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Abstract
Background Enzalutamide, a next-generation antiandrogen agent, is approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). While enzalutamide has been shown to improve time to progression and extend overall survival in men with CRPC, the majority of patients ultimately develop resistance to treatment. Immunotherapy approaches have shown limited clinical benefit in this patient population; understanding resistance mechanisms could help develop novel and more effective treatments for CRPC. One of the mechanisms involved in tumor resistance to various therapeutics is tumor phenotypic plasticity, whereby carcinoma cells acquire mesenchymal features with or without the loss of classical epithelial characteristics. This work investigated a potential link between enzalutamide resistance, tumor phenotypic plasticity, and resistance to immune-mediated lysis in prostate cancer.
Methods Models of prostate cancer resistant to enzalutamide were established by long-term exposure of human prostate cancer cell lines to the drug in culture. Tumor cells were evaluated for phenotypic features in vitro and in vivo, as well as for sensitivity to immune effector cell-mediated cytotoxicity.
Results Resistance to enzalutamide was associated with gain of mesenchymal tumor features, upregulation of estrogen receptor expression, and significantly reduced tumor susceptibility to natural killer (NK)-mediated lysis, an effect that was associated with decreased tumor/NK cell conjugate formation with enzalutamide-resistant cells. Fulvestrant, a selective estrogen receptor degrader, restored the formation of target/NK cell conjugates and increased susceptibility to NK cell lysis in vitro. In vivo, fulvestrant demonstrated antitumor activity against enzalutamide-resistant cells, an effect that was associated with activation of NK cells.
Conclusion NK cells are emerging as a promising therapeutic approach in prostate cancer. Modifying tumor plasticity via blockade of estrogen receptor with fulvestrant may offer an opportunity for immune intervention via NK cell-based approaches in enzalutamide-resistant CRPC.
- Immune Evation
- Prostatic Neoplasms
- Killer Cells, Natural
Data availability statement
Data are available upon reasonable request.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Enzalutamide has been shown to improve time to progression and extend overall survival in men with castration-resistant prostate cancer (CRPC); however, the majority of patients ultimately develop resistance. While immunotherapy approaches with immune-checkpoint inhibitors have been successful across many tumor types, little benefit has been achieved in CRPC. Thus, novel therapeutic approaches for this patient population are needed.
WHAT THIS STUDY ADDS
This study demonstrates that enzalutamide resistance in prostate cancer significantly decreases tumor susceptibility to natural killer (NK)-mediated lysis, while also increasing mesenchymal tumor features and the expression of estrogen receptors. Using fulvestrant, a selective estrogen receptor degrader, NK-cell lysis can be restored to levels observed with non-resistant prostate cancer cells.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
T cell centric immunotherapies, including the use of checkpoint inhibitors, have demonstrated little therapeutic effect in CRPC. As NK cell therapies are emerging as a potential alternative in this poorly T-cell infiltrated tumor type, the results of this study support future exploration of combinations of fulvestrant with NK-based therapies for the treatment of advanced prostate cancer, including the use of cytokines, adoptively transferred memory NK cells, or chimeric antigen receptor(CAR)-NKs.
Background
Prostate cancer is the most commonly diagnosed and the second leading cause of cancer death in men in the USA with over 30,000 deaths estimated in 2023.1 The standard first-line treatment for prostate cancer is androgen-deprivation therapy (ADT), which can be achieved via pharmacological or surgical castration. Following an initial response to ADT, a significant proportion of patients experience disease progression developing castration-resistant prostate cancer (CRPC). The androgen receptor (AR) antagonist enzalutamide is one of the treatment modalities currently available for men with CRPC; it has been shown to extend survival when administered prior to or after chemotherapy.2 3 The clinical benefit of enzalutamide, however, is limited by the presence of primary or acquired tumor resistance mechanisms, which are either dependent on AR signaling, including AR amplification and overexpression, the presence of AR splice variants or mutations, and altered steroidogenesis, or independent of AR signaling, such as overexpression and upregulation of the glucocorticoid receptor, changes in lineage plasticity, and tumor phenotypic plasticity.4 5
Lineage plasticity in prostate cancer refers to the conversion from adenocarcinoma to neuroendocrine prostate cancer, a phenomenon that is proposed to take place in about 20% of men with CRPC and is associated with lack of responses to AR inhibition.6 7 Distinct from lineage plasticity that involves the acquisition of neuroendocrine features, a number of reports in the literature have shown that epithelial prostate cancer cells exposed to ADT or AR inhibitors also can undergo a phenotypic conversion towards a more mesenchymal cell state (ie, tumor phenotypic plasticity by means of an epithelial-mesenchymal transition, EMT). This phenotypic conversion involves the loss of cell adhesions, polarity, and epithelial markers, including E-cadherin and cytokeratin, and the acquisition of markers commonly found in mesenchymal cells such as vimentin and N-cadherin, altogether potentially enabling cell migration and invasion.8–10 In several studies, prostate cancer cell lines undergoing androgen deprivation or treatment with enzalutamide either in vitro or in vivo showed upregulation of the mesenchymal markers vimentin, N-cadherin, and fibronectin.11–14 Using clinical samples, a study comparing the expression of the EMT transcriptional regulator ZEB1 across normal prostate, hormone-naïve localized prostate cancer, CRPC, and metastatic lesions revealed that ZEB1 expression is increased with disease progression, with the highest levels observed in CRPC and metastases.15 In the same study, expression of mesenchymal vimentin in localized prostate cancer was shown to be predictive of shorter recurrence-free survival, while expression of epithelial E-cadherin in CRPC was associated with longer overall survival.15 Altogether, these studies indicate that tumor phenotypic plasticity in prostate cancer is a phenomenon associated with cancer progression and resistance to AR inhibition.
Our laboratory has previously shown that tumor phenotypic plasticity can decrease tumor susceptibility to immune cell-mediated lysis16–19; however, the link between enzalutamide resistance, tumor cell plasticity, and resistance to immune-mediated lysis has not yet been investigated. In this study, we interrogated two enzalutamide-resistant prostate cancer models for their susceptibility to immune effector-mediated lysis. Enzalutamide resistance was associated with a significant decrease of tumor cell susceptibility to natural killer (NK) lysis, a phenomenon that was not observed with effector T cells. RNA sequencing (RNA-seq) and real-time PCR analyses of enzalutamide-resistant cells showed a significant upregulation in the expression of estrogen receptors. Previous studies have linked estrogen receptor signaling to the progression of prostate cancer20; to investigate their role in decreased susceptibility to NK lysis, this work explored the use of fulvestrant, which is a selective estrogen receptor degrader approved for the treatment of hormone-receptor positive metastatic breast cancer in postmenopausal women. Fulvestrant inhibits the dimerization of estrogen receptors and their translocation into the nucleus, while also promoting their proteasomal degradation.21 We demonstrate here that blockade of estrogen receptor signaling with fulvestrant is able to restore sensitivity of enzalutamide-resistant prostate cancer cells to NK lysis in vitro. In vivo, fulvestrant showed antitumor activity against xenografts of enzalutamide-resistant cells growing in nude mice. These data support future investigations of the combination of NK-based immunotherapies with fulvestrant in enzalutamide-resistant prostate cancer.
Methods
Cell lines and reagents
Human LNCAP and MDA-PCa 2b cells were obtained and cultured as recommended by the American Type Culture Collection (ATCC, Manassas, Virginia, USA). LNCAP cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Omega Scientific, Tarzana, California, USA); MDA-PCa 2b cells were grown in Ham’s F-12 medium supplemented with 20% non-heat-inactivated FBS (ATCC), 10 ng/mL mouse epidermal growth factor (Corning Life Sciences, Corning, New York, USA), 0.005 mM phosphoethanolamine, 100 pg/mL hydrocortisone, 45 nM sodium selenite, 25 ng/mL cholera toxin (Sigma, St. Louis, Missouri, USA), and 0.005 mg/mL human recombinant insulin (Life Technologies, Carlsbad, California, USA). Cells were tested with a MycoAlert Mycoplasma Detection Kit (Lonza, Rockville, Maryland, USA) and used at low passage number from the date of acquisition. The identity of the LNCAP and MDA-PCa 2b cells was verified by short tandem repeat analysis (GeneCopoeia, Rockville, Maryland, USA). To generate models of enzalutamide resistance, LNCAP and MDA-PCa 2b cells were grown in the presence of 10 µM enzalutamide (Selleckchem, Houston, Texas, USA) continuously for ≥5 weeks. Fresh media containing enzalutamide was changed two times per week. Resistance was then confirmed via a proliferation assay in the presence of enzalutamide, as indicated below. When indicated, the enzalutamide-resistant cells were treated with 50 nM fulvestrant (Selleckchem) added to the culture medium for 72 hours prior to assays.
