Durable Response of Enzalutamide-resistant Prostate Cancer to Supraphysiological Testosterone Is Associated with a Multifaceted Growth Suppression and Impaired DNA Damage Response Transcriptomic Program in Patient-derived Xenografts

doi:10.1016/j.eururo.2019.05.042

European Urology

Volume 77, Issue 2, February 2020, Pages 144-155

https://doi.org/10.1016/j.eururo.2019.05.042 Get rights and content

Abstract

Background

Androgen deprivation therapy improves the survival of castration-resistant prostate cancer (CRPC) patients, yet ultimately fails with debilitating side effects. Supraphysiological testosterone (SPT)-based therapy produces clinical responses with improved quality of life in a subset of patients. Currently, no information defines a durable response to SPT.

Objective

To identify key molecular phenotypes underlying SPT response to improve patient selection and guide combination treatment to achieve a durable response.

Design, setting, and participants

A patient-derived xenograft (PDX) preclinical trial was performed with 13 CRPC PDXs to identify molecular features associated with SPT response. Comprehensive intratumoral androgen, tumor growth, and integrated transcriptomic and protein analyses were performed in three PDXs resistant to the newer androgen receptor (AR) pathway inhibitor enzalutamide (ENZ) to define SPT response and resistance.

Intervention

Testosterone cypionate.

Outcome measurements and statistical analysis

SPT efficacy was evaluated by PDX growth, prostate-specific antigen (PSA) change, and survival. Intratumoral androgens were analyzed using mass spectrometry. Global transcriptome analysis was performed using RNA sequencing, and confirmed by quantitative real-time polymerase chain reaction and immunohistochemistry. Log-rank and Mann-Whitney tests were used for survival and molecular analyses, respectively.

Results and limitations

A durable SPT responder was identified, presenting robust repressions of ARv7 and E2F transcriptional outputs, and a DNA damage response (DDR) transcriptomic program that were altogether restored upon SPT resistance in the transient responder. ENZ rechallenge of SPT-relapsed PDXs resulted in PSA decreases but tumor progression.

Conclusions

SPT produces a durable response in AR-pathway inhibitor ENZ CRPC that is associated with sustained suppression of ARv7 and E2F transcriptional outputs, and the DDR transcriptome, highlighting the potential of combination treatments that maintain suppression of these programs to drive a durable response to SPT.

Patient summary

Patients with ENZ-resistant prostate cancer have very limited treatment options. Supraphysiological testosterone presents a prominent option for improved quality of life and a potential durable response in patients with sustained suppression on ARv7/E2F transcriptional outputs and DNA repair program.

Introduction

Men with castration-resistant prostate cancer (CRPC) who progress on androgen deprivation therapy (ADT) and newer androgen receptor (AR) pathway inhibitors (ARIs) abiraterone acetate (AA) and enzalutamide (ENZ) have poor outcomes and quality of life (QoL), and limited treatment options. As CRPC develops, AR and AR variants (eg, ARv7) are often adaptively upregulated [1], [2], and androgen signaling remains active even upon resistance to ARIs [2], [3], [4]. While upregulation of AR signaling supports CRPC growth, it paradoxically creates new therapeutic vulnerabilities. Supraphysiological testosterone (SPT) has shown efficacy in prostate cancer cell models [5] and in a subset of patients with improved QoL after ADT [6], [7], [8]. However, not all patients respond, and SPT resistance inevitably develops. Currently, there is no information defining a durable response to SPT.

The objectives of this study are to (1) identify molecular features associated with SPT response using a CRPC patient-derived xenograft (PDX) preclinical trial and (2) characterize SPT durable response phenotype using PDX models that exhibit de novo resistance to the newer ARI ENZ (ENZR, representing ∼40% of CRPC patients [9], [10]). We identified that high AR and ARv7 expression, and a positive ARv7 correlation with E2F score directed SPT response; the SPT durable response was associated with sustained repressions of the ARv7/E2F transcriptional outputs and the DNA damage response (DDR) transcriptome. Together, this work supports SPT therapy in currently incurable ENZR CRPC and highlights the opportunity for SPT-based combination therapies to achieve a durable response.

