Single-cell analysis of treatment-resistant prostate cancer: Implications of cell state changes for cell surface antigen-targeted therapies.

Targeting cell surface molecules using radioligand and antibody-based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)-a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single-cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate-resistant PRAD and NEPC than previously anticipated but also found that the expression of molecules targeted therapeutically, namely PSMA, STEAP1, STEAP2, TROP2, CEACAM5, and DLL3, varied within a subset of gene-regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to current and future antigen-directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types.

Single Cell Analysis of Treatment-Resistant Prostate Cancer: Implications of Cell State Changes for Cell Surface Antigen Targeted Therapies.

Targeting cell surface molecules using radioligand and antibody-based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)--a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis (TMA) on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate-resistant PRAD and NEPC than previously anticipated, but also found that the expression of molecules targeted therapeutically, namely PSMA, STEAP1, STEAP2, TROP2, CEACAM5, and DLL3, varied within a subset of gene-regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer (SCLC) subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to novel antigen-directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types.

Targeting prostate tumor low-molecular weight tyrosine phosphatase for oxidation-sensitizing therapy.

Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr270. PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.

Allosteric interactions prime androgen receptor dimerization and activation.

The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.

Author Correction: FOXA1 mutations alter pioneering activity, differentiation and prostate cancer phenotypes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tumor Microenvironment-Derived NRG1 Promotes Antiandrogen Resistance in Prostate Cancer.

Despite the development of second-generation antiandrogens, acquired resistance to hormone therapy remains a major challenge in treating advanced prostate cancer. We find that cancer-associated fibroblasts (CAFs) can promote antiandrogen resistance in mouse models and in prostate organoid cultures. We identify neuregulin 1 (NRG1) in CAF supernatant, which promotes resistance in tumor cells through activation of HER3. Pharmacological blockade of the NRG1/HER3 axis using clinical-grade blocking antibodies re-sensitizes tumors to hormone deprivation in vitro and in vivo. Furthermore, patients with castration-resistant prostate cancer with increased tumor NRG1 activity have an inferior response to second-generation antiandrogen therapy. This work reveals a paracrine mechanism of antiandrogen resistance in prostate cancer amenable to clinical testing using available targeted therapies.

Somatic Tissue Engineering in Mouse Models Reveals an Actionable Role for WNT Pathway Alterations in Prostate Cancer Metastasis.

To study genetic factors influencing the progression and therapeutic responses of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation-based genetically engineered mouse models (EPO-GEMM) recapitulate features of traditional germline models and, by modeling genetic factors linked to late-stage human disease, can produce tumors that are metastatic and castration-resistant. A subset of tumors with Trp53 alterations acquired spontaneous WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Using the EPO-GEMM approach and an orthogonal organoid-based model, we show that WNT pathway activation drives metastatic disease that is sensitive to pharmacologic WNT pathway inhibition. Thus, by leveraging EPO-GEMMs, we reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as therapeutic target in metastatic prostate cancer. SIGNIFICANCE: Our understanding of the factors driving metastatic prostate cancer is limited by the paucity of models of late-stage disease. Here, we develop EPO-GEMMs of prostate cancer and use them to identify and validate the WNT pathway as an actionable driver of aggressive metastatic disease.This article is highlighted in the In This Issue feature, p. 890.

Modeling biological and genetic diversity in upper tract urothelial carcinoma with patient derived xenografts.

Treatment paradigms for patients with upper tract urothelial carcinoma (UTUC) are typically extrapolated from studies of bladder cancer despite their distinct clinical and molecular characteristics. The advancement of UTUC research is hampered by the lack of disease-specific models. Here, we report the establishment of patient derived xenograft (PDX) and cell line models that reflect the genomic and biological heterogeneity of the human disease. Models demonstrate high genomic concordance with the corresponding patient tumors, with invasive tumors more likely to successfully engraft. Treatment of PDX models with chemotherapy recapitulates responses observed in patients. Analysis of a HER2 S310F-mutant PDX suggests that an antibody drug conjugate targeting HER2 would have superior efficacy versus selective HER2 kinase inhibitors. In sum, the biological and phenotypic concordance between patient and PDXs suggest that these models could facilitate studies of intrinsic and acquired resistance and the development of personalized medicine strategies for UTUC patients.

