Androgen deprivation therapy and second-generation androgen receptor signaling inhibitors such as enzalutamide are standard treatments for advanced/metastatic prostate cancer. Unfortunately, most men develop resistance and relapse; signaling via insulin-like growth factor (IGF) has been implicated in castration-resistant prostate cancer. We evaluated the antitumor activity of xentuzumab (IGF ligand–neutralizing antibody), alone and in combination with enzalutamide, in prostate cancer cell lines (VCaP, DuCaP, MDA PCa 2b, LNCaP, and PC-3) using established in vitro assays, and in vivo, using LuCaP 96CR, a prostate cancer patient-derived xenograft (PDX) model. Xentuzumab + enzalutamide reduced the viability of phosphatase and tensin homolog (PTEN)-expressing VCaP, DuCaP, and MDA PCa 2b cells more than either single agent, and increased antiproliferative activity and apoptosis induction in VCaP. Xentuzumab or xentuzumab + enzalutamide inhibited IGF type 1 receptor and AKT serine/threonine kinase (AKT) phosphorylation in VCaP, DuCaP, and MDA PCa 2b cells; xentuzumab had no effect on AKT phosphorylation and proliferation in PTEN-null LNCaP or PC-3 cells. Knockdown of PTEN led to loss of antiproliferative activity of xentuzumab and reduced activity of xentuzumab + enzalutamide in VCaP cells. Xentuzumab + enzalutamide inhibited the growth of castration-resistant LuCaP 96CR PDX with acquired resistance to enzalutamide, and improved survival in vivo. The data suggest that xentuzumab + enzalutamide combination therapy may overcome castration resistance and could be effective in patients who are resistant to enzalutamide alone. PTEN status as a biomarker of responsiveness to combination therapy needs further investigation.
Worldwide, prostate cancer is the second most commonly diagnosed cancer and the fifth most common cause of cancer-related death among men, with 1,276,106 new cases and 358,989 deaths estimated in 2018 (1). The growth and survival of prostate cancer cells is androgen dependent, and androgen deprivation therapy is standard treatment for advanced and metastatic prostate cancer (2). Unfortunately, most men relapse within 2–3 years despite achieving castrate serum androgen levels, and progress to a castration-resistant state that precedes lethality (3, 4). The androgen receptor (AR) is an important mediator of castration resistance (3), as is the AR splice variant, AR variant 7 (AR-V7; ref. 5), with AR signaling and transcriptional activity generally persisting in castration-resistant prostate cancer (CRPC), despite castrate levels of testosterone (2).
The second-generation androgen signaling inhibitor enzalutamide (Xtandi®; MDV-3100) is a potent androgen antagonist. Enzalutamide inhibits ligand binding to the AR, subsequent AR nuclear translocation, recruitment of AR coactivators and AR binding to DNA, and prevents transcription of essential genes that promote tumor growth (ref. 6; https://www.medicines.org.uk/emc/product/10318/smpc). While enzalutamide has shown efficacy in CRPC, most patients develop resistance and some exhibit de novo resistance (7).
Signaling through the insulin-like growth factor (IGF) axis can contribute to castration-resistant disease via upregulation of IGF-binding proteins and/or recovery of IGF type 1 receptor (IGF-1R) expression after castration (8). IGF-mediated phosphorylation or dephosphorylation of specific AR sites may increase AR nuclear localization or decrease nuclear export, thereby enhancing AR signaling (9). Moreover, IGF induces transactivation of AR in the presence and absence of androgen (8, 9). Thus, there is a mechanistic rationale for combining IGF-targeted and antiandrogen therapy to attenuate tumor progression and improve survival in patients with CRPC.
Xentuzumab (BI 836845; see ref. 10 and Supplementary Materials and Methods for full sequence and structure) is a humanized monoclonal antibody (mAb) that binds IGF-1 and IGF-2, and neutralizes proliferative/antiapoptotic signaling via IGF-1R and insulin receptor isoform A (INSR-A), without interfering with the metabolic effects of the INSR-B isoform or insulin (11). Xentuzumab has demonstrated potent antiproliferative activity in preclinical studies (11), as well as manageable tolerability and encouraging single-agent activity in phase I trials in patients with advanced solid tumors (12, 13).
The objectives of this study were to investigate the in vitro and in vivo antitumor activity of xentuzumab in combination with enzalutamide in models of human prostate cancer.
