Shihua Sun

Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant

Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (ARv567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, ARv567es functioned as a constitutively active receptor, increased expression of full-length AR (ARfl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with ARv567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of ARv567es to ARfl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected ARv567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand.

Androgen Receptor Variants Occur Frequently in Castration Resistant Prostate Cancer Metastases

Background Although androgens are depleted in castration resistant prostate cancer (CRPC), metastases still express nuclear androgen receptor (AR) and androgen regulated genes. We recently reported that C-terminal truncated constitutively active AR splice variants contribute to CRPC development. Since specific antibodies detecting all C-terminal truncated AR variants are not available, our aim was to develop an approach to assess the prevalence and function of AR variants in prostate cancer (PCa). Methodology/Principal Findings Using 2 antibodies against different regions of AR protein (N- or C-terminus), we successfully showed the existence of AR variant in the LuCaP 86.2 xenograft. To evaluate the prevalence of AR variants in human PCa tissue, we used this method on tissue microarrays including 50 primary PCa and 162 metastatic CRPC tissues. RT-PCR was used to confirm AR variants. We observed a significant decrease in nuclear C-terminal AR staining in CRPC but no difference between N- and C-terminal AR nuclear staining in primary PCa. The expression of the AR regulated proteins PSA and PSMA were marginally affected by the decrease in C-terminal staining in CRPC samples. These data suggest that there is an increase in the prevalence of AR variants in CRPC based on our ability to differentiate nuclear AR expression using N- and C-terminal AR antibodies. These findings were validated using RT-PCR. Importantly, the loss of C-terminal immunoreactivity and the identification of AR variants were different depending on the site of metastasis in the same patient. Conclusions We successfully developed a novel immunohistochemical approach which was used to ascertain the prevalence of AR variants in a large number of primary PCa and metastatic CRPC. Our results showed a snapshot of overall high frequency of C-terminal truncated AR splice variants and site specific AR loss in CRPC, which could have utility in stratifying patients for AR targeted therapeutics.

Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer

Prostate cancer growth depends on androgen receptor signaling. Androgen ablation therapy induces expression of constitutively active androgen receptor splice variants that drive disease progression. Taxanes are a standard of care therapy in castration-resistant prostate cancer (CRPC); however, mechanisms underlying the clinical activity of taxanes are poorly understood. Recent work suggests that the microtubule network of prostate cells is critical for androgen receptor nuclear translocation and activity. In this study, we used a set of androgen receptor deletion mutants to identify the microtubule-binding domain of the androgen receptor, which encompasses the DNA binding domain plus hinge region. We report that two clinically relevant androgen receptor splice variants, ARv567 and ARv7, differentially associate with microtubules and dynein motor protein, thereby resulting in differential taxane sensitivity in vitro and in vivo. ARv7, which lacks the hinge region, did not co-sediment with microtubules or coprecipitate with dynein motor protein, unlike ARv567. Mechanistic investigations revealed that the nuclear accumulation and transcriptional activity of ARv7 was unaffected by taxane treatment. In contrast, the microtubule-interacting splice variant ARv567 was sensitive to taxane-induced microtubule stabilization. In ARv567-expressing LuCap86.2 tumor xenografts, docetaxel treatment was highly efficacious, whereas ARv7-expressing LuCap23.1 tumor xenografts displayed docetaxel resistance. Our results suggest that androgen receptor variants that accumulate in CRPC cells utilize distinct pathways of nuclear import that affect the antitumor efficacy of taxanes, suggesting a mechanistic rationale to customize treatments for patients with CRPC, which might improve outcomes.

Mechanisms of the androgen receptor splicing in prostate cancer cells

Prostate tumors develop resistance to androgen deprivation therapy (ADT) by multiple mechanisms, one of which is to express constitutively active androgen receptor (AR) splice variants lacking the ligand-binding domain. AR splice variant 7 (AR-V7, also termed AR3) is the most abundantly expressed variant that drives prostate tumor progression under ADT conditions. However, the molecular mechanism by which AR-V7 is generated remains unclear. In this manuscript, we demonstrated that RNA splicing of AR-V7 in response to ADT was closely associated with AR gene transcription initiation and elongation rates. Enhanced AR gene transcription by ADT provides a prerequisite condition that further increases the interactions between AR pre-mRNA and splicing factors. Under ADT conditions, recruitment of several RNA splicing factors to the 3′ splicing site for AR-V7 was increased. We identified two RNA splicing enhancers and their binding proteins (U2AF65 and ASF/SF2) that had critical roles in splicing AR pre-mRNA into AR-V7. These data indicate that ADT-induced AR gene transcription rate and splicing factor recruitment to AR pre-mRNA contribute to the enhanced AR-V7 levels in prostate cancer cells.

Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer

Background The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. Objective To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. Design, setting, and participants Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. Outcome measurements and statistical analysis Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. Results and limitations Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5–90) compared to CRPC (HS 135, IQR 80–157.5; p < 0.0001), and in biopsy tissue taken before (HS 80, IQR 30–136.3) compared to after (HS 140, IQR 105–167.5; p = 0.007) abiraterone or enzalutamide treatment. Lower nuclear AR-V7 expression at CRPC biopsy was associated with longer OS (hazard ratio 1.012, 95% confidence interval 1.004–1.020; p = 0.003). While this monoclonal antibody primarily binds to AR-V7 in PC biopsy tissue, it may also bind to other proteins. Conclusions We provide the first evidence that nuclear AR-V7 expression increases with emerging CRPC and is prognostic for OS, unlike antibody staining for the AR N-terminal domain. These data indicate that AR-V7 is important in CRPC disease biology; agents targeting AR splice variants are needed to test this hypothesis and further improve patient outcome from CRPC. Patient summary In this study we found that levels of the protein AR-V7 were higher in patients with advanced prostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time.

Tumor Suppressor BRCA1 Is Expressed in Prostate Cancer and Controls Insulin-like Growth Factor I Receptor (IGF-IR) Gene Transcription in an Androgen Receptor–Dependent Manner

Purpose: The insulin-like growth factor (IGF) system plays an important role in prostate cancer. The BRCA1 gene encodes a transcription factor with tumor suppressor activity. The involvement of BRCA1 in prostate cancer, however, has not yet been elucidated. The purpose of the present study was to examine the functional correlations between BRCA1 and the IGF system in prostate cancer. Experimental Design: An immunohistochemical analysis of BRCA1 was done on tissue microarrays comprising 203 primary prostate cancer specimens. In addition, BRCA1 levels were measured in prostate cancer xenografts and in cell lines representing early stages (P69 cells) and advanced stages (M12 cells) of the disease. The ability of BRCA1 to regulate IGF-I receptor (IGF-IR) expression was studied by coexpression experiments using a BRCA1 expression vector along with an IGF-IR promoter-luciferase reporter. Results: We found significantly elevated BRCA1 levels in prostate cancer in comparison with histologically normal prostate tissue (P < 0.001). In addition, an inverse correlation between BRCA1 and IGF-IR levels was observed in the androgen receptor (AR)–negative prostate cancer–derived P69 and M12 cell lines. Coexpression experiments in M12 cells revealed that BRCA1 was able to suppress IGF-IR promoter activity and endogenous IGF-IR levels. On the other hand, BRCA1 enhanced IGF-IR levels in LNCaP C4-2 cells expressing an endogenous AR. Conclusions: We provide evidence that BRCA1 differentially regulates IGF-IR expression in AR-positive and AR-negative prostate cancer cells. The mechanism of action of BRCA1 involves modulation of IGF-IR gene transcription. In addition, immunohistochemical data are consistent with a potential survival role of BRCA1 in prostate cancer.

Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression.

Transforming Growth Factor-β–Stimulated Clone-22 Is an Androgen-Regulated Gene That Enhances Apoptosis in Prostate Cancer following Insulin-Like Growth Factor-I Receptor Inhibition

Purpose: Inhibition of insulin-like growth factor (IGF) signaling using the human IGF-I receptor monoclonal antibody A12 is most effective at inducing apoptosis in prostate cancer xenografts in the presence of androgen. We undertook this study to determine mechanisms for increased apoptosis by A12 in the presence of androgens. Experimental Methods: The castrate-resistant human xenograft LuCaP 35 V was implanted into intact or castrate severe combined immunodeficient mice and treated with A12 weekly. After 6 weeks of tumor growth, animals were sacrificed and tumors were removed and analyzed for cell cycle distribution/apoptosis and cDNA arrays were done. Results: In castrate mice, the tumors were delayed in G2 with no apoptosis; in contrast, tumors from intact mice underwent apoptosis with either G1 or G2 delay. Transforming growth factor-β–stimulated clone-22 (TSC-22) was significantly elevated in tumors from the intact mice compared with castrate mice, especially in those tumors with the highest levels of apoptosis. To further determine the function of TSC-22, we transfected various human prostate cancer cell lines with a plasmid expressing TSC-22. Cell lines overexpressing TSC-22 showed an increase in apoptosis and a delay in G1. When these cell lines were placed subcutaneously in athymic nude mice, a decreased number of animals formed tumors and the rate of tumor growth was decreased compared with control tumors. Conclusions: These data indicate that IGF-I receptor inhibition in the presence of androgen has an enhanced effect on decreasing tumor growth, in part, through increased expression of the tumor suppressor gene TSC-22. (Clin Cancer Res 2009;15(24):7634–41)