Section snippets

Patient-derived xenografts

Animal procedures were performed in accordance with NIH guidelines and approved by the University of Washington Institutional Animal Care and Use Committee. The CRPC PDX preclinical trial, ENZR PDX studies, and the ENZ rechallenge study were performed in castrated male CB17 SCID mice (Supplementary material). Testosterone (T) cypionate (1 mg) or vehicle was administered every 2 wk via intramuscular injection. Animals were sacrificed 5 days after SPT (D5) and at the end of study (EOS) for molecular

SPT preclinical trial using CRPC PDXs

We have developed a large series of CRPC PDXs representing heterogeneity of clinical specimens [3], [13], [17], [18], [19]. Here, we conducted a preclinical trial of SPT using 13 CRPC PDXs (Fig. 1A and Supplementary Table 2, PDX information). Thirty-one percent of PDXs (four of 13) responded to SPT (Fig. 1B). At baseline, SPT responders exhibited lower proliferation and E2F signaling scores (Fig. 1C and D), and higher AR expression and AR activity score (Fig. 1E and F [20]). Responders also

Discussion

This study provides evidence of the preclinical efficacy of continuous SPT, and demonstrates that high AR and ARv7 signaling, and the positive association of ARv7 with the E2F score underlie SPT responsiveness. For the first time, we identify a durable response phenotype associated with sustained repressions of ARv7 and E2F transcriptional outputs, and the DDR transcriptomic program. The robust repression of ARv7/E2F/DDR transcriptional programs highlights the clinical opportunity of

Conclusions

We provided preclinical evidence highlighting ENZR CRPC addicted to ARv7- and E2F-regulated growth and/or DDR deficiency as SPT responders. We further provide tissue-based evidence for the bioavailability of intratumoral T, and identified that the SPT durable response was associated with sustained repressions of ARv7 and E2F transcriptional outputs, and an impaired DDR program. This study also provides the first-in-field rationale to support potential combination treatment with ARv7-targeting

References (44)

P. Mathew
Prolonged control of progressive castration-resistant metastatic prostate cancer with testosterone replacement therapy: the case for a prospective trial
Ann Oncol
(2008)
H.I. Scher et al.
Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study
Lancet
(2010)
N. Sharma et al.
Control of the p53-p21CIP1 axis by E2f1, E2f2, and E2f3 is essential for G1/S progression and cellular transformation
J Biol Chem
(2006)
C. Cai et al.
Androgen receptor gene expression in prostate cancer is directly suppressed by the androgen receptor through recruitment of lysine-specific demethylase 1
Cancer Cell
(2011)
B.A. Teply et al.
Bipolar androgen therapy in men with metastatic castration-resistant prostate cancer after progression on enzalutamide: an open-label, phase 2, multicohort study
Lancet Oncol
(2018)
B.A. Teply et al.
Extreme response to high-dose testosterone in BRCA2- and ATM-mutated prostate cancer
Eur Urol
(2017)
C.D. Chen et al.
Molecular determinants of resistance to antiandrogen therapy
Nat Med
(2004)
E.S. Antonarakis et al.
AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer
N Engl J Med
(2014)
H.M. Lam et al.
Characterization of an abiraterone ultraresponsive phenotype in castration-resistant prostate cancer patient-derived xenografts
Clin Cancer Res
(2017)
H.M. Lam et al.
Supraphysiological testosterone therapy as treatment for castration-resistant prostate cancer
Front Oncol
(2018)

O.S. Mohammad et al.

Supraphysiologic testosterone therapy in the treatment of prostate cancer: models, mechanisms and questions

Cancers (Basel)

(2017)

M.F. Sarosdy

Testosterone replacement for hypogonadism after treatment of early prostate cancer with brachytherapy

Cancer

(2007)

R.L. Leibowitz et al.

Testosterone replacement in prostate cancer survivors with hypogonadal symptoms

BJU Int

(2010)

H.I. Scher et al.

Increased survival with enzalutamide in prostate cancer after chemotherapy

N Engl J Med

(2012)

E.A. Mostaghel et al.