Materials and Methods
Xentuzumab was produced at Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany. Enzalutamide was synthesized at TCG Lifesciences Ltd, India. For in vivo studies, enzalutamide was formulated with 0.5% Natrosol™, and the suspension for dosing was stored in the dark at room temperature for up to 5 days; xentuzumab was administered as provided.
Human prostate cancer cell lines, LNCaP (clone FGC), MDA PCa 2b, PC-3, and VCaP were obtained from American Type Culture Collection. DuCaP cells were kindly provided by Prof. Dr. Helmut Klocker (University Hospital Innsbruck, Austria). Research resource identifiers are provided in the Supplementary Data. All cell lines were further authenticated by short tandem repeat analysis at Boehringer Ingelheim prior to use, and were regularly tested for Mycoplasma (MycoAlert Mycoplasma Detection Kit, Lonza; see Supplementary Table S1). New batches of cell lines were regularly thawed, that is, after approximately 20 passages/2–3 months in culture. See Supplementary Materials and Methods for details of cell culture conditions.
All investigated cell lines express IGF-1R; AR expression was detectable in all except PC-3 cells. VCaP and DuCaP are phosphatase and tensin homolog (PTEN) wild-type, MDA PCa 2b cells harbor a heterozygous PTEN mutation, and LNCaP and PC-3 are PTEN null. PTEN protein was expressed by VCaP, DuCaP, and MDA PCa 2b, but not by LNCaP or PC-3 (Supplementary Fig. S1A).
Cell viability and proliferation assays
Cell viability was measured using the CellTiter-Glo® Luminescent Assay (Promega). Briefly, cells were seeded onto nontransparent 96-well plates, and treated with indicated concentrations of xentuzumab and enzalutamide in medium supplemented with fetal bovine serum (FBS) the following day. All samples were tested with three technical replicates. Plates were incubated for 5 days (DuCaP and MDA PCa 2b), 7 days (LNCaP), or 10 days (VCaP and PC-3). For 10-day treatment, xentuzumab and enzalutamide administration was repeated after 5 days. At the time of first drug administration, a “time zero” (T0) untreated cell plate was measured. Luminescence intensity was read using a microplate reader. For data analysis, the mean value from triplicate wells was taken and fitted by iterative calculations using a sigmoidal curve analysis program (Graph Pad Prism) with variable Hill slope.
In addition, the effect of drug treatment on the proliferation of VCaP cells was assessed using a thymidine incorporation assay. Cells were treated with indicated concentrations of xentuzumab and enzalutamide in medium containing FBS for 96 hours. 3H-thymidine (0.4 μCi/well) was added for the last 24 hours. Thymidine incorporation was determined using a liquid scintillation counter (Wallac 1450 MicroBeta TriLux, PerkinElmer).
Western blot, Simple Western™, and qPCR analyses
For analysis of IGF signaling and apoptosis markers, prostate cancer cells were seeded in medium supplemented with 10% FBS, incubated overnight, and treated with the indicated concentrations of xentuzumab, enzalutamide, or both.
For analysis of AR signaling markers, VCaP cells were seeded in medium with 10% charcoal-stripped serum, incubated overnight, and treated with xentuzumab, enzalutamide, or both for 24 hours (qPCR) or 48 hours (Western blots). Synthetic androgen R1881 (0.1 nmol/L) was added 2 hours after treatment start.
Cells were lysed at various time points post treatment and analyzed by Western blot, Simple Western™, or qPCR analyses (see Supplementary Materials and Methods and Supplementary Table S2 for further details).
VCaP cells were treated with xentuzumab (1 μmol/L), enzalutamide (10 μmol/L), or both, and incubated at 37°C for 24, 48, and 72 hours. Cells were fixed in ice-cold 70% ethanol for ≥2 hours at 4°C, washed in phosphate-buffered saline, and incubated in hypotonic buffer solution [0.1% sodium citrate, 0.1% (v/v) triton-X100, 100 μg/mL DNase-free RNase A] and propidium iodide (10 μg/mL) for 30 minutes at room temperature in the dark. Cells were analyzed by flow cytometry on the fluorescence-activated cell sorting (FACS) Canto II Flow Cytometer (Becton Dickinson). Data were evaluated with FACS Diva Software (Becton Dickinson).
