CANCER RESEARCH | CANCER BIOLOGY

Androgen Receptor Variants Confer Castration

Resistance in Prostate Cancer by Counteracting
Antiandrogen-Induced Ferroptosis
Rui Sun1,2,3, Binyuan Yan3,4, Hao Li3, Donglin Ding3, Liguo Wang5, Jun Pang4, Dingwei Ye1,2, and
Haojie Huang3,6,7

ABSTRACT
◥
Androgen receptor (AR) inhibition by androgen deprivation (AR-V) preferentially bound the SLC7A11 enhancer and upregu-
and/or antiandrogen administration is the mainstay therapy for lated SLC7A11 expression, thereby conferring resistance to ferrop-
advanced prostate cancer. However, most prostate cancers ulti- tosis induced by ENZ treatment. However, this effect was abolished
mately become resistant to these therapies, indicating the impor- following downregulation of AR-Vs using the dual CBP/p300 and

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

tance of identifying mechanisms driving resistance to improve BET inhibitor NEO2734. These findings reveal ferroptosis induc-
patient outcomes. Here we demonstrated that acute treatment with tion as an anticancer mechanism of antiandrogens and SLC7A11 as
the antiandrogen enzalutamide (ENZ) decreased glutathione (GSH) a direct target gene of AR-FL and AR-Vs. AR-V-mediated SLC7A11
production, increased lipid peroxidation, and induced ferroptosis in expression represents a mechanism coupling ferroptosis resistance
prostate cancer cells. Consistently, meta-analysis of transcriptomic to prostate cancer progression.
data linked the androgen-AR axis to metabolism-related biological
processes, including lipid metabolism. The cystine transporter gene Significance: Upregulation of SLC7A11 can be induced by
SLC7A11 was a key AR target, and full-length AR (AR-FL) trans- androgen receptor variants to inhibit antiandrogen-induced pros-
activated SLC7A11 transcription by directly occupying the tate cancer cell ferroptosis and to drive castration resistance in
SLC7A11 promoter and putative enhancer regions. AR variants prostate cancer.

Introduction mediated cystine uptake, triggers ferroptosis (3). Intercellular cystine is

rapidly converted to cysteine, which subsequently serves as the rate-
Ferroptosis is a unique form of cell death that is biochemically and
limiting precursor for glutathione synthesis. Glutathione peroxidase 4
morphologically distinct from other types of cell death such as
(GPX4) utilizes reduced glutathione (GSH) to decrease lipid peroxides
apoptosis, necrosis, autophagy, and pyroptosis (1–3). Ferroptosis is
to lipid alcohols to protect cells against membrane lipid peroxidation
known to be caused by lipid peroxidation and intracellular iron-
and inhibit ferroptosis (10, 11). It is well established that cell death
mediated membrane oxidative damage induced by cystine
plays important roles in tumor suppression (12, 13). Ferroptosis can be
depletion (4–6). Solute carrier family 7 member 11 (SLC7A11) is a
regulated at epigenetic, transcriptional, posttranscriptional, and post-
subunit of the cystine/glutamate antiporter system Xc
and is the
translational levels. Activation of ferroptosis results in inhibition of
major transporter of extracellular cystine (7–9). Cystine depletion
certain cancer types including prostate cancer (14). The roles and
or treatment of chemicals such as erastin, which blocks SLC7A11-
regulatory mechanisms of ferroptosis in prostate cancer progression
remain poorly understood.
Prostate cancer is the leading cause of cancer-related mortality in
1
Department of Urology, Fudan University Shanghai Cancer Center, Shanghai, men in Western countries. Androgen receptor (AR) is the critical
China. 2Department of Oncology, Shanghai Medical College, Fudan University, transcription factor that is required for normal prostate
Shanghai, China. 3Department of Biochemistry and Molecular Biology, Mayo Clinic development (15–17). AR also plays a pivotal role in prostate cancer
College of Medicine and Science, Rochester, Minnesota. 4Department of Urology, development and progression. Androgen signaling pathway inhibition
Kidney and Urology Center, The Seventh Affiliated Hospital, Sun Yat-sen Uni- is only effective in AR-positive prostate cancer, and hormone na€ve
versity, Shenzhen, China. 5Division of Biomedical Statistics and Informatics, Mayo
patients with prostate cancer have benefitted largely from androgen
Clinic College of Medicine and Science, Rochester, Minnesota. 6Department of
Urology, Mayo Clinic College of Medicine and Science, Rochester, Minnesota. deprivation therapies (ADT; refs. 18, 19). However, most prostate
7
Mayo Clinic Comprehensive Cancer Center, Mayo Clinic College of Medicine and cancer cases eventually progress into castration-resistant prostate
Science, Rochester, Minnesota. cancer (CRPC). The underlying mechanisms remain elusive, and it
R. Sun and B. Yan contributed equally to this article. is unclear whether castration resistance is related to deregulation of
ferroptosis in prostate cancer.
Corresponding Authors: Dingwei Ye, 270 Dong’an Road, Shanghai, 200032,
China. E-mail: [email protected]; and Haojie Huang, Gugg 1311B, 200
AR variants (AR-V) have been implicated in the development of
First Street SW, Rochester, MN 55905. E-mail: [email protected] CRPC (20, 21). AR-Vs can be derived through mechanisms such as
intronic polyadenylation-coupled alternative splicing of the AR
Cancer Res 2023;83:3192–204
gene (22, 23). AR-Vs encode various truncated AR proteins partially
doi: 10.1158/0008-5472.CAN-23-0285
or completely lacking a functional ligand-binding domain in the C
This open access article is distributed under the Creative Commons Attribution- terminus (20). Increasing evidence indicates that AR-Vs are con-
NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license. stitutively active in the absence of androgens or in the present of

2023 The Authors; Published by the American Association for Cancer Research antiandrogens, thus promoting prostate cancer progression by

AACRJournals.org | 3192
 Androgen Receptor Variants Inhibit Ferroptosis

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

Figure 1.
AR antagonists promote ferroptosis in prostate cancer cells. A–I, LNCaP and C4–2 cells treated with vehicle (mock) or ENZ, vehicle (mock) or ARV110, or transfected
with nonspecific shRNA (shNS) or AR-specific shRNAs (shAR#1 and #2). At 48 hours after treatment, cells were subjected to measurement of lipid peroxidation by
C11-BODIPY staining and flow cytometry (A–C), and measurement of intracellular levels of GSH (D–F) and LDH (G–I). Experiments were repeated three times
independently and similar results were obtained. J–L, Cell viability in LNCaP and C4–2 cells cultured in regular or cystine-low (2 mmol/L) medium in the presence or
absence of Ferr-1 and treated with vehicle or ENZ (J), ARV110 (K), or infected with lentivirus expressing nonspecific shRNA (shNS) or AR-specific shRNAs (L) for
72 hours.

