate cancer model systems, was required for cell proliferation, enhanced ARs transcriptional activity, and colocated with AR at specific sites on chromatin to regulate genes relevant to disease progression. GRHL2 is itself an AR-regulated gene, creating a positive feedback loop between the two factors. The link between GRHL2 and AR also applied to constitutively active truncated AR variants (ARV), as GRHL2 interacted with and regulated ARVs and vice versa. These oncogenic functions of GRHL2 were counterbalanced by its ability to suppress epithelial-mesenchymal transition and cell invasion. Mechanistic evidence suggested that AR assisted GRHL2 in maintaining the epithelial phenotype. In summary, this study has identified a new AR coregulator with a multifaceted role in prostate cancer, functioning as an enhancer of the oncogenic AR signaling pathway but also as a suppressor of metastasis-related phenotypes. Cancer Res; 77(13); 3417-30. ©2017 AACR.

Patient-derived Models of Abiraterone- and Enzalutamide-resistant Prostate Cancer Reveal Sensitivity to Ribosome-directed Therapy

BACKGROUND The intractability of castration-resistant prostate cancer (CRPC) is exacerbated by tumour heterogeneity, including diverse alterations to the androgen receptor (AR) axis and AR-independent phenotypes. The availability of additional models encompassing this heterogeneity would facilitate the identification of more effective therapies for CRPC. OBJECTIVE To discover therapeutic strategies by exploiting patient-derived models that exemplify the heterogeneity of CRPC. DESIGN, SETTING, AND PARTICIPANTS Four new patient-derived xenografts (PDXs) were established from independent metastases of two patients and characterised using integrative genomics. A panel of rationally selected drugs was tested using an innovative ex vivo PDX culture system. INTERVENTION The following drugs were evaluated: AR signalling inhibitors (enzalutamide and galeterone), a PARP inhibitor (talazoparib), a chemotherapeutic (cisplatin), a CDK4/6 inhibitor (ribociclib), bromodomain and extraterminal (BET) protein inhibitors (iBET151 and JQ1), and inhibitors of ribosome biogenesis/function (RNA polymerase I inhibitor CX-5461 and pan-PIM kinase inhibitor CX-6258). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Drug efficacy in ex vivo cultures of PDX tissues was evaluated using immunohistochemistry for Ki67 and cleaved caspase-3 levels. Candidate drugs were also tested for antitumour efficacy in vivo, with tumour volume being the primary endpoint. Two-tailed t tests were used to compare drug and control treatments. RESULTS AND LIMITATIONS Integrative genomics revealed that the new PDXs exhibited heterogeneous mechanisms of resistance, including known and novel AR mutations, genomic structural rearrangements of the AR gene, and a neuroendocrine-like AR-null phenotype. Despite their heterogeneity, all models were sensitive to the combination of ribosome-targeting agents CX-5461 and CX-6258. CONCLUSIONS This study demonstrates that ribosome-targeting drugs may be effective against diverse CRPC subtypes including AR-null disease, and highlights the potential of contemporary patient-derived models to prioritise treatment strategies for clinical translation. PATIENT SUMMARY Diverse types of therapy-resistant prostate cancers are sensitive to a new combination of drugs that inhibit protein synthesis pathways in cancer cells.

Abstract B047: Cyclin-dependent kinase 2 regulates androgen receptor activity in estrogen receptor negative breast cancer

There are limited treatment options for estrogen receptor (ER)-negative breast cancer, a disease subtype that is highly aggressive and associated with poor clinical outcome. The androgen receptor (AR) is expressed in approximately 30% of ER-negative breast cancers and is emerging as a viable therapeutic target. AR activity is influenced by numerous intracellular signalling pathways which post-translationally modify the AR protein to finely regulate its stability and transcriptional output. This study is investigating the functional interaction between cyclin dependent kinases (CDKs) and AR in breast cancer. We hypothesize that CDK2 is required for optimal AR activity and androgen-regulated cell growth in ER-negative breast cancer cells and that inhibition of CDK2 may be an effective approach to reduce ER-negative breast cancer cell growth. AR-positive, ER-negative MDA-MB-453 breast cancer cells were treated with the androgen 5α-dihydrotestosterone (DHT) in the presence or absence of the CDK2 inhibitors roscovitine, NU6102 or CVT-313. The effects on AR target genes as well as total and phosphorylated AR were assessed by quantitative RT-PCR and Western blotting while MTT assay, trypan blue dye exclusion and flow cytometry were used to examine cell proliferation and viability. DHT induced expression of the AR target genes FKBP5, c1ORF116, SEC14L2 and RANBP3L however each of the CDK2 inhibitors completely blocked the stimulatory effect of DHT on these genes. The CDK2 inhibitors also reduced the ability of DHT to stabilize AR protein and induce Ser81-AR phosphorylation. Basal and DHT-induced cell proliferation was blocked by the CDK2 inhibitors, and this was associated with cell cycle arrest and cell death. The results suggest that AR requires CDK2 for optimal transcriptional activity and proliferative effects in breast cancer cells. This may have important therapeutic implications for ER-negative, AR-positive breast cancers. Citation Format: Luke A. Proctor, Adrienne R. Hanson, Miriam S. Butler, Wayne D. Tilley, Nicole L. Moore. Cyclin-dependent kinase 2 regulates androgen receptor activity in estrogen receptor negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B047.