FOXA1 mutations alter pioneering activity, differentiation and prostate cancer phenotypes.

Mutations in the transcription factor FOXA1 define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown1-9. Here, by annotating the landscape of FOXA1 mutations from 3,086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (around 50% of all mutations) and the highly conserved DNA-contact residue R219 (around 5% of all mutations). Wing2 mutations are detected in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumours with neuroendocrine histology. Interrogation of the biological properties of wild-type FOXA1 and fourteen FOXA1 mutants reveals gain of function in mouse prostate organoid proliferation assays. Twelve of these mutants, as well as wild-type FOXA1, promoted an exaggerated pro-luminal differentiation program, whereas two different R219 mutants blocked luminal differentiation and activated a mesenchymal and neuroendocrine transcriptional program. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) of wild-type FOXA1 and representative Wing2 and R219 mutants revealed marked, mutant-specific changes in open chromatin at thousands of genomic loci and exposed sites of FOXA1 binding and associated increases in gene expression. Of note, ATAC-seq peaks in cells expressing R219 mutants lacked the canonical core FOXA1-binding motifs (GTAAAC/T) but were enriched for a related, non-canonical motif (GTAAAG/A), which was preferentially activated by R219-mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its pioneering function and perturb normal luminal epithelial differentiation programs, providing further support for the role of lineage plasticity in cancer progression.

GREB1 amplifies androgen receptor output in human prostate cancer and contributes to antiandrogen resistance.

Genomic amplification of the androgen receptor (AR) is an established mechanism of antiandrogen resistance in prostate cancer. Here, we show that the magnitude of AR signaling output, independent of AR genomic alteration or expression level, also contributes to antiandrogen resistance, through upregulation of the coactivator GREB1. We demonstrate 100-fold heterogeneity in AR output within human prostate cancer cell lines and show that cells with high AR output have reduced sensitivity to enzalutamide. Through transcriptomic and shRNA knockdown studies, together with analysis of clinical datasets, we identify GREB1 as a gene responsible for high AR output. We show that GREB1 is an AR target gene that amplifies AR output by enhancing AR DNA binding and promoting EP300 recruitment. GREB1 knockdown in high AR output cells restores enzalutamide sensitivity in vivo. Thus, GREB1 is a candidate driver of enzalutamide resistance through a novel feed forward mechanism.

Harnessing Androgen Receptor Pathway Activation for Targeted Alpha Particle Radioimmunotherapy of Breast Cancer.

PURPOSE: The impact of androgen receptor (AR) activity in breast cancer biology is unclear. We characterized and tested a novel therapy to an AR-governed target in breast cancer.Experimental Design: We evaluated the expression of prototypical AR gene products human kallikrein 2 (hK2) and PSA in breast cancer models. We screened 13 well-characterized breast cancer cell lines for hK2 and PSA production upon in vitro hormone stimulation by testosterone [dihydrotestosterone (DHT)]. AR-positive lines were further evaluated by exposure to estrogen (17ß-Estradiol) and the synthetic progestin D-Norgestrel. We then evaluated an anti-hK2-targeted radiotherapy platform (hu11B6), labeled with alpha (α)-particle emitting Actinium-225, to specifically treat AR-expressing breast cancer xenografts under hormone stimulation. RESULTS: D-Norgestrel and DHT activated the AR pathway, while 17ß-Estradiol did not. Competitive binding for AR protein showed similar affinity between DHT and D-Norgestrel, indicating direct AR-ligand interaction. In vivo production of hK2 was sufficient to achieve site-specific delivery of therapeutic radionuclide to tumor tissue at >20-fold over background muscle uptake; effecting long-term local tumor control. CONCLUSIONS: [225Ac]hu11B6 targeted radiotherapy was potentiated by DHT and by D-Norgestrel in murine xenograft models of breast cancer. AR activity in breast cancer correlates with kallikrein-related peptidase-2 and can be activated by D-Norgestrel, a common contraceptive, and AR induction can be harnessed for hK2-targeted breast cancer α-emitter radiotherapy.

Feed-forward alpha particle radiotherapy ablates androgen receptor-addicted prostate cancer.