AACRJournals.org Cancer Res; 83(19) October 1, 2023 3193

 Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

CANCER RESEARCH
3194 Cancer Res; 83(19) October 1, 2023
Sun et al.
 Androgen Receptor Variants Inhibit Ferroptosis

conferring antiandrogen resistance (21, 24, 25). Previous studies de-identified from patient information. All participants provided
have shown that androgen deprivation or treatment of antiandro- written informed consent. This study was conducted in accordance
gen such as enzalutamide (ENZ) induces AR-V expression in with the guidelines and tenets of the Declaration of Helsinki.
prostate cancer cells in vitro and in vivo (22, 24).
In this study, we identified the SLC7A11 gene as a bona fide Transfection and stable cell-line generation
transactivation target of AR in prostate cancer cells. Inhibition of Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific)
the AR by ENZ impaired GSH production and sensitized prostate was used to transfect prostate cancer cells with siRNAs. Lentivirus
cancer cells to ferroptosis by repressing SLC7A11 expression. transduction system was utilized to generate stable cell lines with
However, sustained expression of AR-Vs in ENZ-treated cells specific gene knockdown. PEI was used to transfect shRNA plasmids
drove SLC7A11 expression and thereby promoted castration- together with lentivirus package plasmids (PSPAX2 and PMD2.G)
resistance due to ferroptosis inhibition. Cotreatment of CBP/ into HEK293T cells. Forty-eight hours after transfection, supernatant
p300 and BET dual inhibitor NEO2734 inhibited AR-V expression containing viruses was collected, filtered, and utilized to infect indi-
and overcame resistance to ferroptosis and ENZ in prostate cancer cated cells. Polybrene (8 mg/mL) was added to the viral supernatant to
cells. increase the infection efficiency. Forty-eight hours after infection,
culture medium was replaced with fresh medium, and puromycin
(1 mg/mL) was administrated for cell selection. Sequence information
Materials and Methods of siRNAs and shRNAs is provided in Supplementary Table S1.

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

Cell lines, cell culture, plasmids, and chemicals
LNCaP, 22Rv1, and HEK293T cell lines were purchased from the Lipid peroxidation assay
ATCC. C4–2 cell line was purchased from Uro Corporation. LNCaP, Cells were incubated in a 60 mm dish containing 5 mmol/L BODIPY
C4–2, and 22Rv1 cells were cultured in RPMI1640 cell culture medium 581/591 C11 dye (Cayman, 27086) for 25 minutes. Cells were washed
(Corning) containing 10% FBS, together with 100 mg/mL streptomycin with PBS, trypsinized and subjected to flow cytometry analysis using
and 100 U/mL penicillin. HEK293T cells were cultured in DMEM flow cytometer as described previously (28).
cell culture medium (Corning) containing 10% FBS, together with
100 mg/mL streptomycin and 100 U/mL penicillin. Cells were cultured Western blotting and IHC
in incubator with 37 C and 5% CO2. All cell lines were authenticated Detailed information regarding antibodies used for Western blotting
by short tandem repeat profiling and tested as Mycoplasma free. and IHC is provided in Supplementary Table S2. For Western blotting
FLAG-AR expression vector was generated as described previous- analysis, cells were lysed in modified RIPA buffer (50 mmol/L Tris-HCl
ly (26). To generate HA-SLC7A11 expression vector, SLC7A11 pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmol/L
cDNA was amplified by PCR using indicated primers (SLC7A11-F: NaCl, 0.1% SDS, and 1 mmol/L EDTA) supplemented with 1% protease
50 -CGGTCGACCATGGTCAGAAAGCCTGTTGTGTCCA CC-30 , inhibitor cocktail. Protein concentration was determined using DC
SLC7A11-R: 50 -CAGCGGCCGCTTATAACTTATCTTCTTCTGG- protein assay reagent (Bio-Rad). Cell extracts were supplemented with
TACAACTTCC AGTAT-30 ) and subcloned into the pCMV-HA 10% DTT (Thermo Fisher Scientific) and boiled at 95 C for 3 minutes.
vector. Ferroptosis inhibitor Ferr-1 and deferoxamine (DFO) were Samples were subjected to SDS-PAGE (Bio-Rad) separation, and the
obtained from Cayman Chemical. ENZ and Bavdegalutamide ARV110 gels were further transferred to nitrocellulose (NC) membranes
were obtained from TargetMOL. dihydrotestosterone (DHT) was (Thermo Fisher Scientific). After transferring, the NC membranes were
purchased from Selleckchem. Z-VAD-FMK was obtained from blocked in 5% nonfat milk (Bio-Rad) for 1 hour at room temperature
MedChemExpress. NEO2734 was kindly provided by Epigenetix and incubated with the indicated primary antibodies at 4 C overnight.
Inc. The activity of NEO2734 in inhibition of BET as well as CBP/p300 Next day, the NC membranes were washed with 1 TBST for 10
bromodomains was initially confirmed with the BROMOscan plat- minutes three times and incubated with matched secondary antibody
form (DiscoverX/Eurofins; ref. 27). for 1 hour at room temperature. The membranes were washed with 1
TBST for 10 minutes three times. Finally, the signals were developed
Prostate cancer patient samples with SuperSignal West Pico Luminal Enhancer Solution (Thermo
Prostate cancer specimens used for IHC were obtained from Fisher Scientific) on autoradiography films (HyBlot CL).
the tissue registry of Mayo Clinic. The studies were approved by For IHC staining, prostate cancer patient specimens and xenograft
the Institute Review Board of Mayo Clinic. All specimens were tumor samples were fixed by formalin and embedded in paraffin. Four-