Resistance to CYP17A1 inhibition with abiraterone in castration-resistant prostate cancer: induction of steroidogenesis and androgen receptor splice variants

Clin Cancer Res

(2011)

H.M. Lam et al.

disrupts HNF4α-regulated gene networks linking to prostate preneoplasia and immune disruption in noble rats

Endocrinology

(2016)

N. Ruppender et al.

Cellular adhesion promotes prostate cancer cells escape from dormancy

PLoS One

(2015)

A. Subramanian et al.

Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles

Proc Natl Acad Sci USA

(2005)

H.M. Nguyen et al.

Cabozantinib inhibits growth of androgen-sensitive and castration-resistant prostate cancer and affects bone remodeling

PLoS One

(2013)

T.M. Therneau et al.

Modeling survival data: extending the Cox model

(2000)

H.M. Nguyen et al.

LuCaP prostate cancer patient-derived xenografts reflect the molecular heterogeneity of advanced disease and serve as models for evaluating cancer therapeutics

Prostate

(2017)

E.G. Bluemn et al.

Androgen receptor pathway-independent prostate cancer is sustained through FGF signaling

Cancer Cell

(2017)

Cited by (39)

Metastatic Bladder Cancer Expression and Subcellular Localization of Nectin-4 and Trop-2 in Variant Histology: A Rapid Autopsy Study
2023, Clinical Genitourinary Cancer
Nectin-4 and Trop-2 are transmembrane targets of FDA-approved antibody-drug conjugates (ADC) Enfortumab-vedotin (EV) and Sacituzumab govitecan (SG), respectively, for the treatment of metastatic urothelial carcinoma (mUC). The expression and role of Nectin-4 and Trop-2 in mUC variant histology is poorly described.
We evaluate membranous and cytoplasmic protein expression, and mRNA levels of Nectin-4 and Trop-2 within matched primary and metastatic mUC samples to determine heterogeneity of ADC targets in mUC variants.
Patients with mUC were consented for rapid autopsy immediately after death. Tissues from matched primary and metastatic lesions were collected. A total of 67 specimens from 20 patients were analyzed: 27 were UC, 17 plasmacytoid (PUC), 18 UC with squamous differentiation (UCSD), and 5 neuroendocrine (NE); 10 from primary and 57 from metastatic sites. All histology except NE expressed moderate-high levels of Nectin-4 and Trop-2 by both immunohistochemistry and RNAseq. Nectin-4 demonstrated prominent cytoplasmic staining in metastatic PUC and UCSD. Trop-2 demonstrated strong cytoplasmic and membrane staining in primary and metastatic tumors. Interestingly, Nectin-4 and Trop-2 expression are positively correlated at both mRNA and protein levels.
UC and non-NE variants express notable level of Nectin-4 and Trop-2 in both primary and metastatic lesions. Membrane staining of Nectin-4 and Trop-2 is present but cytoplasmic staining is a more common event in both mUC and mUC variant histology. These findings support evaluation of EV and SG in heavily treated variant histology BC and urge attention on the clinical relevance of cytoplasmic localization of ADC targets.
A kinome-wide CRISPR screen identifies CK1α as a target to overcome enzalutamide resistance of prostate cancer
2023, Cell Reports Medicine
Enzalutamide (ENZA), a second-generation androgen receptor antagonist, has significantly increased progression-free and overall survival of patients with metastatic prostate cancer (PCa). However, resistance remains a prominent obstacle in treatment. Utilizing a kinome-wide CRISPR-Cas9 knockout screen, we identified casein kinase 1α (CK1α) as a therapeutic target to overcome ENZA resistance. Depletion or pharmacologic inhibition of CK1α enhanced ENZA efficacy in ENZA-resistant cells and patient-derived xenografts. Mechanistically, CK1α phosphorylates the serine residue S1270 and modulates the protein abundance of ataxia telangiectasia mutated (ATM), a primary initiator of DNA double-strand break (DSB)-response signaling, which is compromised in ENZA-resistant cells and patients. Inhibition of CK1α stabilizes ATM, resulting in the restoration of DSB signaling, and thus increases ENZA-induced cell death and growth arrest. Our study details a therapeutic approach for ENZA-resistant PCa and characterizes a particular perspective for the function of CK1α in the regulation of DNA-damage response.
Understanding and targeting prostate cancer cell heterogeneity and plasticity
2022, Seminars in Cancer Biology
Prostate cancer (PCa) is a prevalent malignancy that occurs primarily in old males. Prostate tumors in different patients manifest significant inter-patient heterogeneity with respect to histo-morphological presentations and molecular architecture. An individual patient tumor also harbors genetically distinct clones in which PCa cells display intra-tumor heterogeneity in molecular features and phenotypic marker expression. This inherent PCa cell heterogeneity, e.g., in the expression of androgen receptor (AR), constitutes a barrier to the long-term therapeutic efficacy of AR-targeting therapies. Furthermore, tumor progression as well as therapeutic treatments induce PCa cell plasticity such that AR-positive PCa cells may turn into AR-negative cells and prostate tumors may switch lineage identity from adenocarcinomas to neuroendocrine-like tumors. This induced PCa cell plasticity similarly confers resistance to AR-targeting and other therapies. In this review, I first discuss PCa from the perspective of an abnormal organ development and deregulated cellular differentiation, and discuss the luminal progenitor cells as the likely cells of origin for PCa. I then focus on intrinsic PCa cell heterogeneity in treatment-naïve tumors with the presence of prostate cancer stem cells (PCSCs). I further elaborate on PCa cell plasticity induced by genetic alterations and therapeutic interventions, and present potential strategies to therapeutically tackle PCa cell heterogeneity and plasticity. My discussions will make it clear that, to achieve enduring clinical efficacy, both intrinsic PCa cell heterogeneity and induced PCa cell plasticity need to be targeted with novel combinatorial approaches.