Caspase 3/7 activity
VCaP cells were treated with xentuzumab (0.1 μmol/L or 1 μmol/L), enzalutamide (1 μmol/L or 10 μmol/L), or xentuzumab + enzalutamide and incubated at 37°C for 96 hours. Caspase 3/7 activity was assayed in at least triplicate using the IncuCyte® Caspase 3/7 Reagent (Essen Bioscience), according to the manufacturer's instructions. Kinetic measures of the number of caspase 3/7-positive cells were recorded over time and plotted as signals per mm2.
In vivo study in LuCaP 96CR patient-derived xenograft model
Fox Chase CB17 severe combined immunodeficiency (SCID; CB17/lcr-Prkdc scid/lcrlcoCrl) male mice (4 to 8 weeks old; Charles River Laboratories) were castrated and implanted with LuCaP 96CR (castration resistant) tumor fragments 2 weeks later (see Supplementary Materials and Methods). Once tumor volumes exceeded 101 mm3, animals were randomized to receive Natrosol™ vehicle control (p.o.; n = 15), enzalutamide (30 mg/kg p.o.; 5 days on, 2 days off, for 10 cycles; n = 15), or enzalutamide (dosing regimen as previously noted) plus xentuzumab (200 mg/kg i.p., once weekly for 10 cycles; n = 14). Serum prostate-specific antigen (PSA [KLK3; kallikrein related peptidase 3]) was measured using the Architect Total PSA Assay (Abbott Laboratories). Animals were sacrificed either after 10 weeks of treatment (6 hours after the last dose), when tumors had exceeded 1,000 mm3, when weight loss exceeded 20%, or when otherwise compromised [as defined by the University of Washington Institutional Animal Care and Use Committee protocol (Seattle, WA)]. Tumors were then harvested and processed for analysis (see Supplementary Materials and Methods). All animal studies were approved by the internal ethics committee and the local governmental committee.
Additional materials and methods
Details of analysis of PTEN status of LuCaP 96CR, RNA interference, statistical analysis of in vitro assays, RNA isolation and qPCR analysis of tumor samples, RNA-Seq, gene set enrichment analysis (GSEA), and immunohistochemistry (IHC) are described in the Supplementary Materials and Methods. RNA-Seq and microarray data for the LuCaP 96CR patient-derived xenograft (PDX) model are deposited in the Gene Expression Omnibus (GSE124704, GSM2462884, GSM2462771, and GSM2462795).
Availability of materials and data
All materials, data, and protocols described in the article will be made available upon reasonable request, if the request is made within 6 years of publication. All data sets that were analyzed in the article will be made available upon request to the Editors and peer-reviewers, and to the community at the time of publication.
Antiproliferative effect of IGF and AR signaling blockade in prostate cancer cell lines
The xentuzumab + enzalutamide combination was tested in a set of prostate cancer cell lines. The viability of VCaP, DuCaP, and MDA PCa 2b cells was reduced by single-agent xentuzumab or enzalutamide in a dose-dependent manner, after 5 or 10 days of incubation. Combination treatment further reduced cell viability versus the single agents (as shown in Fig. 1A–C, these reductions were statistically significant). In contrast, in LNCaP cells, xentuzumab + enzalutamide provided no greater inhibition than enzalutamide alone, and single-agent xentuzumab exhibited no antiproliferative effects (Fig. 1D); both agents (alone and combined) were inactive in PC-3 cells (Fig. 1E). The greater antiproliferative effect of combination treatment was confirmed by measuring thymidine incorporation in VCaP cells (Fig. 1F). The notion that addition of xentuzumab enhanced the activity of enzalutamide in three PTEN wild-type cell lines but not in PTEN-null LNCaP cells prompted us to investigate the effect of PTEN knockdown in VCaP cells. RNA interference–mediated silencing resulted in complete loss of antiproliferative activity of xentuzumab, and reduced the activity of xentuzumab + enzalutamide to the level observed with enzalutamide alone, demonstrating that functional PTEN is required for combinatorial activity in the VCaP cell line (Fig. 1G; Supplementary Fig. S2A and S2B).