Figure 2.
Genome-wide analyses link AR to metabolism-related biological processes. A, Heatmap showing expression of genes downregulated (left) and upregulated (right)
by DHT treatment relative to mock in LNCaP cells cultured in medium containing charcoal-stripped serum (CSS). B, GO enrichment analysis of the 415 upregulated
genes [log2 (fold change) > 1] using the tools from the GSEA website (https://www.gsea-msigdb.org/gsea/msigdb/human/annotate.jsp). Top 10 annotation clusters
are shown according to their enrichment scores [
log10 (P value)]. C, Diagram showing the role of SLC7A11 in the context of ferroptosis regulation. D, UCSC Genome
Browser screenshot of RNA-seq data showing SLC7A11 expression levels in LNCaP (GSM2432771 vs. GSM2432769) and C4–2 cells (GSM2432783 vs. GSM2432781)
cultured in CSS medium and treated with or without DHT (10 nmol/L) for 24 hours. E and F, qRT-PCR (E) and Western blot (F) analysis of SLC7A11 expression in LNCaP
and C4–2 cells treated as in D. G–I, UCSC screenshot of RNA-seq data (G) showing SLC7A11 expression levels in LNCaP cells (GSM6132392 vs. GSM6132398) treated
with or without ENZ (10 mmol/L) for 72 hours and qRT-PCR (H) and WB (I) analysis of SLC7A11 expression in LNCaP and C4–2 cells with the same treatments as in G.
J and K, Western blot (J) and qRT-PCR (K) analysis of SLC7A11 protein (J) and mRNA expression (K) in LNCaP and C4–2 cells infected with lentivirus expressing
nonspecific shRNA (shNS) or AR-specific shRNAs for 72 hours. L and M, qRT-PCR (L) and Western blot (M) analysis of LuCaP35 xenograft tumors from male mice
(n ¼ 5 mice/group) treated with sham castration or castration for 1 week. N, The dot plots showing the comparison of expression levels of AR (left) and SLC7A11 (right)
between primary tissues and corresponding metastatic prostate cancer patient samples (GSE32269). O, The scatter plots showing the positive correlation between
AR and SLC7A11 mRNA expression in GSE32269 dataset. P and Q, Representative images (P) and quantitative data (Q) for SLC7A11 protein IHC staining in a group of
primary tissues (n ¼ 20) and metastatic CRPC samples (n ¼ 20).

AACRJournals.org Cancer Res; 83(19) October 1, 2023 3195

 Sun et al.

micrometer-thick sections were cut from the samples and mounted osmium acid for 2 hours. Cell sections were dehydrated in ethanol and
onto slides. Antigen retrieval and immuno-staining were performed as embedded in acetone. The sections (50–60 nm) were stained with 3%
described previously (29). IHC staining was scored based on the “most uranium acetate and lead citrate. TEM images were captured using the
common” criteria. Staining score ¼ staining intensity  staining transmission electron microscope (JEM-1011).
positivity. Staining intensity was graded into four categories: 0, 1, 2,
and 3. Specifically, 0 ¼ no staining, 1 ¼ weak staining (staining obvious Quantitative reverse transcription and PCR
only at 400), 2 ¼ medium staining (staining obvious at 100 but not Total RNA was extracted from cells using TRIzol reagent (Ambion)
40), and 3 ¼ strong staining (staining obvious at 40). For staining and reversely transcribed into cDNA using the GoScript Kit (Pro-
positivity, 0 ¼ no positive cells, 1 ¼ 10% or less of positive cells, 2 ¼ mega). The SYBR Green Mix (Bio-Rad) and CFX96 Real-Time System
11% to 50% positive cells, 3 ¼ 51% to 70% positive cells, 4 ¼ 71% or (Bio-Rad) were utilized to conduct the real-time PCR according to
more positive cells. manufacturer’s instruction. Expression of ACTB housekeeping gene
was used as an internal control, and data were present as mean  SD.
Biochemical experiments Sequence information for primers used for qRT-PCR is provided in
To assess intracellular GSH concentration, GSH assay (Cayman Supplementary Table S3.
Chemical) was performed according to manufacturer’s specifications.
In brief, cell lysate supernatant from indicated treatments were reacted Quantitative chromatin immunoprecipitation PCR
with monochlorobimane to generate a highly fluorescent product that Chromatin immunoprecipitation (ChIP) experiments were per-

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

can be measured using excitation and emission wavelengths of 380 and formed as described previously (22). In brief, chromatin was cross-
480 nm, respectively. linked for 10 minutes at room temperature with 11% formaldehyde/
To assess cytotoxicity, LDH assay (Eton Bioscience) was performed PBS solution added to cell culture medium. Cross-linked chromatin
according to the manufacturer’s introductions. In brief, LNCaP and was sonicated, diluted, and immunoprecipitated with Protein G-
C4–2 cells were cultured in 6-well adherent plates treated with ENZ or plus Agarose beads (Bio-Rad) prebound with antibody at 4 C
AR PROTAC ARV110 (1 mmol/L) for 48 hours. Lactic acid from overnight. Precipitated protein–DNA complexes were eluted, and
indicated culture media was oxidized by enzyme reactions to yield cross-linking was reversed at 65 C for 16 hours. DNA fragments
color product, which can be measured at 570 nm for colorimetric assay. were purified and analyzed by qPCR. qChIP-PCR data were ana-
lyzed as % input after normalizing each ChIP DNA fraction’s Ct
Transmission electron microscopy value to the input DNA fraction’s Ct value. Sequence information
Indicated prostate cancer cells were fixed with 2.5% glutaraldehyde for primers used for qChIP-PCR is provided in Supplementary
in phosphoric acid buffer for 2 hours, followed by after fixation in 1% Table S3.

Figure 3.
AR promotes SLC7A11 expression at transcriptional level. A, Meta-analysis of ChIP data of transcription factors that potentially bind to the SLC7A11 gene locus using
the online-based platform Cistrome software (http://dbtoolkit.cistrome.org). B and C, UCSC screenshot of AR ChIP-Seq showing AR occupancy at the SLC7A11 loci in
LNCaP and C4–2 cells cultured in CSS medium and treated with or without DHT (B), and qChIP-PCR analysis of AR binding at the SLC7A11 promoter and enhancer
regions in LNCaP and C4–2 (C) cells with the same treatment as in B. D and E, AR ChIP-Seq occupancy profiles at the SLC7A11 loci in C4–2 cells treated with or without
ENZ (D). qChIP-PCR confirming AR binding at the SLC7A11 promoter and enhancer region was abolished in C4–2 cells (E) with the same treatment as in D.