Proliferation assay
Tumor cells were plated in black wall 96-well plates (CellBind, Corning Life Sciences) at 3×103 cells per well in media as indicated above. Enzalutamide was added to the media at concentrations ranging from 0–20 µM (LNCAP) or 0–10 µM (MDA-PCa 2b), and fulvestrant was added at a concentration of 50 nM when indicated. Cells were imaged using the IncuCyte Live-Cell Analysis System (Sartorius, Bohemia, New York, USA) with a 10× objective every 12 hours for 7–10 days. IncuCyte AI Confluence Analysis was used to quantify the percentage of confluence within individual wells over time. Each cell line was normalized to a consistent starting point (1% confluence) using values from untreated tumor wells at time 0, and proliferation curves were prepared with GraphPad Prism V.9.5.0 (GraphPad Software, La Jolla, California, USA). Data were analyzed by two-way analysis of variance (ANOVA).
Cell viability assay
To evaluate the ability of fulvestrant to modulate tumor cell viability, tumor cells were seeded at 1000 cells per well in a flat bottom 96-well plate (CellBind, Corning Life Sciences) in growth media. Fulvestrant (20–50 nM) was added directly to the wells. After 72 hours, an MTT assay was conducted using the Cell Proliferation Kit I (MTT, Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s recommendations. Cell viability (%) was calculated as: (optical density (OD) in wells of interest / average OD in untreated wells) × 100.
Immunoblot
Tumor cells were lysed with RIPA buffer (Santa Cruz Biotechnology, Dallas, Texas, USA) containing 1 mM PMSF (Santa Cruz). Protein concentration was quantified using a BCA protein assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA); proteins (20–30 µg) were resolved on Bolt 4–12% Bis-Tris Plus Gels (Invitrogen, Carlsbad, California, USA) and transferred onto nitrocellulose membranes via the iBlot 2 Transfer System (Invitrogen). Membranes were blocked with 5% Blotting-Grade Blocker (Bio-Rad, Hercules, California, USA) in Tris-buffered saline plus 0.1% Tween (KD Medical, Columbia, Maryland, USA) (TBST) for 1 hour at room temperature and probed overnight with primary antibodies at 4°C. Primary antibodies included androgen receptor (Clone Ar441, 1:100, Invitrogen), PSA/KLK3 (Clone D11E1, 1:1000, Cell Signaling Technology), vimentin (polyclonal GTX100619, 1:5000, GeneTex), E-cadherin (clone 36, 1:1000, BD Biosciences) and GAPDH (Clone 0411, 1:10,000, Santa Cruz). Blots were probed with secondary antibodies conjugated to IRDye 680 or IRDye 800 (LI-COR Biosciences, Lincoln, Nebraska, USA) for 1 hour at room temperature and visualized on the Odyssey Infrared Imaging System (LI-COR Biosciences). ImageJ analysis software was used to measure the mean fluorescence intensity of the antibody stainings. The fluorescence intensities were normalized to their corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) staining intensity and then to the wildtype cell line for the LNCAP and MDA-PCa 2b pairs.
Cytotoxicity assays
NK cells were isolated from healthy donor peripheral blood mononuclear cells (PBMC) by using a magnetic NK Cell Isolation Kit (Miltenyi Biotec, Auburn, California, USA). The target cells were harvested, labeled with 10 µM calcein-AM (Invitrogen) for 20 min at 37°C, washed, and plated at 5×103 cells per well in 384-well flat bottom culture plates. NK effector cells from various donors were added to the target cells at effector-to-target (E:T) ratios of 20:1 and 10:1, as indicated. After a 4-hour incubation, calcein-AMPOS cells were quantified using a Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, Massachusetts, USA). Percent lysis was calculated as follows: % lysis = (1 − (cell count in well of interest / tumor only average cell count)) × 100. When indicated, effector NK cells were incubated for 48 hours with 50 nM fulvestrant prior to the cytotoxic assay, and fulvestrant (50 nM) was also added throughout the duration of the assay.
Generation of brachyury-reactive T cells
PBMC collected from an HLA-A2 healthy donor were plated at 2.5×106 cells per well in a 24-well plate along with 1 µg/mL of the HLA-A2-restricted brachyury agonist peptide, WLLPGTSTV. On days 3 and 5 of culture, 10 ng/mL of human interleukin (IL)-7 and IL-15 (PeproTech, Cranbury, New Jersey, USA) were added. One week following stimulation, cultures were washed and rested for 5 days, followed by restimulation with the brachyury agonist peptide for 4 hours. Interferon (IFN)-γ producing cells were labeled using an IFN-γ secretion detection kit (Miltenyi Biotec). CD8POS/IFN-γPOS cells were enriched using an MA900 Multi-Application Cell Sorter (Sony Biotechnology, San Jose, California, USA). Purified cells were rapidly expanded and stimulated using ImmunoCult Human CD3/CD28/CD2 T Cell Activator (Stem Cell Technologies, Cambridge, Massachusetts, USA); T cells were used for lytic assays 3 days following stimulation.
Real-time PCR
Total RNA from tumor cells was isolated using the RNeasy Mini Kit (QIAGEN, Germantown, Maryland, USA). RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and complementary DNA (cDNA) was purified via the QIAquick PCR Purification Kit (QIAGEN). All kits were used according to the manufacturer’s instructions. cDNA (20 ng) was amplified in triplicate using FastStart Essential DNA Probes Master (Roche Diagnostics, Indianapolis, Indiana, USA) in a LightCycler 96 (Roche, Basel, Switzerland) instrument. The following gene expression probes were used (Thermo Fisher Scientific): AR (Hs00171172_m1), KLK3 (Hs02576545_m1), VIM (Hs00958116_m1), FN1 (Hs00415006_m1), ZEB1 (Hs00232783_m1), ESR1 (Hs001711860_m1), ESR2 (Hs011100353_m1), and GAPDH Control Mix (4325792). Normalized expression of each target gene was calculated relative to GAPDH as 2−ΔCt.
Flow cytometry
Tumor cells or NK cells were stained for cell surface expression markers in 96-well round bottom plates in phosphate buffered saline (PBS) buffer containing 4% FBS. Antibodies used were purchased from BioLegend (San Diego, California, USA), BD Biosciences (San Jose, California, USA), R&D Systems (Minneapolis, Minnesota, USA), or Thermo Fisher Scientific and were used according to the manufacturers’ recommendation. Fluorescently-labeled antibodies included human ULBP-2/5/6 (clone 165903, R&D Systems), HLA-A/B/C (clone G46-2.6, BD), B7-H6 (clone JAM1EW, Thermo Fisher Scientific), CD16 (clone 3G8, BioLegend), and CD56 (clone HCD56, BioLegend), murine pan-H2 (clone M1/42, BioLegend), CD45 (clone 30-F11, BioLegend), CD49b (clone DX5, BioLegend), PD-1 (clone 29F.1A12, BioLegend), and IFN-γ (clone XMG1.2, BioLegend), and human/murine Granzyme B (clone QA18A28, BioLegend). For ex vivo flow analyses, tumors were weighed, mechanically dissociated, and incubated in RPMI containing 5% FBS, 1 mg/mL collagenase type I and IV, and 40 U/mL DNase I at 37°C for 30 min shaking at a speed of 300 rpm. Tumors were then passed through a 70 µm filter to obtain a single-cell suspension for analysis by flow cytometry. Whole spleens were processed by being crushed through a 70 µm filter followed by red cell lysis via ammonium-chloride-potassium (ACK) lysis buffer incubation on ice. In xenograft experiments LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used to identify live cells, and murine NK cells were defined as mCD45POS/mCD49bPOS. All cytometry data were acquired using the Attune NxT Flow Cytometer (Thermo Fisher Scientific), and data were analyzed via FlowJo Software (FlowJo, Ashland, Oregon, USA).