Inhibitory effects on IGF-1R and AR signaling
Xentuzumab inhibited IGF-1R phosphorylation in all five cell lines (Fig. 2; Supplementary Fig. S1B–S1E). Both xentuzumab and combination treatment suppressed AKT serine/threonine kinase (AKT) phosphorylation in VCaP (Fig. 2), DuCaP, and MDA PCa 2b (Supplementary Fig. S1B and S1C), but not in LNCaP or PC-3 PTEN null (Supplementary Fig. S1D and S1E). Enzalutamide had little effect on IGF-1R and AKT phosphorylation in any of the cell lines.
VCaP cells were selected to further investigate the effects of xentuzumab + enzalutamide; these cells recapitulate the relative expression levels of full-length AR (AR-FL) and AR-Vs observed in clinical CRPC specimens, that is, detectable but lower levels of AR-Vs than AR-FL (14).
Xentuzumab alone did not affect expression of AR-regulated genes in VCaP cells. In line with its mechanism of action, enzalutamide (±xentuzumab) markedly reduced transcript and protein levels of AR-regulated target genes, namely PSA, FK506 binding protein 5 [FKBP5 (FKBP prolyl isomerase 5)], transmembrane serine protease 2 (TMPRSS2), and ETS-related gene [ERG (ETS transcription factor ERG); Supplementary Fig. S3A]. Treatment with xentuzumab + enzalutamide led to the most pronounced reduction in expression of the cell-cycle genes, ubiquitin-conjugating enzyme E2 C (UBE2C), cyclin-dependent kinase 1 (CDK1), and cell-division cycle protein 20 (CDC20; Supplementary Fig. S3B).
Effect of IGF and AR signaling blockade on cell-cycle profile and induction of apoptosis
Xentuzumab increased the sub-G1 cell population (indicative of apoptotic cells) over time compared with untreated VCaP controls (25% vs. 10% after 3 days); xentuzumab + enzalutamide further enhanced this population to 44% after 3 days (Fig. 3). Enzalutamide alone had only minor effects on the cell-cycle profile of VCaP cells. Xentuzumab produced a minor (with 0.1 μmol/L) or moderate (with 1 μmol/L) increase in cleaved caspase 3/7, while enzalutamide (1 and 10 μmol/L) did not affect caspase 3/7 activity over time versus control (Fig. 4A and C; Supplementary Fig. S4A). In combination, xentuzumab + enzalutamide significantly enhanced caspase activity compared with either single agent. Cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP) was only apparent after combination treatment (Fig. 4B and C).
To further elucidate the mechanism of increased apoptosis, treatment effects on regulators of apoptosis were investigated. Xentuzumab and xentuzumab + enzalutamide inhibited the phosphorylation of well-established direct substrates of AKT, namely the Forkhead box O (FoxO) transcription factors, FoxO3a and FoxO1 (important regulators of expression of genes involved in apoptosis; Fig. 4C), and the BH3-only proapoptotic protein, Bcl2-associated agonist of cell death (Bad; Fig. 4D). Minor treatment effects were observed on total protein expression levels of FoxOs, and other pro- and anti-apoptotic factors, Bcl-2-like protein 4 [Bax (BCL2 associated X, apoptosis regulator)], Bcl-2 antagonist/killer (Bak), Bcl-2-like protein 11 [Bim (BCL2L11; BCL2 like 11)], and B-cell lymphoma extra-large (Bcl-XL; Supplementary Fig. S4B).
In vivo activity of xentuzumab and enzalutamide
The antitumor activity of xentuzumab + enzalutamide was evaluated using the LuCaP 96CR PDX model, which mimics CRPC with acquired resistance to the standard-of-care agent, enzalutamide. Whereas, as expected, enzalutamide monotherapy did not inhibit tumor growth, addition of xentuzumab resensitized tumors to enzalutamide and resulted in significant reductions in tumor volume (Fig. 5A and B). All mice treated with the combination were alive at the end of the study (>10 weeks), representing a significant survival improvement versus enzalutamide alone (Fig. 5C). Treatment with enzalutamide or the combination for 2 weeks resulted in a trend toward decreased serum PSA versus control (Fig. 5D). At sacrifice, the reduction in PSA with combination treatment was statistically significant versus control (∼65%, P = 0.033), but not versus enzalutamide alone (P = 0.079).