3196 Cancer Res; 83(19) October 1, 2023 CANCER RESEARCH

 Androgen Receptor Variants Inhibit Ferroptosis

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

Figure 4.
Role of SLC7A11 in AR-mediated prostate cancer cell growth. A–D, LNCaP and C4–2 cells transfected with shNS or shSLC7A11 and stable cell lines were used for
Western blot analysis (A), MTS assays (B), and colony formation assays, followed by photographing (C) and quantification (D). Three biological replicates were
analyzed. ERK2 was used as a loading control. E–H, LNCaP cells were infected with lentivirus expressing the indicated shRNAs or expression vectors for 72 hours and
subjected to Western blot analysis (E), MTS assays (F), and colony formation assays, followed by photographing (G) and quantification (H). Three biological
replicates were analyzed. I–L, LNCaP cells infected with lentivirus expressing the indicated plasmids were treated with or without ENZ and subjected to Western blot
analysis (I), MTS assays (J), and colony formation assays, followed by photographing (K) and quantification (L). Three biological replicates were analyzed.

Bioinformatic analysis a 96-well plate with a density of 1,500 cells per well. At the indicated
The raw reads of RNA-seq were aligned to the human genome time points, 10 mL CellTiter 96R Aqueous One Solution reagent
reference (hg19) using STAR 2.7.10. The bam2wig.py in RSeQC was (Promega) was added to cells. After incubating at 37 C incubator for
used to transform the bam files to wiggle format, which was transferred 2 hours, cell growth was measured in a microplate reader with
to bigwig format to be visualized in the UCSC Genome Browser. absorbance at 490 nm.

MTS cell proliferation assay Colony formation assay

Cell proliferation was measured using MTS assay (Promega) Cells were resuspended in fresh culture medium, seeded in triplicate
according to manufacturer’s instruction. Briefly, cells were seeded in with a density of 2,000 cells/well for 22Rv1, 1,500 cells/well for LNCaP

AACRJournals.org Cancer Res; 83(19) October 1, 2023 3197

 Sun et al.

and C4–2 in six-well plates, and cultured under normal growth (1 mm3) and injected subcutaneously into 6-week-old male mice.
conditions for 10 to 14 days. Colonies were washed three times in After xenografts reached a size of 200 mm3, animals were randomly
PBS, and fixed with 4% paraformaldehyde for 0.5 hours. The colonies divided into two groups. One group was treated with sham castration
were further stained by 0.5% crystal violet for 1 hour and washed with and the other group was castrated surgically. After 1 week, tumors were
water twice to remove the crystal violet. The number of colonies in each harvested and proceeded for Western blot and qRT-PCR.
well was counted using an inverted microscope.
Statistical analysis
Generation of xenografts and PDX tumors and drug treatment All data are presented as the mean  SE from three or more
The animal studies were approved by the Institutional Animal Care independent experiments. Differences between two groups were ana-
and Use Committee at the Mayo Clinic. C4–2 xenograft experiments lyzed using unpaired Student t test. Survival curves were obtained
were performed as described previously (26). Briefly, C4–2 cells using the Kaplan–Meier method, and the log-rank test was used to test
(3  106) were mixed with Matrigel [in 50 mL of 1  PBS plus 50 mL the difference in survival curves. P values <0.05 were considered
of Matrigel (BD Biosciences)] and injected subcutaneously into statistically significant.
the right flank of 6-week-old male mice. After xenografts reached a
size of 200 mm3, animals were randomly divided into two groups for Data availability
treatment with vehicle or ENZ (10 mg/kg daily). 22Rv1 cells (3  106) The next-generation sequencing data from The Cancer Genome
were mixed with Matrigel [in 50 mL of 1  PBS plus 50 mL of Matrigel Atlas (TCGA), Stand Up To Cancer (SU2C), and Neuroendocrine

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

(BD Biosciences)] and injected subcutaneously into the right flank of Prostate Cancer (NEPC) cohort were obtained and analyzed via
6-week-old male mice. After xenografts reached a size of 100 mm3 cBioPortal (https://www.cbioportal.org/). The microarray data from
and animals were randomly divided into different groups for treatment the primary and mCRPC cohort were obtained from the Gene
daily with vehicle, ENZ (10 mg/kg), ARV110 (5 mg/kg), NEO2734 Expression Omnibus database with the accession number GSE32269.
(10 mg/kg), or combination of ENZ and NEO2734. Treatments were All data supporting the findings of this study are available within the
administrated 5 days per week by intraperitoneal injection and tumor article and the Supplementary Information files. The relevant reagents
volumes were measured using a digital caliper. LuCaP35 PDX was such as plasmids are available from the corresponding authors upon
provided by E. Corey from the University of Washington. For the request. All other raw data are available upon request from the
LuCaP35 PDX study, PDX tumors were divided into small pieces corresponding authors.

Figure 5.
SLC7A11 attenuates ferroptosis induced by AR antagonist. A–C, C4–2 cells infected with lentivirus expressing the indicated shRNAs and/or expression vectors and
cultured in regular or cystine-low medium (2 mmol/L) for 48 hours and subjected to MTS assays (A), measurement of intracellular GSH levels (B), and detection of lipid
peroxidation by flow cytometry after C11-BODIPY staining (C). Three biological replicates were analyzed. D, C4–2 cells treated as indicated were subjected to
transmission electron microscopy. White arrows, mitochondria with obvious cristae. Red arrows, shrunken mitochondria. Scale bars, left, 2 mm; right, 500 nm.
Experiment was repeated three times independently with similar results. E and F, Representative images (E) and quantitative data (F) for IHC staining of 4HNE in
C4–2 xenograft tumors in mice treated with or without ENZ for 20 days.