Tumor-NK cell conjugate assay
NK cells were isolated from PBMC of healthy donors as described above and cultured overnight in RPMI containing 10% human AB serum. NK cells and tumor cells were stained with 5 µM CellTrace carboxyfluorescein succinimidyl ester (CFSE) or 5 µM CellTrace Violet (CTV) (Thermo Fisher Scientific), respectively, following the manufacturer’s recommendations. Cells were counted and plated at 1×104 tumor cells and 2×105 NK cells per well (E:T ratio of 20:1) into round bottom 96-well plates, centrifuged at 500×g for 3 min, and then allowed to incubate at 37°C for 4 hours. Data were acquired using the Attune NxT Flow Cytometer (Thermo Fisher Scientific), and cells were enumerated by 123count eBeads (Thermo Fisher Scientific) according to the manufacturer’s instructions; data were evaluated with FlowJo Software. Samples were initially gated on cells based on forward scatter (FSC) × side scatter (SSC), and subsequently, conjugates were identified as CFSEPOS/CTVPOS; all samples were assessed in triplicate and findings were evaluated using NK cells from four independent donors.
Immunofluorescence-based staining and in situ hybridization
Xenograft tumor tissues were fixed in Z-fix (Anatech, Battle Creek, Michigan, USA), embedded in paraffin, sectioned, and mounted onto glass slides (American HistoLabs, Gaithersburg, Maryland, USA). Human prostate cancer tissue microarrays (PR483d and PR551) were purchased from US Biomax (Derwood, Maryland, USA). PR483d was used to identify AR and ESR1 expression while PR551 was used to identify CD56 and CD45 expression in prostate cancer tissues. Slides were deparaffinized with a xylene substitute and rehydrated with ethanol gradients, followed by antigen retrieval using pH6 or, in the case of ESR1, pH9 buffer (Akoya Biosciences, Marlborough, Massachusetts, USA). Slides were then blocked with BLOXALL solution (Vector Laboratories, Burlingame, California, USA) for 10 min, washed in TBST, and incubated for 1 hour at room temperature with primary antibodies diluted in Renaissance Background Reducing Buffer (BioCare Medical, Pacheco, California, USA). Primary antibodies included: E-cadherin (clone 36/E-Cadherin, 1:100, BD Biosciences), androgen receptor (clone AR411, 1:50, Invitrogen), PSA (clone D11E1, 1:1000, Cell Signaling), vimentin (polyclonal GTX100619, 1:500, GeneTex), ESR1 (clone E115, 1:200, Abcam), CD45 (clone HI30, 1:100, Thermo Fisher Scientific), CD56 (clone MRQ-42, 1:200, Sigma), and cytokeratin (clone 2A4, 1:100, Abcam). ImmPress-HRP species-specific IgG Polymer reagents (Vector Laboratories) or Opal Anti-Ms+Rb HRP (Akoya Biosciences) were used as secondary antibodies; Opal fluorophores (Opal-480, 520, 570, 620 and 690, Akoya Biosciences) were used for signal detection, according to the manufacturer’s protocol.
For immunofluorescence-based staining of tumor cells in culture, cells were grown in BioCoat Poly-D-Lysine 8-well culture slides (Corning Life Sciences), fixed in 3% paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pennsylvania, USA) for 10 min at room temperature, and washed twice with PBS. Cells were permeabilized with PBS supplemented with 0.05% Triton X-100 (Thermo Fisher Scientific) for 20 min at room temperature, followed by blocking in a solution of PBS supplemented with 10% goat sera and 1% BSA for 1 hour at room temperature. Primary antibodies diluted in PBS supplemented with 1% BSA were incubated overnight at 4°C; secondary antibodies included Alexa Fluor 532 goat anti-rabbit IgG (Life Technologies) and Alexa Fluor 647 goat anti-mouse IgG (Invitrogen).
Slide scanning, image capturing, and image analysis of stained tissues and chamber slides were performed on an Axio Scan.Z1 and Zen Blue software (Zeiss, Oberkochen, Germany). The entire tumor section, tissue core, or culture chamber were scanned with a 20× objective and used for analysis. To quantify normalized fluorescence intensity of E-cadherin and vimentin expression on tumor cells stained in chamber slides, six regions of interest (ROI) were quantified for total fluorescence intensity of the respective marker and divided by the number of DAPI-positive nuclei present in the ROI. To quantify expression of AR, PSA, E-cadherin, vimentin, and ESR1 in xenograft tissues, four ROI per tumor section were analyzed for mean fluorescence intensity (MFI) for each respective marker; a total of three tumors were analyzed. To quantify percent positive signal of vimentin in tumor xenografts, four ROI per tumor were quantified for the percent area of positive vimentin signal in relation to the area of the entire ROI; a total of two tumors were analyzed. For all quantifications, ROI with no obvious signs of necrosis or tissue degradation were selected randomly for analysis, and all ROI were equivalently sized within a single tumor tissue.
For in situ hybridization staining, tumor cells were grown and fixed in chamber slides as described above, and RNAscope technology (Advanced Cell Diagnostics, Newark, California, USA) was used following the manufacturer’s protocol. A TBXT (brachyury) or a negative control probe was used for signal detection, followed by cell staining with Alexa Fluor 488 Phalloidin (Thermo Fisher) according to the manufacturer’s protocol. Image capturing was performed on an Axio Observer 7 with a 20× objective and Zen Blue Software (Zeiss). All slides were counterstained with DAPI (1:1000, Invitrogen) and mounted using ProLong Diamond mountant (Invitrogen).
Bulk RNA-seq analysis
Total RNA from LNCAP and LNCAP-EnzaR tumor cells grown in culture was prepared using the RNeasy Mini Kit (QIAGEN) and analyzed for integrity on an Agilent TapeStation (Agilent Technologies, Santa Clara, California, USA). Samples with an RNA integrity number greater than 8.0 were sequenced by the Novogene UC Davis Sequencing Center (Novogene, Sacramento, California, USA) using the NovaSeq6000 Illumina platform. Raw read counts were further analyzed using the NIH Integrated Data Analysis Platform. Genes containing low raw counts (counts per million (CPM) <0.5) across three or more of the samples were filtered out before proceeding with further analysis; counts were then normalized to library size as CPM. Following transformation of the data to log2CPM, an analysis of differential expression of genes (DEG) was conducted and the results were visualized on a volcano plot using a p value threshold of 0.05 and a fold-change threshold of 2.
Gene expression omnibus data
A previously published database (GSE197780) from the Gene Expression Omnibus was used to compare the expression of selected genes in primary prostate cancer tissues before and after 3 months of neoadjuvant enzalutamide therapy.22 RNA-seq data were downloaded as normalized log2-transformed read counts. Graphs were created and analyzed using GraphPad Prism V.9.5.0. A paired two-tailed Student’s t-test was used to compare expression of each indicated gene in pre-enzalutamide versus post-enzalutamide samples.
Mice, tumor inoculation, and treatment
All animal procedures reported in this study were approved by the NCI Animal Care and Use Committee and in accordance with federal regulatory requirements and standards. All components of the intramural NIH ACU program are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. NSG-MHC I/II KO male mice (NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-Ab1em1Mvw H2-D1tm1Bpe Il2rgtm1Wjl/SzJ) were obtained from the Jackson Laboratory and bred in-house; 8 week-old female athymic nude (NU/NU) mice were obtained from the NCI Frederick Cancer Research Facility. Mice were injected subcutaneously (s.c.) in the right flank with 2×106 LNCAP or LNCAP-EnzaR cells mixed at a 1:1 (in the NSG mice) or 3:1 (in the NU/NU mice) ratio with Matrigel (Corning Life Sciences, Corning, New York, USA). When indicated, mice were injected with 20 mg/kg of fulvestrant intramuscularly (i.m.) one to two times weekly, as indicated in the figure legends. Tumors were measured with a Vernier caliper two to three times per week. Tumor volume = (short diameter2 × long diameter)/2.