Abstract 1194: Neutralization of IGF-I and -II ligands with the fully humanized bispecific monoclonal antibody xentuzumab inhibits AR-V7-induced enzalutamide resistance in castration-resistant prostate cancer

Background: Recent data from multiple investigators have shown that an increase in constitutively active androgen receptor (AR) variants such as AR-V7 is associated with more aggressive prostate cancer and a significantly shortened overall survival. Additionally, expression of AR-V7 in circulating tumor cells or increased expression in metastatic tissue is a biomarker for resistance to currently available anti-androgen therapies, including enzalutamide (ENZA) and abiraterone, resulting in castration resistant prostate cancer (CRPC). Insulin-like growth factor (IGF) signaling pathways have been incriminated as mechanisms of resistance but little is known about the potential role of IGFs in CRPC growth in relation to AR variants. In this preclinical study we examined the effects of IGF-I and -II inhibition in LuCaP 96CR, an ENZA resistant prostate cancer patient-derived xenograft. Hypothesis: IGF-I and -II neutralization will block AR-V7 activity and inhibit castration-resistant prostate cancer growth. Study procedures: LuCaP 96CR was implanted s.c. into castrate SCID mice (15 mice per group). When tumors exceeded 150mm3 animals were randomized into groups: 1) Control; 2) ENZA (50 mg/kg, QD, po), 3) xentuzumab (BI 836845[1], 200 mg/kg QW IP) in combination with ENZA. At the end of the study, tumors were collected for preparation of RNA and protein lysates and histology. Results: ENZA did not show significant inhibitory effects on LuCaP 96CR, but the combination of xentuzumab and ENZA resulted in significant tumor inhibition (p Summary: These data show that addition of xentuzumab provides tumor inhibition of ENZA-resistant CRPC. Interestingly, associated with a significant decrease in tumor growth, this treatment resulted in increased AR-V7 mRNA and protein expression but no significant increases in downstream markers of AR-V7 activity, UBE2C and UGT2B17. Conclusion: Our results suggest that the IGF-I and -II neutralizing antibody xentuzumab may reverse AR-V7-mediated ENZA resistance. Citation Format: Gang Liu, Holly M. Nguyen, Kristine van Maltzan, Shihua Sun, Cynthia Sprenger, Ulrike Weyer-Czernilofsky, Eva Corey, Stephen R. Plymate. Neutralization of IGF-I and -II ligands with the fully humanized bispecific monoclonal antibody xentuzumab inhibits AR-V7-induced enzalutamide resistance in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1194. doi:10.1158/1538-7445.AM2017-1194

Abstract 4965: Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic prostate cancer

BACKGROUND: The androgen-receptor splice variant 7 (AR-V7) has been implicated in the development of castration resistant prostate cancer (CRPC) and resistance to current therapies including enzalutamide and abiraterone. AR-V7 mRNA expression in circulating tumour cells of patients with CRPC correlated with treatment resistance. However, the importance of AR-V7 has been questioned in light of low AR-V7 mRNA levels relative to the full-length androgen receptor in CRPC and it is critically important to develop validated assays that confirm AR-V7 protein levels and its clinical importance in patients with CRPC. METHODS: Following validation of a monoclonal antibody, immunohistochemical analysis of nuclear AR-V7 (alongside a nuclear AR N-terminal domain antibody; AR-NTD) was performed in a patient cohort identified with matched therapy-naive hormone-sensitive primary prostate cancer (HSPC) and CRPC. We determined the levels of nuclear AR-V7 as patients progressed from HSPC to CRPC. We also determined if AR-V7 expression levels associated with overall survival from time of CRPC biopsy. RESULTS: In our patient cohort (n = 39), nuclear AR-V7 (p = CONCLUSION: We provide first evidence that expression of nuclear AR-V7 protein not only increases with emerging treatment resistance in CRPC but also is associated with overall survival from time of CRPC biopsy. These data support AR-V7 protein being key to CRPC progression and that agents targeting AR splice variants will be important to improve patient outcome in CRPC. Citation Format: Daniel Nava Rodrigues, Jon Welti, Adam Sharp, Shihua Sun, Ruth Riisnaes, Ines Figueiredo, Zafeiris Zafeiriou, Pasquale Rescigno, Johann S. de Bono, Stephen R. Plymate. Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4965.