3198 Cancer Res; 83(19) October 1, 2023 CANCER RESEARCH

 Androgen Receptor Variants Inhibit Ferroptosis

Results (DFO) but not apoptosis inhibitor (Z-VAD-FMK) largely blocked

ENZ-induced loss of cell viability (Supplementary Fig. S1E–S1G). In
AR inhibition suppresses GSH production and promotes
contrast, treatment with the ferroptosis inducer erastin largely
ferroptosis in prostate cancer
enhanced cell viability inhibition induced by ENZ, ARV110, or AR
Androgen ablation has long been documented to induce cell death
shRNAs (Supplementary Fig. S1H–S1J). To further validate these
such as apoptosis in the normal prostate and prostate cancer (30).
observations, we cultured LNCaP and C4–2 cells with regular or
Prostate cancer is a malignancy having a high-rate lipid metabo-
cystine-low medium and measured cell viability. We demonstrated
lism (31). We therefore sought to determine whether AR inhibition
that decrease in cystine concentration reduced viability in LNCaP and
induces lipid peroxidation and ferroptosis in prostate cancer cells. As
C4–2 cells and this effect was largely enhanced by ENZ, ARV110, or
expected, inhibition of AR by ENZ decreased expression of KLK3, a
AR shRNAs (Fig. 1J–L). Notably, cystine reduction-induced cell
well-studied AR target gene in both LNCaP and its castration-resistant
viability loss was reversed by Ferr-1 (Fig. 1J–L). Together, these data
derivative C4–2 cells (Supplementary Fig. S1A). Notably, ENZ treat-
indicate that AR inhibition induces ferroptosis in prostate cancer cells.
ment increased lipid peroxidation in these two cell lines (Fig. 1A).
Similar results were obtained by treatment with either ARV110, a
Identification of SLC7A11 as an androgen-induced gene
degrader of full-length AR (AR-FL; ref. 32) or two independent AR-
To define the molecular mechanism by which the androgen-AR axis
specific small hairpin RNAs (shRNA; Fig. 1B and C; Supplementary
regulates ferroptosis in prostate cancer cells, we performed meta-
Fig. S1B–S1D). Both pharmacologic inhibition and genetic depletion
analysis of RNA-seq data generated from LNCaP cells cultured in

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

of AR also decreased GSH but increased soluble enzyme lactate
androgen-depleted (charcoal-stripped serum or CSS) medium and
dehydrogenase (LDH) levels, indicating the compromise of cell mem-
treated with or without DHT (33). We found that lipid metabolic
ber integrity in LNCaP and C4–2 cell lines (Fig. 1D–I). Treatment with
process genes were among the top groups of genes upregulated by
ferroptosis inhibitors such as ferrostatin (Ferr-1) or iron chelator
DHT [log2 (fold change) ≥ 1; Fig. 2A and B). Importantly, meta-

Figure 6.
AR-Vs promote SLC7A11 expression and suppress ferroptosis. A, UCSC screenshot of ChIP-seq data of AR and H3K4me1 showing AR occupancy and the level of
H3K4me1 at the SLC7A11 gene locus in 22Rv1 cells in which endogenous AR-FL and AR-Vs were specifically knocked down individually. B, qChIP-PCR confirming the
binding of AR-FL and AR-Vs at the SLC7A11 promoter and enhancer regions in 22Rv1 cells. C and D, 22Rv1 cells transfected with nonspecific siRNA (siNS), siRNA
specific for AR-FL (siAR-FL), and siRNAs specific for AR-Vs (siAR-Vs, including siAR-V1, V3, V4, and V7) and cultured in CSS medium were subjected to Western blot
(C) and qRT-PCR (D) analysis. ERK2 was used as a loading control. E and F, 22Rv1 cells transfected with indicated siRNAs and cultured in regular or cystine-low CSS
medium in the presence or absence of Ferr-1 were subjected to MTS (E) and FACS analysis (F) after cells were stained with propidium iodide (PI).

AACRJournals.org Cancer Res; 83(19) October 1, 2023 3199

 Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

CANCER RESEARCH
3200 Cancer Res; 83(19) October 1, 2023
Sun et al.
 Androgen Receptor Variants Inhibit Ferroptosis

analysis of RNA-seq data suggests that SLC7A11, a known ferroptosis scription factors, including those highly relevant in prostate cancer
inhibitory gene (Fig. 2C), is a putative androgen-upregulated gene such as AR, p300, FOXA1, and FOXA2, revealed that AR was one of
(Fig. 2A and D). Androgen regulation of SLC7A11 mRNA expression the top 20 transcription factors with a strong potential to transactivate
was further validated by qRT-PCR and Western blot analysis in both expression of the SLC7A11 gene (Fig. 3A), suggesting that upon
LNCaP and C4–2 cell lines (Fig. 2E and F). In contrast, RNA-seq activation AR might regulate SLC7A11 expression by directly binding
analysis showed that SLC7A11 mRNA expression was downregulated to this gene locus. In support of this notion, analysis of the AR ChIP-
by ENZ treatment in LNCaP (Fig. 2G), and this result was further seq data we generated previously (38) revealed that DHT treatment
confirmed by qRT-PCR and Western blot analysis in LNCaP and C4–2 markedly increased AR occupancy at promoter (P) and non-promoter
cell lines (Fig. 2H and I). Furthermore, we found that knockdown of [e.g., putative enhancer (E)] regions of SLC7A11 locus in both LNCaP
AR by two independent shRNAs also decreased SLC7A11 expression at and C4–2 cell lines (Fig. 3B). Androgen-induced AR binding in these
both protein and mRNA levels in LNCaP and C4–2 cells (Fig. 2J genomic regions was further confirmed by qChIP-PCR in these two
and K). Although DHT or ENZ treatment had no obvious effect on cell lines (Fig. 3C). In contrast, ENZ treatment abolished AR occu-
mRNA expression of GPX4, another key regulator of ferroptosis pancy at the SLC7A11 gene locus (Fig. 3D and E). These data indicate
(Supplementary Fig. S2A–S2C), DHT increased and ENZ decreased that AR promotes SLC7A11 expression through occupancy at different
GPX4 expression at the protein level (Supplementary Fig. S2D and regions in this gene locus and AR binding can be abrogated by
S2E). These findings are consistent with a previous report that cystine inhibition of the androgen/AR axis.
uptake caused by upregulation of SLC7A11 only increases GPX4