Statistical analyses
Graphs were generated and data were analyzed using GraphPad Prism V.9.5.0. Analyses of tumor growth curves were conducted using two-way ANOVA. Statistical differences between two sets of data were determined using an unpaired or paired two-tailed Student’s t-test, as indicated in the figure legends. One-way ANOVA with Tukey’s post hoc test was used to determine statistical differences among three or more sets of data. Data points in graphs represent the mean±SD or SEM where indicated. Asterisks indicate statistically significant values indicated as follows: ns, not significant; *p≤0.05; **p≤0.01; ***p≤0.001, ****p≤0.0001.
Results
Enzalutamide-resistant prostate cancer cells display phenotypic plasticity
To model enzalutamide resistance in vitro, LNCAP cells (ARPOS/PSAPOS) were continuously exposed to culture medium supplemented with 10 µM enzalutamide for ≥5 weeks (figure 1A). A proliferation assay was then conducted to interrogate the susceptibility of LNCAP and LNCAP enzalutamide-resistant cells (LNCAP-EnzaR) to the cytostatic effect of enzalutamide. While the proliferation of LNCAP cells was significantly reduced at both doses of enzalutamide evaluated over the duration of a 180-hour assay, the proliferation of LNCAP-EnzaR cells was not negatively affected by addition of 10 or 20 µM enzalutamide (figure 1B), with 10 µM enzalutamide increasing rather than decreasing the proliferation of the resistant cells. Real-time PCR and protein analyses revealed a marked modulation of androgen receptor (AR) expression, which was significantly increased at the messenger RNA (mRNA) level (figure 1C) but reduced (~30% reduction) at the protein level (figure 1D) in LNCAP-EnzaR compared with control LNCAP cells. In agreement with the loss of AR protein, expression of the main AR-target, prostate-specific antigen (PSA), was significantly reduced in LNCAP-EnzaR cells both at the mRNA (KLK3, figure 1C) and protein levels (figure 1D, ~80% reduction), compared with LNCAP cells. Acquisition of resistance to enzalutamide also led to remarkable changes in the phenotype of the tumor cells, which exhibited a significant increase of mRNA encoding for mesenchymal vimentin (VIM), fibronectin (FN1), and the transcriptional regulator ZEB1 above the levels observed with control LNCAP cells (figure 1E). Immunofluorescence-based antibody staining of LNCAP and LNCAP-EnzaR cells grown on chamber slides, as well as the accordant quantification of signal, showed nearly a complete loss of epithelial E-cadherin expression with a corresponding decrease in cell-to-cell contacts (figure 1F,G) and a significant increase in expression of mesenchymal vimentin in the enzalutamide-resistant cells (figure 1F,G, and online supplemental figure 1).
Supplemental material
Enzalutamide-resistant prostate cancer cells display phenotypic plasticity. (A) Schematic representing the development of LNCAP enzalutamide-resistant cells. LNCAP cells (ARPOS/PSAPOS) were cultured for 5 weeks or longer in media containing enzalutamide (10 µM). (B) Proliferation of LNCAP (top panel) and LNCAP-EnzaR cells (bottom panel) in the presence of indicated concentrations of enzalutamide. Error bars indicate mean±SEM for technical replicates; ****p<0.0001 by two-way analysis of variance (ANOVA). (C) Expression of androgen receptor (AR) and KLK3 mRNA (encoding for PSA) via real-time PCR. (D) Expression of AR (110 kDa, arrowhead) and PSA (29 kDa, arrowhead) proteins via immunoblot analysis. A single membrane was used for AR and PSA detection in two different IRDye channels; GAPDH used as a control was detected subsequently on the same membrane. Numbers below the blots indicate the expression of each protein of interest normalized to that of parental LNCAP, as detailed in the Methods. (E) mRNA expression of vimentin (VIM), fibronectin (FN1), and ZEB1 by real-time PCR. (F) OPAL immunofluorescence-based detection of epithelial E-cadherin (red) and mesenchymal vimentin (white) in LNCAP and LNCAP-EnzaR cells grown in chamber slides. DAPI (blue) was used to stain nuclei; scale bars: 50 µm; 20× objective with digital zoom. (G) Normalized fluorescence intensity for E-cadherin and vimentin expression in LNCAP and LNCAP-EnzaR cells calculated as described in the Methods; six regions of interest (ROI) were randomly selected; horizontal lines display the mean. (H) NSG MHC-class I/II deficient mice were injected subcutaneously (s.c.) in the right flank with LNCAP or LNCAP-EnzaR cells. Shown is the average tumor growth curves for each cell line. No statistical significance (ns) was observed in two-way ANOVA comparison. (I) OPAL immunofluorescence-based detection of AR (red), PSA (white), E-cadherin (red), and vimentin (white) in LNCAP and LNCAP-EnzaR tissues collected on day 27. DAPI (blue) was used to stain nuclei; scale bars: 50 µm; 20× objective with digital zoom. (J) Mean fluorescence intensity for each indicated marker in LNCAP and LNCAP-EnzaR tumors calculated as described in the Methods; four ROI were randomly selected per tumor section; n=3 tumors per group were evaluated; horizontal lines display the mean. Error bars indicate mean±SD for technical replicates; ***p<0.001 and ****p<0.0001 by two-tailed Student’s t-test in (C, E, G, J). LNCAP-EnzaR, LNCAP enzalutamide-resistant cells; mRNA, messenger RNA; PSA, prostate-specific antigen; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
To establish whether the phenotypic changes observed in vitro could be sustained in vivo, LNCAP and LNCAP-EnzaR cells were grown as xenografts in NSG MHC-class I/II deficient male mice. As shown in figure 1H, both cell lines grew at similar rates in immunodeficient mice. Immunofluorescence-based staining and quantification of signal in tumors collected 27 days post-implantation showed a significant decrease of AR and PSA expression in LNCAP-EnzaR compared with LNCAP tumors (figure 1I,J, and online supplemental figure 2), indicating that the resistant phenotype is maintained over the ~30 day length of the study. Regarding features of phenotypic plasticity, although the level of E-cadherin expression was similar between LNCAP and LNCAP-EnzaR tumors (figure 1J), the distribution and localization of E-cadherin to areas of cell–cell contacts was partially disrupted in LNCAP-EnzaR compared with LNCAP xenografts (figure 1I). Expression of mesenchymal vimentin was significantly increased in enzalutamide resistant LNCAP tumors (figure 1I,J).