Analysis of tumor samples
At the end of the LuCaP 96CR efficacy study (at sacrifice), tumors were harvested (6 hours after the last dose) and processed for analysis. Enzalutamide significantly increased AR-FL mRNA and protein versus control, and only moderate further increases occurred with combination therapy (Supplementary Fig. S5A and S5C). In contrast, AR-V7 mRNA was significantly increased by xentuzumab + enzalutamide compared with enzalutamide alone (Supplementary Fig. S5A), and this finding was confirmed at the protein level using IHC (Supplementary Fig. S5B). Of note, compared with control, combination treatment significantly reduced transcripts of UBE2C [a mitotic (M)-phase cell-cycle gene regulated by AR-V7], while enzalutamide alone had no significant effect (Fig. 6A).
RNA-Seq and GSEA analyses of enzalutamide- and combination-treated LuCaP 96CR tumors (Fig. 6B–E; Supplementary Table S3) showed that enzalutamide-activated or -suppressed targets were further altered when xentuzumab was added; eight genes were upregulated and 29 genes downregulated (Fig. 6B). The GSEA results of all C2-canonical pathways with false discovery rate (FDR) < 0.05 (133 negatively; 81 positively enriched) are detailed in Supplementary Table S3; all Hallmark pathways with FDR < 0.05 (27 negatively; six positively enriched) are shown in Fig. 6C. Compared with enzalutamide alone, the hypoxia-inducible factor-1α [HIF-1-α (HIF1A; hypoxia-inducible factor-1 subunit alpha)] transcription factor network (Fig. 6D) and the closely-related hypoxia gene set (Fig. 6C) were significantly downregulated by the combination, as was the androgen response gene set (Fig. 6E). Other negatively enriched pathways included extracellular matrix, adhesion, epithelial–mesenchymal transition networks, and signaling pathways such as Wnt and Notch (Fig. 6C; Supplementary Table S3). Positively enriched pathways included E2F transcription factors, MYC targets Version 1 and 2, and the G2–M checkpoint (Fig. 6C; Supplementary Table S3).
The lack of a durable response to androgen- or AR-targeted therapy is a major challenge in CRPC management (7). Transactivation of AR by growth factors and cytokines is one of many mechanisms that drive progression to castration resistance (15). IGF-1R–mediated activation of the PI3K/AKT pathway is one of the proposed mechanisms for AR transactivation under castrated conditions (15), the presence of which supports cotargeting of the AR and IGF-1R pathways in patients with CRPC.
Our in vitro data show that xentuzumab + enzalutamide reduces proliferation more effectively than either treatment alone in PTEN-positive prostate cancer cells. In VCaP cells, requirement of functional PTEN for combinatorial antiproliferative activity was demonstrated by PTEN knockdown. LuCaP 96CR harbors loss of one copy of the PTEN gene, and a deletion of exon 3 on the other allele, but it is possible that PTEN in LuCaP 96CR may still retain functional phosphatase activity, because exon 3 loss does not disrupt the active site (Supplementary Fig. S6). Interestingly, a clinical trial of the IGF-1R–targeting mAb, ganitumab, failed to establish a correlation between clinical benefit and PTEN status (but most tumor samples were PTEN positive; ref. 16). Likewise, suppression of IGF-1R expression with an antisense oligonucleotide reduced proliferation and increased apoptosis of PTEN-null PC-3 and LNCaP cells (17). The mechanisms of increased apoptosis after knockdown of IGF-1R in PTEN-null cell lines are not yet understood, particularly as IGF-1/2 neutralization does not result in similar effects. These differences may be explained by the distinct capability of IGF-1R–targeted agents and IGF ligand–blocking antibodies to affect proliferative and survival signaling downstream of homo- and heterodimers of the IGF/INSR family (18). Our finding that xentuzumab failed to inhibit AKT phosphorylation in LNCaP or PC-3 PTEN-null cells suggests that the lack of antiproliferative activity may reflect the failure of xentuzumab to inhibit downstream IGF ligand–independent signaling in these cells. Taken together, the impact of PTEN status on response to xentuzumab + enzalutamide in CRPC requires further investigation.