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

protein synthesis, but not GPX4 gene mRNA expression (34), indi- AR inhibits ferroptosis through upregulation of SLC7A11
cating that antiandrogens regulate ferroptosis via modulating expres- SLC7A11 imports extracellular cystine into cells for glutathione
sion of both SLC7A11 and GPX4, two key regulators of ferroptosis. synthesis in the cytoplasm, and GPX4 utilizes glutathione to detoxify
Both qRT-PCR and Western blot analyses further showed that lipid peroxide to protect cells from ferroptosis (28). To investigate
SLC7A11 expression was significantly downregulated in hormone- the impact of SLC7A11 on prostate cancer cell growth and survival,
sensitive LuCaP35 patient-derived xenografts (PDX) after castration we performed MTS assay and showed that SLC7A11 depletion
of mice (Fig. 2L and M; Supplementary Fig. S2F–S2I). significantly suppresses cell growth (Fig. 4A and B). In contrast,
AR signaling plays a central role in prostate cancer progression and SLC7A11 overexpression significantly increased cell viability in dif-
aberrant AR mRNA expression positively correlates with shorter ferent prostate cancer cell lines (Supplementary Fig. S3A and S3B).
disease-free survival of TCGA patients (Supplementary Fig. S2J). Colony growth assay further demonstrated that SLC7A11 knockdown
SLC7A11 expression was higher in prostate tumors compared with decreased colony numbers compared with control cells (Fig. 4C
matched normal prostatic tissues in the TCGA cohort (Supplementary and D). Furthermore, we showed that restoration of SLC7A11 in
Fig. S2K). SLC7A11 level was positively associated with AR expression AR-depleted cells largely restored cell viability (Fig. 4E–H). Impor-
in TCGA prostate cancer specimens and higher SLC7A11 expression tantly, forced expression of SLC7A11 overcame, at least in part, ENZ-
correlated with shorter survival of patients (Supplementary Fig. S2L induced inhibition of cell viability and growth (Fig. 4I–L). AR
and S2M). SLC7A11 expression was much lower in AR-negative NEPC depletion-induced loss of viability in LNCaP and C4–2 cells cultured
samples (35) compared with AR-positive CRPC samples (Supplemen- in cystine-low medium was reversed by restored expression of either
tary Fig. S2N; ref. 36). To investigate SLC7A11 expression in CRPC, we AR or SLC7A11 (Supplementary Fig. S4A–S4D).
analyzed the microarray data (GSE32269) from paired primary pros- We further demonstrated that AR depletion-induced inhibition of
tate cancer and metastatic CRPC samples (37). We found that expres- cell viability and reduction in GSH cellular level were reversed by
sion of both AR and SLC7A11 mRNA was higher in CRPC samples forced expression of SLC7A11 (Fig. 5A–C). Transmission electron
compared with primary tumors (Fig. 2N) and that SLC7A11 also microscopy (TEM) analysis revealed that ENZ-treated C4–2 cells
positively correlated with AR expression in this cohort (Fig. 2O). We exhibited shrunken mitochondria with increased membrane density,
further performed IHC analysis of SLC7A11 protein expression in a morphologic feature of ferroptosis in comparison to control cells
patient samples. We confirmed that SLC7A11 level was higher in (Fig. 5D; ref. 28). Because ferroptosis is characterized by overproduc-
CRPC samples compared with primary tumors (Fig. 2P and Q). tion of lipid peroxidation, we performed 4-Hydroxynonenal (4HNE)
Together, the results from cultured cell lines, PDX tumors and patient IHC analysis to determine lipid peroxidation levels in C4–2 xenograft
samples reveal that SLC7A11 is a target gene transcriptionally upre- tumors treated with or without ENZ. We demonstrated that ENZ
gulated by the androgen signaling. treatment largely decreased tumor volume and SLC7A11 expression
but induced 4HNE level in C4–2 xenografts (Fig. 5E and F; Supple-
Identification of SLC7A11 as an AR-binding target gene mentary Fig. S5A–S5C). These data suggest that ferroptosis is respon-
Consistent with gene expression data, meta-analysis of chromatin sible, at least in part, for AR inhibition-induced cell death in vitro and
immunoprecipitation sequencing (ChIP-seq) data of specific tran- in vivo.

Figure 7.
Dual inhibitor NEO2734 cotreatment overcomes AR-V-mediated resistance to ferroptosis and ENZ in prostate cancer. A–C, 22Rv1 cells treated with vehicle or
different doses of NEO2734 for 24 hours were subjected to WB (A) and qRT-PCR analysis of AR-FL and AR-V7 (B), and SLC7A11 mRNA expression (C). D, 22Rv1 cells
treated with vehicle or different doses of NEO2734 for 36 hours were subjected to the detection of lipid peroxidation by flow cytometry after C11-BODIPY staining.
E–G, 22Rv1 cells were treated as indicated and subjected to WB analysis at 48 hours posttreatment (E), colony formation assay after 12 days of treatment followed by
colony photographing (F) and quantification (G). H–J, 22Rv1 cells infected with lentivirus expressing empty vector or HA-SLC7A11 and stable cells were subjected to
WB analysis (H) and treated with vehicle or ENZ plus NEO2734 followed by colony formation assay for 12 days. Colonies were photographed (I) and quantified (J) at
the end of treatment. K–M, Mice with 22Rv1 xenograft tumors were treated with the indicated drugs and tumor volumes were measured at the indicated
time points (K) and tumors were photographed (L) and weighted (M) at the end of drug treatment (day 21). Data shown as mean  SD (n ¼ 6 replicates/group).
N–P, Representative images (N) and quantitative data for IHC staining of 4HNE (O) and SLC7A11 (P) proteins in 22Rv1 xenograft tumors.

AACRJournals.org Cancer Res; 83(19) October 1, 2023 3201

 Sun et al.