A second model of enzalutamide-resistant prostate cancer exhibits phenotypic plasticity. (A) Schematic representing the development of MDA-PCa 2b enzalutamide-resistant cells. MDA-PCa 2b cells (ARPOS/PSAPOS) were cultured for 5 weeks or longer in media containing 10 µM enzalutamide. (B) Proliferation of MDA-PCa 2b (top panel) and MDA-PCa 2b-EnzaR cells (bottom panel) in the presence of 10 µM enzalutamide. Error bars indicate mean±SEM for technical replicates; ***p<0.001, ****p<0.0001 by two-way ANOVA. (C, E) Expression of indicated markers via real-time PCR. (D, F) Expression of AR (110 kDa, arrowhead) and PSA (29 kDa, arrowhead) (D) or E-cadherin (120 kDa, arrowhead) and vimentin (57 kDa, arrowhead) (F) via immunoblot analysis. A single membrane was used for AR and PSA (D) or E-cadherin and vimentin (F) detection in two different IRDye channels; GAPDH used as a control was detected subsequently on each of the corresponding membranes. Numbers below the blots indicate the expression of each protein of interest normalized to the parental MDA-PCa 2b cells, as detailed in the Methods. Error bars indicate mean±SD for technical replicates; **p<0.01, ***p<0.001, and ****p<0.0001 by two-tailed Student’s t-test in (C, E). ANOVA, analysis of variance; AR, androgen receptor; PSA, prostate-specific antigen; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
An additional enzalutamide-resistant cell line model was generated by exposing ARPOS/PSAPOS MDA-PCa 2b cells to media supplemented with 10 µM enzalutamide for ≥5 weeks (figure 2A). Significant reduction of cell proliferation was observed with MDA-PCa 2b cells in response to enzalutamide, while the effect was less pronounced (although statistically significant) with MDA-PCa 2b-EnzaR cells (figure 2B). Paralleling observations with the LNCAP cell pair, resistance to enzalutamide led to a significant increase of AR expression at the mRNA level (figure 2C), while the expression of AR protein decreased by ~80% in MDA-PCa 2b-EnzaR compared with parental cells (figure 2D). A decrease of PSA/KLK3 mRNA was also observed with MDA-PCa 2b-EnzaR cells (figure 2C), while reduction of PSA protein (figure 2D) was not observed with this cell line. Real-time PCR analyses showed similar phenotypic changes to those observed with the LNCAP pair, with increased expression of VIM, FN1, and ZEB1 in the enzalutamide-resistant MDA-PCa 2b cells (figure 2E). In addition, expression of E-cadherin protein was reduced by ~50% and vimentin protein was increased ~3.8 fold in MDA-PCa 2b-EnzaR versus the parental cells (figure 2F). Altogether, these data indicated that acquisition of resistance to enzalutamide in two independent human prostate cancer cell models led to tumor phenotypic changes characteristic of an epithelial-mesenchymal switch, a phenomenon commonly associated with tumor resistance to numerous therapeutics.
Resistance to enzalutamide reduces susceptibility to NK-mediated lysis
To assess whether enzalutamide-resistance could potentially impact the response of prostate cancer cells to immune-mediated attack, flow cytometry analysis of surface markers relevant to immune effector lysis was conducted, including expression of classical MHC-class I molecules (HLA-A/B/C), ligands for NK-activation MIC-A/B, ULBP-2/5/6, and B7-H6. Among all the markers evaluated, expression of HLA-A/B/C was found to be high in both LNCAP and LNCAP-EnzaR cells, with significantly higher MFI observed in enzalutamide-resistant versus parental cells (figure 3A). Expression of B7-H6, the cognate ligand for the activating NK receptor NKp30, was low in LNCAP cells and had a moderate increase at the MFI level in the LNCAP-EnzaR cell line (figure 3B). Similarly, the NK-activating ligands ULBP-2/5/6 were significantly increased in LNCAP-EnzaR compared with the LNCAP cells, both at the level of percent positive cells and the MFI (figure 3C).
Resistance to enzalutamide significantly reduces susceptibility to NK lysis. (A–C) Representative flow cytometry overlay histograms depicting surface expression of HLA-A/B/C, B7-H6, and ULBP-2/5/6 in LNCAP and LNCAP-EnzaR cells. Dotted vertical line indicates the positive/negative gating based on the isotype control. Plots below the histograms show the quantification of the percentage of positive cells and the mean fluorescence intensity (MFI) for each marker in three replicate assays performed on tumor cells collected at different passage numbers in culture. *p<0.05 by two-tailed Student’s t-test comparing LNCAP versus LNCAP-EnzaR; ns, non-significant; horizontal lines display the mean. (D) Expression of TBXT (brachyury) mRNA determined by in situ hybridization (pink dots) in LNCAP and LNCAP-EnzaR cells grown in chamber slides. Tumor cells were stained with phalloidin (green) and DAPI (blue nuclei). Scale bars: 20 µm; 20× objective with digital zoom. (E) LNCAP and LNCAP-EnzaR cells were used as targets for brachyury-reactive T cells. Lysis was conducted over 5-hour and 24-hour time periods at a 20:1 effector-to-target (E:T) ratio. No statistical significance (ns) was observed in two-way ANOVA comparison. Data are representative of two independent experiments. (F) Lysis assay using purified NK cells isolated from healthy donor PBMC against LNCAP and LNCAP-EnzaR cells. Shown are eight donors; dotted lines connecting values for LNCAP and LNCAP-EnzaR cells indicate individual donors. Lysis was conducted over a 4-hour period; E:T ratios are indicated. (G) NK-mediated lysis of MDA-PCa 2b and MDA-PCa 2b-EnzaR cells using purified NK cells (E:T ratio 20:1) isolated from four healthy donor PBMC. Dotted lines connecting values indicate individual donors. Lysis was conducted over a 4-hour time period; **p<0.01, and ***p<0.001 by paired two-tailed Student’s t-test comparing parental versus EnzaR cells (F, G). (H) Representative flow cytometry overlay histograms depicting surface expression of HLA-A/B/C in MDA-PCa 2b and MDA-PCa 2b-EnzaR cells. Dotted vertical line indicates the positive/negative gating based on the isotype control. Numbers indicate the percentage of positive cells and the MFI (in parentheses). ANOVA, analysis of variance; LNCAP-EnzaR, LNCAP enzalutamide-resistant cells; mRNA, messenger RNA; NK, natural killer; PBMC, peripheral blood mononuclear cell.
As the increase of MHC-class I expression could lead to enhanced T-cell lysis, LNCAP and LNCAP-EnzaR cells were first evaluated for their susceptibility to lysis by T cells specific for the tumor-associated antigen brachyury (TBXT). figure 3D and online supplemental figure 3 demonstrate similar levels of expression of brachyury in both cell lines, as determined by in situ hybridization; as shown in figure 3E, acquisition of resistance to enzalutamide had no impact on cancer cell susceptibility to T-cell lysis. The increase of MHC-class I molecules, which bind to NK-inhibitory receptors, together with the increase in B7-H6 and ULBP-2/5/6 expression, which are ligands for activating NK receptors, led to the hypothesis that enzalutamide-resistance may modulate tumor responses to NK-cell lysis. As shown in figure 3F with NK cells isolated from PBMC from eight different healthy donors, LNCAP-EnzaR cells were significantly less susceptible to NK lysis when compared with the parental LNCAP cells at both E:T ratios evaluated (10:1 and 20:1). In addition to LNCAP cells, the loss of tumor susceptibility to NK-mediated lysis in the context of enzalutamide resistance was corroborated with the MDA-PCa 2b-EnzaR cells, which also demonstrated a decrease in susceptibility to NK-mediated lysis when using NK cells derived from four healthy donor PBMC (figure 3G). Flow cytometry analysis of the MDA-PCa 2b pair showed markedly higher expression of HLA-A/B/C in MDA-PCa 2b-EnzaR cells compared with the parental counterparts as well (figure 3H).
Upregulation of estrogen receptors in enzalutamide-resistant prostate cancer cells
To investigate potential mechanisms associated with decreased susceptibility to NK-mediated lysis in the context of enzalutamide resistance, RNA-seq analysis was conducted on RNA prepared from LNCAP and LNCAP-EnzaR cells grown in culture. A DEG analysis showed 795 genes downregulated and 762 genes upregulated in LNCAP-EnzaR cells, using a p value threshold of 0.05 and a fold-change threshold of 2.0 (figure 4A). Among the top 20 genes upregulated in LNCAP-EnzaR cells (online supplemental table 1A,B, respectively) was ESR1, which encodes for the hormone receptor estrogen receptor alpha. The upregulation of ESR1 in cells resistant to enzalutamide was confirmed via real-time PCR; LNCAP-EnzaR and MDA-PCa 2b-EnzaR cells showed a significantly higher expression of ESR1 mRNA and a small, statistically significant increase in ESR2 mRNA levels, compared with their corresponding parental cells (figure 4B,C). The increased expression of ESR1 was also observed at the protein level; as shown in figure 4D,E, and online supplemental figure 4A, ESR1 expression was significantly increased in xenografts of LNCAP-EnzaR cells growing in NSG MHC-class I/II deficient mice, compared with LNCAP tumors.