Xentuzumab + enzalutamide treatment was shown to exert its combinatorial activity through enhanced tumor cell apoptosis. These findings are in line with the key role of IGF signaling in conferring a survival response that acts as a resistance mechanism, limiting the effectiveness of cytotoxic or targeted anticancer agents. Mechanistically, xentuzumab induced the intrinsic apoptotic pathway by affecting the phosphorylation status of AKT and downstream proapoptotic regulators, BH3-only protein, Bad, and transcription factors, FoxO3a and FoxO1, on residues reported to be AKT substrates. Specifically, inhibition of Bad phosphorylation on serine residues S136 and S112 by xentuzumab and xentuzumab + enzalutamide is in line with reports that suppression of Bad activity by IGF-1R/AKT signaling involves phosphorylation on these residues (19, 20), and inhibition of FoxO protein phosphorylation occurred at threonine and serine residues regulated by AKT (FoxO3a, T32 and S253 and FoxO1, T24; refs. 21, 22). Total protein levels of apoptotic regulators were largely unaffected in our studies, which is concordant with published evidence suggesting that other pathways, such as p53 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), play important roles in regulating gene expression of pro- and antiapoptotic factors (23–25).
The observation that progression of enzalutamide-resistant LuCaP 96CR PDX was inhibited by the combination treatment versus controls, together with the survival benefit with the combination versus single-agent enzalutamide, supports available evidence that the augmented antitumor effects of IGF- and AR-targeted combinations translate into enhanced in vivo efficacy (8, 26). For example, the anti-IGF-1R antibody, A12, enhanced tumor regression in vivo and prolonged survival after castration (8). In our study, the in vivo antitumor efficacy of combination treatment was not accompanied by significant suppression of serum PSA levels versus enzalutamide alone, suggesting that serum PSA level may not be an appropriate indicator of xentuzumab activity in CRPC clinical trials.
In LuCaP 96CR tumor samples taken at the end of the efficacy study, increased AR-V7 expression levels were observed in the xentuzumab + enzalutamide group compared with the enzalutamide-only group. Interestingly, clinical data have shown an upregulation of AR-V7 as a treatment response to enzalutamide or abiraterone acetate in patients with CRPC (27). While the mechanism behind the increase in AR-V7 upon xentuzumab + enzalutamide treatment in our study remains unclear, we speculate that it likewise reflects an adaptive response upon therapy. Regardless of upregulated AR-V7 at study end, a downregulation of AR response pathways (GSEA) and of UBE2C expression (qPCR) in the presence of xentuzumab + enzalutamide compared with enzalutamide alone was demonstrated. Suppression of the M-phase checkpoint UBE2C, on one hand, reflects antiproliferative activity elicited by xentuzumab + enzalutamide (consistent with the reduction in tumor growth), but, in addition, may indicate reduced transcriptional activity of AR-V7. UBE2C is reported to be a representative target gene of AR-V7 under androgen-depleted conditions (28, 29), with its expression being specifically regulated by AR-V7 and not by AR-FL, both in prostate cancer cells and in tissues from patients with CRPC (30). In light of the significant tumor growth delay and clear survival benefit observed with combination treatment at the end of the experiment, the upregulation of E2F and MYC targets revealed by GSEA seems counterintuitive. Our interpretation is that, after long-term treatment for up to 10 weeks, tumor samples are not representative of acute pharmacodynamic effects of the drugs but, rather, mirror a steady state, reflecting compensatory mechanisms. E2F and MYC are not bona fide oncogenic drivers, but their upregulation may support tumor cell survival at this late time point. Investigation of counter-regulatory mechanisms in response to xentuzumab + enzalutamide treatment has important translational relevance and will be a focus of future studies.
Interestingly, the HIF-1α/hypoxia pathway was one of the pathways downregulated most by enzalutamide + xentuzumab. IGF-1R signaling regulates HIF-1α and protects tumor cells from the negative effects of hypoxia (31). Consequently, downregulation of the HIF-1α pathway may contribute to antitumor efficacy of xentuzumab + enzalutamide combination therapy.
Our results support two potential strategies for more effective inhibition of the IGF/IGF-1R signaling axis in CRPC. The first strategy is that of concurrent administration of IGF-1/-2 ligand–neutralizing antibodies such as xentuzumab with androgen signaling inhibitors, the objective being to prevent the IGF-stimulated resistance pathway from driving prostate cancer progression, by inhibiting AR-V7 transcriptional activity (among other mechanisms). Indeed, it was recently shown that IGF-1R is a primary target of both the AR-FL and AR-V7 cistrome (32). The marked increase in AR-V7 following enzalutamide treatment is an important and frequent mechanism of resistance to androgen-directed therapies; AR-V7 is expressed in less than 1% of primary prostate cancer (castration sensitive) but at least 75% of castration-resistant tumors (27). Our results showed reduced expression of AR-V7–regulated genes following combination treatment, consistent with the previous findings that inhibition of IGF-1R by the tyrosine kinase inhibitors NVP-AEW541 and AG1024 led to downregulation of AR-V7 transcriptional activity in prostate cancer cells (33). The second potential strategy employs a more precision-based approach, using candidate biomarkers such as PTEN and AR-V7 to aid patient enrichment.