AR-Vs suppress ferroptosis by inducing SLC7A11 expression

AR-Vs, which either partially or completely lack a functional ligand
Discussion
binding domain, confer constitutive AR activity and AR signaling AR-targeted therapy remains the mainstay treatment of advanced
inhibitor resistance in prostate cancer (20, 39). We have shown prostate cancer in patients. Androgen ablation triggers apoptotic cell
previously that AR-Vs bind to and transactivate a unique subset of death in both normal prostate glandular epithelial cells and androgen-
genes that are related to cell-cycle transition and cancer progres- dependent prostate cancer cells (30, 42), and ADT-induced prostate
sion (40). 22Rv1 is a known ENZ-resistant cell line, which expresses cancer cell apoptosis has been implicated in regression of prostate
AR-Vs (39). We found that ENZ treatment failed to suppress SLC7A11 cancer in patients (43). Ferroptosis is a form of cell death that is distinct
mRNA expression in 22Rv1 cells (Supplementary Fig. S6A). By from apoptosis and is an iron-dependent cell death induced by lipid
analyzing the AR-FL and AR-V ChIP-seq data in 22Rv1 cells we peroxidation on cell membrane. Notably, a previous study suggests
generated previously (40), we found that AR-FL bound to both the that ferroptosis induction can be a novel therapeutic strategy for the
promoter and enhancer regions [indicated by high enrichment of treatment of advanced prostate cancer (14). In this study, we provide
histone H3 lysine 4 monomethylation (H3K4me1), an enhancer evidence that antiandrogen treatment or AR protein depletion
histone mark] in the SLC7A11 gene locus in 22Rv1 cells; however, decreased cellular level of GSH and induced ferroptosis of prostate
AR-Vs preferentially bound to the putative enhancer regions cancer cells. Specifically, we show that AR inhibitory agents suppress
(Fig. 6A). AR-FL and AR-V binding in this locus in 22Rv1 cells expression of SLC7A11, an essential negative regulator of ferroptosis.
was further confirmed by qChIP-PCR (Fig. 6B). Previous studies Our current studies hence reveal ferroptosis as a new form of cell death

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

suggest that expression of AR-Vs, but not AR-FL confers ENZ induced by AR ablation therapies (Fig. 8, left).
resistance in 22Rv1 cells (41). In agreement with this report and our Our studies in cell lines, PDX tumors, and patient samples show
finding that ENZ treatment failed to abrogate AR-V occupation and that SLC7A11 is an androgen-regulated gene. By analyzing the AR
suppress SLC7A11 expression in 22Rv1 cells (Supplementary ChIP-seq data, we demonstrate that DHT treatment induces AR-FL
Fig. S6A and S6B), AR-FL knockdown had little or no effect on occupancy at both the promoter and enhancer regions of SLC7A11
SLC7A11 expression in 22Rv1 cells cultured in androgen-depleted gene locus. This observation is corroborated by the finding that ENZ
medium whereas AR-V knockdown largely decreased SLC7A11 treatment eliminated AR-FL occupancy in prostate cancer cells. Our
expression at both mRNA and protein levels (Fig. 6C and D). data reveal that SLC7A11 is a bona fide AR target gene and importantly,
These data suggest that AR-Vs play a pivotal role in upregulating our findings also provide a plausible explanation as to why acute
SLC7A11 expression in ENZ-resistant 22Rv1 cells. Importantly, androgen ablation or antiandrogen treatment induces ferroptosis.
under androgen depletion conditions, AR-V ablation largely sen- Intriguingly, we provide evidence that AR-Vs also occupy at the
sitized 22Rv1 cells to ferroptosis induced by low concentration of SLC7A11 gene locus although it appears that AR-Vs preferentially
cystine (Fig. 6E and F). These data suggest that AR-Vs promote bind to the enhancer region. Importantly, we show that SLC7A11
SLC7A11 expression to suppress ferroptosis, thereby contributing to expression is primarily regulated by AR-Vs under androgen depriva-
ENZ resistance in prostate cancer cells. tion conditions in AR-V-expressing prostate cancer cells. We and
others have shown that androgen depletion or antiandrogen treatment
CBP/p300 and BET dual inhibitor NEO2734 overcomes induces AR-V expression in prostate cancer cells in vitro or xenograft
AR-V–mediated ferroptosis and ENZ resistance in prostate tumors in vivo (22, 24). On the basis of the previous findings and those
cancer from the present study, we envisage a model wherein AR targeting
Our previous studies show that NEO2734, a dual inhibitor of CBP/ therapies induce AR-V expression, which in turn drives SLC7A11
p300 and BET proteins largely decreases AR-FL protein expres- expression in a manner independent of androgens, thereby contrib-
sion (26, 27). Given that AR-FL and AR-Vs share the same regulatory uting tumor progression by suppressing ferroptosis (Fig. 8, right).
elements [such as the promoter and enhancer(s)] of the AR gene (39), We provide further evidence that the effect of AR-Vs on SLC7A11
we sought to determine whether NEO2734 also inhibits AR-V expres- expression and ferroptosis resistance can be therapeutically targeted by
sion. We treated 22Rv1 cells with different doses of NEO2734. We the CBP/p300 and BET dual inhibitor NEO2734, although such effect
demonstrated that NEO2734 inhibited expression of AR-FL and AR- of NEO2734 can be mediated, at least partially by other targets in
vs. at both mRNA and protein levels (Fig. 7A and B). Different from addition to AR since this compound can also inhibit the growth of AR-
ENZ treatment (Supplementary Fig. S6A), NEO2734 treatment sup- negative prostate cancer cells.
pressed SLC7A11 expression in 22Rv1 cells (Fig. 7C). NEO2734 Intriguingly, it has been reported previously that ENZ treatment
treatment also increased lipid ROS in 22Rv1 cells (Fig. 7D). Co- increases SLC7A11 expression in LNCaP cells (44). It is well known
treatment of NEO2734 and ENZ substantially reduced 22Rv1 cell that growth of LNCaP cells is highly sensitive to androgen levels and
viability (Fig. 7E–G). Most importantly, the effect of dual treatments the LNCaP cell line can evolve into different cellular states depending
was largely reversed by restored expression of HA-SLC7A11 (Fig. 7H– on the cell culture conditions. Indeed, a number of androgen refractory
J), suggesting a pivotal role of SLC7A11 downregulation in mediating LNCaP sublines have been derived when LNCaP cells are cultured in
the cell viability loss induced by treatment with NEO2734 and ENZ. various androgen-depleted medium (45, 46). Given that androgen
We also treated 22Rv1 xenograft tumors with ENZ, ARV110, deprivation induces expression of AR-Vs (22, 24) and that AR-Vs can
NEO2734, and the combination of NEO2734 and ENZ. Compared induce SLC7A11 expression in androgen depletion conditions as we
to each individual treatment, coadministration of NEO2734 and demonstrated in the present study, it is not surprising that ENZ
ENZ resulted in much greater inhibition of tumor growth, stronger treatment increases SLC7A11 expression when AR-V expression is
suppression of SLC7A11 expression, and higher level of the lipid induced.
peroxidation indicator 4HNE (Fig. 7K–P). These data indicate that In summary, we demonstrate for the first time that antiandrogen
the CBP/p300 and BET dual inhibitor NEO2734 can inhibit AR-V therapy decreases GSH level, elevates lipid peroxidation, and induces
expression and AR-V-mediated resistance to ENZ and ferroptosis in ferroptosis. We identify the cystine transporter gene SLC7A11 as a
prostate cancer in vitro and in vivo. bona fide AR target gene. Notably, both AR-FL and AR-Vs can