Supplemental material
Enzalutamide increases expression of estrogen receptors in prostate cancer. (A) Volcano plot displaying differentially expressed genes between LNCAP-EnzaR and LNCAP cells; red dots indicate genes upregulated or downregulated >2 fold (p<0.05). The top 10 most upregulated genes in LNCAP-EnzaR versus LNCAP are denoted in blue; ESR1 is marked with a box. (B, C) Expression of ESR1 and ESR2 mRNA by real-time PCR in the LNCAP (B) and MDA-PCa 2b (C) cell pairs. **p<0.01, ***p<0.001, and ****p<0.0001 by two-tailed Student’s t-test. (D) OPAL immunofluorescence-based detection of ESR1 (red) in LNCAP and LNCAP-EnzaR tumors grown s.c. in the flank of NSG MHC-class I/II deficient mice. Tumors were collected on day 27; DAPI (blue) was used to stain nuclei; scale bars: 50 µm; 20× objective with digital zoom. (E) Mean fluorescence intensity calculated as described in the Methods; four ROI were randomly selected per tumor section; n=3 tumors were evaluated; horizontal lines display the mean. ****p<0.0001 by two-tailed Student’s t-test. (F) Representative images of a prostate cancer tissue array containing 40 cases of prostate adenocarcinoma and 8 normal prostate tissues stained for AR (white), ESR1 (red), pan-cytokeratin (CK, green), and DAPI (blue). Scale bars: 20 µm; 20× objective with digital zoom. (G) Scoring of AR and ESR1 expression in the tissue array showing the number of tumor versus normal prostate samples positive (≥5% of cells in core) for the indicated markers. (H) Representative images of a prostate cancer tissue array stained via OPAL multiplex immunofluorescence for markers of immune infiltration (CD45, white) and NK cells (CD56, green). Red arrows indicate CD45POS/CD56POS infiltrating NK cells. Scale bars: 20 µm; 20× objective with digital zoom. (I) Expression of ESR1, ESR2, epithelial CDH1, and mesenchymal CDH2, VIM, and ZEB1 in a previously published RNA-seq database (GSE197780) from the Gene Expression Omnibus comparing primary prostate cancer tissues before (pre, gray symbols) and after 3 months of neoadjuvant enzalutamide therapy (post-enza, pink symbols). Data are presented as normalized log2-transformed read counts. ***p<0.001, ****p<0.0001 for paired two-tailed Student’s t-test. AR, androgen receptor; FC, fold-change; LNCAP-EnzaR, LNCAP enzalutamide-resistant cells; NK, natural killer; RNA-seq, RNA sequencing; s.c., subcutaneously.
Expression of ESR1 in human prostate cancer tissues was evaluated to understand the potential clinical relevance of these findings. A commercial tissue microarray comprised of 8 normal prostate tissues and 40 primary prostate adenocarcinomas was stained for AR, ESR1, and pan-cytokeratin (CK) (figure 4F and online supplemental figure 4B). Scoring of the individual tissue cores revealed 14/40 (35.0%) cases double positive for expression of AR and ESR1 (ARPOS/ESR1POS), 10/40 (25.0%) cases positive for AR and negative for ESR1 (ARPOS/ESR1NEG), 3/40 (7.5%) cases negative for AR and positive for ESR1 (ARNEG/ESR1POS), and 13/40 (32.5%) cases double negative for AR and ESR1 (ARNEG/ESR1NEG) (figure 4F,G, and online supplemental table 2). Additional stainings were conducted in a prostate cancer tissue array using antibodies against CD45 and CD56 for characterization of tumor infiltration with NK cells. Two representative images are shown in figure 4H and online supplemental figure 4C; overall, 5/48 tissues had isolated CD56POS/CD45POS cells located within clusters of CD45POS cells (figure 4H, arrowheads). To further investigate if treatment of patients with cancer with enzalutamide could lead to higher expression of ESR1 in prostate tissues, previously published RNA-seq data available from the Gene Expression Omnibus (GSE197780) was evaluated22; analysis corresponding to primary prostate cancer tissues prior to and after 3 months of neoadjuvant enzalutamide therapy demonstrated that ESR1 and ESR2 were significantly increased in post-enzalutamide samples (figure 4I), compared with prostate cancer tissues obtained prior to therapy. In addition, there was lower expression of epithelial E-cadherin (CDH1) and higher expression of mesenchymal N-cadherin (CDH2), vimentin (VIM), and ZEB1 in samples post-enzalutamide (figure 4I), potentially indicating the occurrence of an epithelial–mesenchymal switch in response to treatment.
Blockade of estrogen receptor signaling with fulvestrant improves susceptibility to NK lysis
The functional role of ESR signaling in enzalutamide-resistant cells was investigated by using fulvestrant, a pure estrogen receptor antagonist. Treatment with fulvestrant did not have a direct effect on the viability of the LNCAP or MDA-PCa 2b cell pairs (online supplemental figure 5A,B), indicating that ESR signaling is not required for the survival of the parental or enzalutamide-resistant cells; additionally, fulvestrant was unable to revert resistance to enzalutamide (online supplemental figure 5C). Our laboratory has previously shown that upregulation of ESR1 in mesenchymal-like lung cancer cells decreases susceptibility to immune effector cell lysis.23 To investigate whether ESR signaling plays a similar role in the context of enzalutamide resistance, the LNCAP-EnzaR (figure 5A) and MDA-PCa 2-EnzaR cells (figure 5B) were pretreated for 72 hours with 50 nM fulvestrant prior to being used as targets for NK cells isolated from healthy donor PBMC. A significant increase in NK lysis was observed with 4/5 donors for the LNCAP-EnzaR (figure 5A) and 2/4 donors for the MDA-PCa 2b-EnzaR model (figure 5B) when the tumor cells were pre-exposed to fulvestrant compared with the untreated cells, at both E:T ratios used. To mimic the potential scenario of fulvestrant treatment in vivo, NK cells isolated from two healthy donor PBMC were pretreated with fulvestrant in culture for 48 hours prior to the lysis assay, in addition to pretreating the LNCAP-EnzaR cells. Fulvestrant was also added throughout the duration of the assay when pretreated NK cells were used. A similar enhancement of NK lysis was observed regardless of NK-cell treatment condition when the LNCAP-EnzaR cells were pretreated with fulvestrant (online supplemental figure 6A), suggesting that the NK cells are not negatively affected by the fulvestrant.
Fulvestrant increases NK lysis of enzalutamide-resistant prostate cancer cells. LNCAP-EnzaR cells (A) and MDA-PCa 2b-EnzaR cells (B) were left untreated or pretreated with fulvestrant (50 nM) for 72 hours prior to being used as targets for NK cells isolated from PBMC from four to five healthy donors per cell line (dotted lines connecting values indicate matched individual donors). Lysis was conducted over a 4-hour period at indicated effector-to-target (E:T) ratios. *p<0.05 by paired two-tailed Student’s t-test comparing untreated versus fulvestrant-treated cells; ns, non-significant. (C, D) LNCAP and LNCAP-EnzaR cells (untreated or treated with 50 nM fulvestrant for 72 hours prior to the assay) were labeled with 5 µM CellTrace Violet; healthy donor NK cells isolated from four donors (DN1–DN4) were labeled with 5 µM CFSE. Target cells and NK cells were co-cultured for 4 hours at an E:T ratio of 20:1. (C) Representative plots demonstrate the gating used to observe the formation of conjugates between each cell line and donor NK cells. (D) Number of conjugates formed by each cell line/donor NK cells, quantified as detailed in the Methods; dotted lines connecting values indicate individual donors. *p<0.05 and ***p<0.001 by one-way ANOVA followed by Tukey’s multiple comparisons post-test. (E) Lysis assay conducted with NK cells isolated from donors DN1-DN4 (used in the panels C, D). Lysis was conducted over a 4-hour period at an E:T ratio of 20:1. *p<0.05 by paired two-tailed Student’s t-test; ns, non-significant. (F) Correlation of MFI of granzyme B expression in CD16POS NK cells isolated from donors DN1–DN4 (used in panels C–E) versus change in % NK-mediated lysis of LNCAP-EnzaR cells treated with fulvestrant. ANOVA, analysis of variance; CFSE, carboxyfluorescein succinimidyl ester; LNCAP-EnzaR, LNCAP enzalutamide-resistant cells; MFI, mean fluorescence intensity; NK, natural killer; PBMC, peripheral blood mononuclear cell.