In conclusion, reciprocal reactivation of IGF and AR signaling in response to AR/IGF inhibitor–mediated feedback provides a rationale for simultaneous targeting of both pathways in AR- and/or IGF-dependent tumors. In our studies, combined treatment with anti-AR and anti-IGF-1/-2 therapies produced concurrent suppression of AR signaling and compensatory IGF/IGF-1R activity. The combination of these effects led to enhanced antiproliferative/proapoptotic effects in vitro, and significant in vivo efficacy in a PDX model of castration- and enzalutamide-resistant prostate cancer. Further in vivo studies will investigate the emergence of counter-regulatory mechanisms arising during treatment, and may identify suitable enrichment biomarkers [e.g., PTEN status, IGF-1/2 levels, TMPRSS2-ERG (T2E) fusion gene expression, or AR-V7 gene expression] for selection of patients who would be most likely to benefit from this combination therapy.
Disclosure of Potential Conflicts of Interest
U. Weyer-Czernilofsky, M.H. Hofmann, P.J. Adam, F. Solca, N. Kraut, and R. Baumgartinger are employees of Boehringer Ingelheim. E. Corey reports receiving a commercial research grant from Boehringer Ingelheim. T. Bogenrieder is a former employee of Boehringer Ingelheim. No potential conflicts of interest were disclosed by the other authors.
Conception and design: U. Weyer-Czernilofsky, M.H. Hofmann, K. Friedbichler, P.J. Adam, F. Solca, E. Corey, T. Bogenrieder
Development of methodology: R. Baumgartinger, E. Corey, G. Liu, S.R. Plymate
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.H. Hofmann, K. Friedbichler, R. Baumgartinger, H.M. Nguyen, E. Corey, G. Liu, C.C. Sprenger, S.R. Plymate
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): U. Weyer-Czernilofsky, M.H. Hofmann, K. Friedbichler, R. Baumgartinger, P.J. Adam, F. Solca, E. Corey, G. Liu, C.C. Sprenger, S.R. Plymate, T. Bogenrieder
Writing, review, and/or revision of the manuscript: U. Weyer-Czernilofsky, M.H. Hofmann, K. Friedbichler, R. Baumgartinger, P.J. Adam, F. Solca, N. Kraut, E. Corey, G. Liu, C.C. Sprenger, S.R. Plymate, T. Bogenrieder
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Baumgartinger
Study supervision: U. Weyer-Czernilofsky, M.H. Hofmann, P.J. Adam, E. Corey, C.C. Sprenger, T. Bogenrieder
U. Weyer-Czernilofsky had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
We thank Dr. Daniel Gerlach (Boehringer Ingelheim RCV GmbH & Co. KG, Vienna, Austria), for support in analysis of the PTEN status of LuCaP 96CR, Daniela Fürweger and Ulrike Nagl (Boehringer Ingelheim RCV GmbH & Co. KG, Vienna, Austria), for performing several of the experiments, and Prof. Dr. Helmut Klocker, University Hospital Innsbruck (Innsbruck, Austria), for provision of DuCaP cells. Medical writing assistance, supported financially by Boehringer Ingelheim, was provided by Fiona Scott, of GeoMed, an Ashfield company, part of UDG Healthcare plc, during the preparation of this article. The studies were supported by Boehringer Ingelheim RCV GmbH & Co. KG and the Veterans Affairs Research Program (to S.R. Plymate). Establishment and maintenance of the LuCaP patient-derived xenografts by E. Corey and H.M. Nguyen was supported by the Pacific Northwest Prostate Cancer SPORE (Grant Reference Number: P50 CA097186), the ProGMap Project (Grant Reference Number: P01 CA163227), the Prostate Cancer Foundation, and the Richard M. Lucas Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.