Human kallikrein peptidase 2 (hK2) is a prostate specific enzyme whose expression is governed by the androgen receptor (AR). AR is the central oncogenic driver of prostate cancer (PCa) and is also a key regulator of DNA repair in cancer. We report an innovative therapeutic strategy that exploits the hormone-DNA repair circuit to enable molecularly-specific alpha particle irradiation of PCa. Alpha-particle irradiation of PCa is prompted by molecularly specific-targeting and internalization of the humanized monoclonal antibody hu11B6 targeting hK2 and further accelerated by inherent DNA-repair that up-regulate hK2 (KLK2) expression in vivo. hu11B6 demonstrates exquisite targeting specificity for KLK2. A single administration of actinium-225 labeled hu11B6 eradicates disease and significantly prolongs survival in animal models. DNA damage arising from alpha particle irradiation induces AR and subsequently KLK2, generating a unique feed-forward mechanism, which increases binding of hu11B6. Imaging data in nonhuman primates support the possibility of utilizing hu11B6 in man.

Androgen Deprivation Therapy Potentiates the Efficacy of Vascular Targeted Photodynamic Therapy of Prostate Cancer Xenografts.

Purpose: WST11 vascular targeted photodynamic therapy (VTP) is a local ablation approach relying upon rapid, free radical-mediated destruction of tumor vasculature. A phase III trial showed that VTP significantly reduced disease progression when compared with active surveillance in patients with low-risk prostate cancer. The aim of this study was to identify a druggable pathway that could be combined with VTP to improve its efficacy and applicability to higher risk prostate cancer tumors.Experimental Design: Transcriptome analysis of VTP-treated tumors (LNCaP-AR xenografts) was used to identify a candidate pathway for combination therapy. The efficacy of the combination therapy was assessed in mice bearing LNCaP-AR or VCaP tumors.Results: Gene set enrichment analysis identifies the enrichment of androgen-responsive gene sets within hours after VTP treatment, suggesting that the androgen receptor (AR) may be a viable target in combination with VTP. We tested this hypothesis in mice bearing LNCaP-AR xenograft tumors by using androgen deprivation therapy (ADT), degarelix, in combination with VTP. Compared with either ADT or VTP alone, a single dose of degarelix in concert with VTP significantly inhibited tumor growth. A sharp decline in serum prostate-specific antigen (PSA) confirmed AR inhibition in this group. Tumors treated by VTP and degarelix displayed intense terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining 7 days after treatment, supporting an increased apoptotic frequency underlying the effect on tumor inhibition.Conclusions: Improvement of local tumor control following androgen deprivation combined with VTP provides the rationale and preliminary protocol parameters for clinical trials in patients presented with locally advanced prostate cancer. Clin Cancer Res; 24(10); 2408-16. ©2018 AACR.

Androgen Receptor Signaling in Salivary Gland Cancer.

Salivary gland cancers comprise a small subset of human malignancies, and are classified into multiple subtypes that exhibit diverse histology, molecular biology and clinical presentation. Local disease is potentially curable with surgery, which may be combined with adjuvant radiotherapy. However, metastatic or unresectable tumors rarely respond to chemotherapy and carry a poorer prognosis. Recent molecular studies have shown evidence of androgen receptor signaling in several types of salivary gland cancer, mainly salivary duct carcinoma. Successful treatment with anti-androgen therapy in other androgen receptor-positive malignancies such as prostate and breast cancer has inspired researchers to investigate this treatment in salivary gland cancer as well. In this review, we describe the prevalence, biology, and therapeutic implications of androgen receptor signaling in salivary gland cancer.

SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer.

Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. We use in vitro and in vivo human prostate cancer models to show that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR)-dependent luminal epithelial cells to AR-independent basal-like cells. This lineage plasticity is enabled by the loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2, and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching.

Internalization of secreted antigen-targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis.

Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen-targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue-specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.

Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer.

During the past 10 years, preclinical studies implicating sustained androgen receptor (AR) signalling as the primary driver of castration-resistant prostate cancer (CRPC) have led to the development of novel agents targeting the AR pathway that are now in widespread clinical use. These drugs prolong the survival of patients with late-stage prostate cancer but are not curative. In this Review, we highlight emerging mechanisms of acquired resistance to these contemporary therapies, which fall into the three broad categories of restored AR signalling, AR bypass signalling and complete AR independence. This diverse range of resistance mechanisms presents new challenges for long-term disease control, which may be addressable through early use of combination therapies guided by recent insights from genomic landscape studies of CRPC.