Antitumor effect of fulvestrant against enzalutamide-resistant prostate cancer cells (A) Average tumor growth and (B) individual tumor volumes of LNCAP-EnzaR cells growing as xenografts in NSG MHC-class I/II deficient mice left untreated versus treated with fulvestrant (20 mg/kg) given intramuscularly (i.m.) on days 18, 26, and 33 (tick marks in panel A). (C) Average tumor growth and (D) individual tumor volumes of LNCAP-EnzaR cells growing as xenografts in nude mice left untreated or administered fulvestrant (20 mg/kg) on days 19, 27, and 32 (tick marks in panel C). n=6–7 mice/group. Error bars in A, C, indicate SEM **p<0.01 by two-way ANOVA. (E–G) LNCAP-EnzaR cells were grown as xenografts in nude mice and left untreated or administered fulvestrant (20 mg/kg) on days 12, 15, 19, 23, and 27. Spleens and tumors were collected from mice on day 29 for further analysis. n=6 mice/group. The percentage of granzyme BPOS/IFN-γPOS NK cells and PD-1POS/IFN-γPOS NK cells are quantified in the (E) spleen and (F) tumor of each mouse. Horizontal lines display the mean; unpaired two-tailed Student’s t-tests demonstrated no significant differences. (G) Correlations of the percentage of granzyme BPOS/IFN-γPOS (left panel) or PD-1POS/IFN-γPOS (right panel) tumor infiltrating NK cells (NK TIL) versus the tumor volume of each mouse measured at the end of study (day 29). (H) Nude mice implanted with LNCAP-EnzaR xenografts were left untreated or administered weekly doses of fulvestrant (20 mg/kg) starting on day 12. Tumors were collected on day 47 for analysis by immunofluorescence-based detection assays for expression of E-cadherin (green), vimentin (red), and DAPI (blue). Shown are ROI from two control and two fulvestrant-treated tumors. White dash–outlined squares identify magnified regions in far-right ROI; scale bars: 50 µm or 20 µm where indicated. (I) Quantification analysis of tumors in (H) for E-cadherin and vimentin expression. Graphs show the fluorescence signal across four ROI randomly selected within each tumor section, n=2 tumors per treatment group; horizontal lines display the mean. P values were calculated by two-tailed Student’s t-test. ANOVA, analysis of variance; LNCAP-EnzaR, LNCAP enzalutamide-resistant cells; NK, natural killer; ROI, regions of interest.
The potential mechanism of fulvestrant-enhanced NK lysis was investigated by first evaluating the expression of ligands for various NK receptors on the surface of LNCAP-EnzaR cells treated with fulvestrant. Representative plots and quantification of expression across five experimental repeats are shown in online supplemental figure 6B-D. Fulvestrant treatment had no significant effect on the expression of the various markers evaluated. Tumor phenotypic plasticity via an EMT has been shown to decrease the expression of adhesion molecules on tumor cells which, in turn, could impair the formation of conjugates with immune effector cells, resulting in impaired lysis. To evaluate whether this mechanism could be responsible for the decreased lysis of enzalutamide-resistant cells and subsequently restored by fulvestrant, a flow-based conjugate formation assay was conducted. LNCAP and LNCAP-EnzaR cells untreated or pretreated with 50 nM fulvestrant for 72 hours prior to the assay were labeled with CellTrace Violet; healthy donor NK cells isolated from four donors were labeled with CFSE. Target cells and NK cells were co-cultured for 4 hours and the formation of conjugates between each cell line and donor NK cells was evaluated via flow cytometry as shown in representative plots in figure 5C. A significant decrease in the number of NK-tumor cell conjugates was observed when comparing untreated LNCAP-EnzaR cells versus the parental LNCAP cells, where a reduction of ~50% was observed with four NK donors evaluated (figure 5D). Fulvestrant pretreatment of LNCAP-EnzaR cells was able to fully reverse this effect and reconstitute the formation of NK-tumor cell conjugates to the levels observed with parental LNCAP cells (figure 5D). The level of lysis obtained with the same four donor-derived NK cells was evaluated in parallel; as shown in figure 5E, NK cells from donors DN1 and DN2 showed increased lysis of LNCAP-EnzaR cells pretreated with fulvestrant, while there was no significant improvement with donors DN3 and DN4. These data indicated that while fulvestrant can revert the defective formation of conjugates characteristic of the LNCAP-EnzaR tumors, lysis might still not proceed with some donor NK cells. Characterization of markers of cytotoxicity via flow cytometry showed that the MFI of granzyme B expression on NK cells directly correlates with the change of lysis in response to fulvestrant (figure 5F). Thus, donor NK cell variability may be responsible for the lack of improved lysis in response to fulvestrant, as observed with some of the donors shown in figure 5A, B and E.
Antitumor activity of fulvestrant against LNCAP-EnzaR cells in vivo
The activity of fulvestrant against LNCAP-EnzaR cells in vivo was first evaluated in the context of NSG MHC-class I/II deficient mice. Treatment of mice bearing LNCAP-EnzaR (s.c.) tumors with 20 mg/kg fulvestrant (i.m.) beginning on day 18 post-tumor implantation and weekly thereafter did not have an impact on the tumor growth rate (figure 6A,B). To evaluate whether fulvestrant could have an effect in the presence of endogenous murine NK cells, additional in vivo experiments were conducted with LNCAP-EnzaR cells grown s.c. in the flank of athymic nude mice. In the presence of NK cells, treatment with 20 mg/kg fulvestrant beginning on day 19 post-tumor implantation and weekly thereafter, showed a significant reduction of tumor burden compared with the untreated group (figure 6C,D), with 1/7 mice being tumor-free in the fulvestrant-treated group. These results led us to investigate the potential effect of fulvestrant on spleen and tumor infiltrating NK cells, as well as the tumor cells. As shown in figure 6E,F, there was a trend towards increased percentage of granzyme BPOS/IFN-γPOS NK cells and PD-1POS/IFN-γPOS NK cells in the spleen and, to a lesser extent, the tumor of mice treated with fulvestrant. Interestingly, a strong negative correlation was observed in fulvestrant-treated mice between the percentage of granzyme BPOS/IFN-γPOS (r=−0.8134, p=0.049, figure 6G, left panel) or PD-1POS/IFN-γPOS (r=−0.8523, p=0.031, figure 6G, right panel) tumor infiltrating NK cells versus the tumor volume of each mouse measured at the end of study, implicating the impact of fulvestrant on NK cells in the antitumor efficacy.
The effect of fulvestrant on tumor cells in vivo was also evaluated. Immunofluorescence-based analysis of EMT markers, including E-cadherin and vimentin, demonstrated that while fulvestrant did not affect the overall level of E-cadherin expression (figure 6H,I, online supplemental figure 7), tumors treated with fulvestrant presented with a more defined localization of E-cadherin to areas of cell–cell contact. In addition, vimentin positivity was reduced in fulvestrant-treated tumors, indicating a potential reversion towards a less mesenchymal phenotype, which, in turn, could improve sensitivity to immune-mediated lysis.
Discussion
Despite recent advances in the treatment of prostate cancer, resistance to AR inhibitors such as enzalutamide remains a significant clinical problem. Understanding what mechanisms are involved and how they impact responses to other therapeutic modalities could help develop novel and more effective treatments for this disease. Here, we demonstrate that resistance of prostate cancer cells to enzalutamide drives tumor phenotypic plasticity, upregulates estrogen receptor expression, and significantly decreases tumor cell susceptibility to lysis by NK effector cells. Treatment with fulvestrant is able to significantly increase tumor cell vulnerability to NK-mediated lysis.