Exploiting the tumor-suppressive activity of the androgen receptor by CDK4/6 inhibition in castration-resistant prostate cancer
2022, Molecular Therapy
Citation Excerpt :
These mice were then treated with two doses of testosterone treatments, both of which were well tolerated by the animals (Figures S9B and S9C). The lower dose of testosterone treatment (40 mg/kg) that was used in the experiment is comparable with the dose used in the previous preclinical studies and clinical trials.8-10 The growth of Rb-proficient C4-2 tumors was markedly repressed by both testosterone treatments (Figure 5A).
The androgen receptor (AR) plays a pivotal role in driving prostate cancer (PCa) development. However, when stimulated by high levels of androgens, AR can also function as a tumor suppressor in PCa cells. While the high-dose testosterone (high-T) treatment is currently being tested in clinical trials of castration-resistant prostate cancer (CRPC), there is still a pressing need to fully understand the underlying mechanism and thus develop treatment strategies to exploit this tumor-suppressive activity of AR. In this study, we demonstrate that retinoblastoma (Rb) family proteins play a central role in maintaining the global chromatin binding and transcriptional repression program of AR and that Rb inactivation desensitizes CRPC to the high-dose testosterone treatment in vitro and in vivo. Using a series of patient-derived xenograft (PDX) CRPC models, we further show that the efficacy of high-T treatment can be fully exploited by a CDK4/6 inhibitor, which strengthens the chromatin binding of the Rb-E2F repressor complex by blocking the hyperphosphorylation of Rb proteins. Overall, our study provides strong mechanistic and preclinical evidence on further developing clinical trials to combine high-T with CDK4/6 inhibitors in treating CRPC.
Molecular and Clinical Characterization of Patients With Metastatic Castration Resistant Prostate Cancer Achieving Deep Responses to Bipolar Androgen Therapy
2022, Clinical Genitourinary Cancer
Bipolar androgen therapy (BAT) is an emerging treatment strategy for men with metastatic castration resistant prostate cancer (mCRPC) whereby serum testosterone is cycled from supraphysiologic to near-castrate levels each month. BAT has been shown to induce clinical responses in a significant proportion of patients, some of which are extreme. We explored the clinical and molecular characteristics of patients with mCRPC who achieved deep responses to BAT.
We conducted a retrospective analysis of patients with mCRPC treated with BAT at Johns Hopkins. We identified 22 of 114 (19%) patients with mCRPC who achieved ≥70% PSA reductions upon treatment with BAT. All patients were treated using 400 mg testosterone cypionate intramuscularly every 28 days, together with continuous LHRH agonist therapy. Clinical-grade DNA sequencing was obtained using commercially available assays.
Somatic next-generation sequencing was obtained for 15 of 22 (68%) patients. Of these 15 extreme PSA responders with sequencing data available, All 15 of 15 (100%) harbored a pathogenic mutation in TP53 and/or a homologous recombination DNA repair (HRD) gene. Among the subset of patients with measureable disease (N = 15), 10 patients (67%) achieved an objective response including one patient with a complete response. The median radiographic progression-free survival of these deep PSA responders was 11.3 months (95% CI, 7.9-25.0 months).
We observed an enrichment of TP53 and HRD mutations in mCRPC patients with extreme PSA responses to BAT, with durability lasting about a year. These data support the hypothesis that BAT may most benefit patients with DNA repair-deficient mCRPC. Further studies of BAT in biomarker-selected mCRPC populations (ie, TP53/HRD-mutated) are warranted.
Androgens in prostate cancer: A tale that never ends
2021, Cancer Letters
Citation Excerpt :
Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising clinical benefits to patients with the BRCA1/2 mutation [138]. It has also been reported that androgens and AR are involved in the regulation of the cell cycle and DNA damage, and that supraphysiologic testosterone has been found to suppress the genes essential for the DNA-damage response [136]. Thus, PARP inhibitors might provide more-potent clinical efficacy in an androgen-rich environment; e.g., in patients before ADT or receiving BAT.
Androgens play an essential role in prostate cancer. Clinical treatments that target steroidogenesis and the androgen receptor (AR) successfully postpone disease progression. Abiraterone and enzalutamide, the next-generation androgen receptor pathway inhibitors (ARPI), emphasize the function of the androgen-AR axis even in castration-resistant prostate cancer (CRPC). However, with the increased incidence in neuroendocrine prostate cancer (NEPC) showing resistance to ARPI, the importance of androgen-AR axis in further disease management remains elusive. Herein we review the steroidogenic pathways associated with different disease stages and discuss the potential targets for disease management after manifesting resistance to abiraterone and enzalutamide.