Annotating STEAP1 regulation in prostate cancer with 89Zr immuno-PET.

UNLABELLED: Antibodies and antibody-drug conjugates targeting the cell surface protein 6 transmembrane epithelial antigen of prostate 1 (STEAP1) are in early clinical development for the treatment of castration-resistant prostate cancer (PCa). In general, antigen expression directly affects the bioactivity of therapeutic antibodies, and the biologic regulation of STEAP1 is unusually complicated in PCa. Paradoxically, STEAP1 can be induced or repressed by the androgen receptor (AR) in different human PCa models, while also expressed in AR-null PCa. Consequently, there is an urgent need to translate diagnostic strategies to establish which regulatory mechanism predominates in patients to situate the appropriate therapy within standard of care therapies inhibiting AR. METHODS: To this end, we prepared and evaluated (89)Zr-labeled MSTP2109A ((89)Zr-2109A), a radiotracer for PET derived from a fully humanized monoclonal antibody to STEAP1 in preclinical PCa models. RESULTS: (89)Zr-2109A specifically localized to the STEAP1-positive human PCa models CWR22Pc, 22Rv1, and PC3. Moreover, (89)Zr-2109A sensitively measured treatment-induced changes (∼66% decline) in STEAP1 expression in CWR22PC in vitro and in vivo, a model we showed to express STEAP1 in an AR-dependent manner. CONCLUSION: These findings highlight the ability of immuno-PET with (89)Zr-2109A to detect acute changes in STEAP1 expression and argue for an expansion of ongoing efforts to image PCa patients with (89)Zr-2109A to maximize the clinical benefit associated with antibodies or antibody-drug conjugates to STEAP1.

Androgen receptor signaling regulates DNA repair in prostate cancers.

UNLABELLED: We demonstrate that the androgen receptor (AR) regulates a transcriptional program of DNA repair genes that promotes prostate cancer radioresistance, providing a potential mechanism by which androgen deprivation therapy synergizes with ionizing radiation. Using a model of castration-resistant prostate cancer, we show that second-generation antiandrogen therapy results in downregulation of DNA repair genes. Next, we demonstrate that primary prostate cancers display a significant spectrum of AR transcriptional output, which correlates with expression of a set of DNA repair genes. Using RNA-seq and ChIP-seq, we define which of these DNA repair genes are both induced by androgen and represent direct AR targets. We establish that prostate cancer cells treated with ionizing radiation plus androgen demonstrate enhanced DNA repair and decreased DNA damage and furthermore that antiandrogen treatment causes increased DNA damage and decreased clonogenic survival. Finally, we demonstrate that antiandrogen treatment results in decreased classical nonhomologous end-joining. SIGNIFICANCE: We demonstrate that the AR regulates a network of DNA repair genes, providing a potential mechanism by which androgen deprivation synergizes with radiotherapy for prostate cancer.

Overcoming mutation-based resistance to antiandrogens with rational drug design.

The second-generation antiandrogen enzalutamide was recently approved for patients with castration-resistant prostate cancer. Despite its success, the duration of response is often limited. For previous antiandrogens, one mechanism of resistance is mutation of the androgen receptor (AR). To prospectively identify AR mutations that might confer resistance to enzalutamide, we performed a reporter-based mutagenesis screen and identified a novel mutation, F876L, which converted enzalutamide into an AR agonist. Ectopic expression of AR F876L rescued the growth inhibition of enzalutamide treatment. Molecular dynamics simulations performed on antiandrogen-AR complexes suggested a mechanism by which the F876L substitution alleviates antagonism through repositioning of the coactivator recruiting helix 12. This model then provided the rationale for a focused chemical screen which, based on existing antiandrogen scaffolds, identified three novel compounds that effectively antagonized AR F876L (and AR WT) to suppress the growth of prostate cancer cells resistant to enzalutamide. DOI:http://dx.doi.org/10.7554/eLife.00499.001.