With the exception of mismatch repair-deficient prostate tumors characterized by high number of mutations and high frequency of neoantigens, which constitute only 3–5% of all prostate cancer cases, immune checkpoint blockade (ICB) therapy has had little meaningful impact in prostate cancer.24 25 The lack of response to ICB is attributed to the overall lack of neoantigens, poor T-cell infiltration, and the presence of an immunosuppressive tumor microenvironment characteristic of prostate cancer.26 27 For tumors that present with an unfavorable environment for T-cell mediated responses, NK cells represent potentially significant effector cells due to their ability to mediate cytotoxicity independently of the presence of MHC and/or antigen on the target cells.28 In the particular case of prostate cancer, NK cells are emerging as a promising alternative to T-cell focused approaches as both clinical and preclinical evidence suggest that NK cells may be essential to prostate cancer control. For example, increased NK-cell infiltration of prostate cancer tissues has been associated with lower risk of tumor progression,29 and the presence of NK cells highly positive for the activating receptors NKp30 and NKp46 in the blood of patients with metastatic prostate cancer has been associated with a longer time to castration resistance and improved survival.29 Based on these observations, one could hypothesize that therapeutics that are able to enhance NK activity may afford better tumor control in prostate cancer than those only focused on T-cell activation.
Using two prostate cancer cell line models of enzalutamide resistance, we demonstrated that resistance to enzalutamide significantly decreases tumor susceptibility to NK-cell lysis, an effect that was not observed with effector T cells. In light of the proposed central role of NK cells in prostate cancer control, we further investigated biomarkers of resistance. Decreased tumor susceptibility to NK-cell lysis may include tumor downregulation of ligands for NK-activating receptors, increased expression of ligands for inhibitory NK receptors,30 or defects in adhesion molecules that preclude the formation of NK/target cell conjugates. In prostate cancer, for example, a study demonstrated the downregulation of the intercellular adhesion molecule 1 on the surface of the cancer cells, which is required for efficient NK-cell activation, as a major mechanism of resistance to NK lysis.31 Here, enzalutamide-resistant prostate cancer cells showed no consistent pattern of upregulation or downregulation of surface ligands that could explain their significantly lower susceptibility to lysis by NK cells; however, we did observe a significant decrease in the number of NK-tumor cell conjugates when comparing enzalutamide-resistant to parental cells. Two prominent features of the enzalutamide-resistant cells observed in this study were the acquisition of a mesenchymal-like tumor phenotype and the overexpression of ESR1 and ESR2. Estrogens are key signaling molecules that regulate many physiological processes such as cell growth, development, and differentiation. Recent discoveries have demonstrated the importance of ESR signaling in prostate cancer progression,32 33 and specific roles have been described for ESR1 and ESR2 in the prostate. ESR1 expression has been observed in stromal cells of the human prostate and shown to indirectly promote the proliferation of cancer cells via soluble factors.20 34 In contrast, ESR2 has been shown to act as a tumor suppressor in epithelial cells of the prostate.33 35 While the role of ESR signaling in the development and progression of prostate cancer has been well-studied, less is known about the role of ESR in the context of enzalutamide resistance.
In a previous study, our laboratory conducted a quantitative high-throughput screening assay to identify drugs that could enhance immune-mediated lysis of lung cancer cells with mesenchymal-like phenotypic features, using a library of approximately 2800 small-molecule compounds. The screening identified fulvestrant as a compound able to increase the sensitivity of mesenchymal-like lung cancer cells to lysis by immune effector NK cells, antigen-specific T cells and TNF-related apoptosis-inducing ligand (TRAIL), while also decreasing the expression of mesenchymal fibronectin, vimentin, and the EMT transcription factor brachyury in the tumor cells.23 In agreement with those observations, here we demonstrated that fulvestrant is able to reconstitute the formation of NK-tumor cell conjugates to levels observed with parental cells, ultimately leading to an increase in the susceptibility of enzalutamide-resistant cells to NK-mediated lysis in vitro. This study also demonstrated the ability of fulvestrant as a monotherapy to delay the growth of enzalutamide-resistant cells in vivo, an effect that was positively correlated with the activated phenotype of NK cells in the tumor. One of the ligands that was increased both at the mRNA and protein level in the fulvestrant-treated LNCAP-EnzaR cells was B7-H6 (NCR3LG1), a ligand for the activating receptor NKp30.36 Our group has recently reported on the central role of the NKp30/B7-H6 axis in small cell lung cancer where it allows for efficient NK-cell mediated lysis.37 Although B7-H6 overexpression has been reported for several tumor types, no studies have shown the relevance of this molecule in prostate cancer.
Fulvestrant has been previously explored as a therapeutic in prostate cancer. Preclinically, treatment of LNCAP cells with fulvestrant has been shown to decrease the expression of AR at the mRNA and protein level and to reduce the proliferation of prostate cancer cells in response to androgens.38 Clinically, fulvestrant was used as a monotherapy in a phase II trial in patients with hormone-refractory prostate cancer; although fulvestrant was safe, it failed to provide clinical or PSA responses in this patient population.39 The data here shows that while blockade of ESR signaling via fulvestrant does not directly decrease cell viability or alleviate enzalutamide resistance in LNCAP-EnzaR cells, fulvestrant pretreatment significantly increases the susceptibility of enzalutamide-resistant cells to NK lysis. Based on these observations, future studies are planned to interrogate combinations of fulvestrant and NK-based therapies, including the use of the IL-15 superagonist N-803, which increases the activation and proliferation of NK cells and CD8 T cells. While the present study has been conducted with human cell line models, future studies will use murine prostate cancer models to evaluate the effect of fulvestrant in combination with NK-based therapeutics in a setting with a fully functioning immune system.
A limitation of this study is that only two cell line models were used owing to the paucity of available ARPOS/PSAPOS prostate models. While the analysis of publicly available RNA-seq data from primary prostate cancer tissues before and after neoadjuvant enzalutamide corroborated the increased expression of estrogen receptors and mesenchymal features in post-enzalutamide samples, additional studies using enzalutamide-resistant patient-derived xenografts are needed to evaluate whether the mechanisms described here can be generalized. In this study, enzalutamide treatment was not incorporated in the in vivo models. Although the characterization of phenotypic features (low AR, low PSA, high vimentin, high ESR1) supported the notion that resistance to enzalutamide was maintained over the length of the in vivo studies, we cannot exclude the possibility that resistance is lost after longer periods of growth in the absence of selective pressure.
Overall, this study demonstrates the ability of fulvestrant to increase the susceptibility of prostate cancer cells resistant to enzalutamide to NK-mediated lysis thus providing rationale for future exploration of combinations of fulvestrant with NK-based immunotherapy approaches, including the use of the IL-15 superagonist N-803, adoptively transferred memory NK cells, high-affinity NK (haNK) cells, programmed death-ligand 1(PD-L1)-targeting haNK cells, or other chimeric antigen receptor(CAR)-NKs for the treatment of advanced prostate cancer, particularly in the case of tumors that no longer respond to enzalutamide treatment.
Supplemental material
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
This study involves the use of de-identified peripheral blood mononuclear cells that were obtained from healthy volunteers who provided written informed consent at the NIH Clinical Center Blood Bank (Clinical Protocol NCT00001846). Participants gave informed consent to participate in the study before taking part.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
MD, KF and LAH contributed equally.
Contributors MD, KF, and LAH contributed equally to this work. MD, KF, LAH, and CP conceptualized and designed the overall study, conducted experiments, acquired and analyzed data. SA, HQ, and DH conducted experiments, acquired and analyzed data. MD, KF, LAH, SA, and CP wrote and edited the manuscript. JS and CP supervised the study. CP is the guarantor of the study.
Funding This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health (NIH), ZIC BC 010937.
Competing interests CP discloses spouse’s employment and holdings in MacroGenics, Inc. The remaining authors do not have any competing interests to disclose.
Provenance and peer review Not commissioned; externally peer reviewed.
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