View all citing articles on Scopus

View full text

Abstract

Background

Objective

Design, setting, and participants

Intervention

Outcome measurements and statistical analysis

Results and limitations

Conclusions

Patient summary

Introduction

Section snippets

Patient-derived xenografts

SPT preclinical trial using CRPC PDXs

Discussion

Conclusions

Ann Oncol

Lancet

J Biol Chem

Cancer Cell

Lancet Oncol

Eur Urol

Molecular determinants of resistance to antiandrogen therapy

Nat Med

AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer

N Engl J Med

Characterization of an abiraterone ultraresponsive phenotype in castration-resistant prostate cancer patient-derived xenografts

Clin Cancer Res

Supraphysiological testosterone therapy as treatment for castration-resistant prostate cancer

Front Oncol

Supraphysiologic testosterone therapy in the treatment of prostate cancer: models, mechanisms and questions

Cancers (Basel)

Testosterone replacement for hypogonadism after treatment of early prostate cancer with brachytherapy

Cancer

Testosterone replacement in prostate cancer survivors with hypogonadal symptoms

BJU Int

Increased survival with enzalutamide in prostate cancer after chemotherapy

N Engl J Med

Resistance to CYP17A1 inhibition with abiraterone in castration-resistant prostate cancer: induction of steroidogenesis and androgen receptor splice variants

Clin Cancer Res

disrupts HNF4α-regulated gene networks linking to prostate preneoplasia and immune disruption in noble rats

Endocrinology

Cellular adhesion promotes prostate cancer cells escape from dormancy

PLoS One

Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles

Proc Natl Acad Sci USA

Cabozantinib inhibits growth of androgen-sensitive and castration-resistant prostate cancer and affects bone remodeling

PLoS One

Modeling survival data: extending the Cox model

LuCaP prostate cancer patient-derived xenografts reflect the molecular heterogeneity of advanced disease and serve as models for evaluating cancer therapeutics

Prostate

Androgen receptor pathway-independent prostate cancer is sustained through FGF signaling

Cancer Cell