3202 Cancer Res; 83(19) October 1, 2023 CANCER RESEARCH

 Androgen Receptor Variants Inhibit Ferroptosis

Figure 8.
Hypothetical working model. AR-FL transactivates
SLC7A11 gene transcription by directly occupying at the
promoter and enhancer regions of SLC7A11 gene. Anti-
androgen treatment suppresses SLC7A11 expression and
induces ferroptosis in prostate cancer cells by inhibiting
AR-FL-mediated transactivation of SLC7A11 expression
(left). In contrast, AR-Vs preferentially bind to the
SLC7A11 gene enhancer and upregulate SLC7A11 expres-
sion, thereby conferring resistance to ferroptosis induced
by ENZ treatment (right). However, the effect of AR-Vs
can be abolished by CBP/p300 and BET dual inhibitor
treatment-mediated inhibition of AR-V expression.

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

transactivate SLC7A11 gene through direct occupancy at the SLC7A11 D. Ye: Conceptualization, supervision, project administration, writing–review and
promoter and/or enhancer regions. Antiandrogen therapy induces editing. H. Huang: Conceptualization, resources, supervision, funding acquisition,
project administration, writing–review and editing.
ferroptosis by blocking the regulation of SLC7A11 expression by
AR-FL; however, this effect is abolished by subsequent induction of
AR-Vs. Most importantly, we show that cotreatment of the CBP/p300
Acknowledgments
and BET dual inhibitor NEO2734 restores antiandrogen-induced The authors thank Dr. B.Q. Huang for assistance in transmission electron
microscopy analysis. This work was supported by the Mayo Clinic Foundation (to
ferroptosis by blocking the undesired expression of AR-Vs. Thus,
H. Huang) and Fudan University Shanghai Cancer Center (to D. Ye).
blocking AR-V-mediated inhibition of ferroptosis represents a new
tactic to overcome ENZ resistance in CRPC. The publication costs of this article were defrayed in part by the payment of
publication fees. Therefore, and solely to indicate this fact, this article is hereby
Authors’ Disclosures marked “advertisement” in accordance with 18 USC section 1734.
No author disclosures were reported.
Note
Authors’ Contributions Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
R. Sun: Conceptualization, data curation, formal analysis, investigation, writing–
original draft. B. Yan: Data curation. H. Li: Data curation, formal analysis, inves-
tigation. D. Ding: Data curation, formal analysis, investigation. L. Wang: Data Received January 26, 2023; revised May 23, 2023; accepted July 28, 2023;
curation, supervision, investigation. J. Pang: Supervision, writing–review and editing. published first August 1, 2023.

References
1. Green DR. The coming decade of cell death research: five riddles. Cell 2019;177: 9. Lim JC, Donaldson PJ. Focus on molecules: the cystine/glutamate exchanger
1094–107. (system x(c)(-)). Exp Eye Res 2011;92:162–3.
2. Koren E, Fuchs Y. Modes of regulated cell death in cancer. Cancer Discov 2021; 10. Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan
11:245–65. VS, et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 2014;156:
3. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. 317–31.
Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012;149: 11. Friedmann Angeli JP, Schneider M, Proneth B, Tyurina YY, Tyurin VA,
1060–72. Hammond VJ, et al. Inactivation of the ferroptosis regulator Gpx4 triggers
4. Tang D, Chen X, Kang R, Kroemer G. Ferroptosis: molecular mechanisms and acute renal failure in mice. Nat Cell Biol 2014;16:1180–91.
health implications. Cell Res 2021;31:107–25. 12. Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis.
5. Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in Nat Rev Cancer 2002;2:277–88.
disease. Nat Rev Mol Cell Biol 2021;22:266–82. 13. Green DR, Evan GI. A matter of life and death. Cancer Cell 2002;1:
6. Chen X, Kang R, Kroemer G, Tang D. Broadening horizons: the role of 19–30.
ferroptosis in cancer. Nat Rev Clin Oncol 2021;18:280–96. 14. Ghoochani A, Hsu EC, Aslan M, Rice MA, Nguyen HM, Brooks JD, et al.
7. Conrad M, Sato H. The oxidative stress-inducible cystine/glutamate anti- Ferroptosis inducers are a novel therapeutic approach for advanced prostate
porter, system x c (-) : cystine supplier and beyond. Amino Acids 2012;42: cancer. Cancer Res 2021;81:1583–94.
231–46. 15. Cai C, He HH, Chen S, Coleman I, Wang H, Fang Z, et al. Androgen receptor
8. Koppula P, Zhang Y, Zhuang L, Gan B. Amino acid transporter SLC7A11/xCT at gene expression in prostate cancer is directly suppressed by the androgen
the crossroads of regulating redox homeostasis and nutrient dependency of receptor through recruitment of lysine-specific demethylase 1. Cancer Cell
cancer. Cancer Commun (Lond) 2018;38:12. 2011;20:457–71.

AACRJournals.org Cancer Res; 83(19) October 1, 2023 3203

 Sun et al.

16. Grossmann ME, Huang H, Tindall DJ. Androgen receptor signaling in andro- 32. Proof-of-concept with PROTACs in prostate cancer. Cancer Discov 2020;10:
gen-refractory prostate cancer. J Natl Cancer Inst 2001;93:1687–97. 1084.
17. Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance to 33. Zhang Y, Pitchiaya S, Cieslik M, Niknafs YS, Tien JC, Hosono Y, et al. Analysis of
androgen receptor inhibitors in prostate cancer. Nat Rev Cancer 2015;15:701–11. the androgen receptor-regulated lncRNA landscape identifies a role for ARLNC1
18. Agus DB, Cordon-Cardo C, Fox W, Drobnjak M, Koff A, Golde DW, et al. in prostate cancer progression. Nat Genet 2018;50:814–24.
Prostate cancer cell cycle regulators: response to androgen withdrawal and 34. Zhang Y, Swanda RV, Nie L, Liu X, Wang C, Lee H, et al. mTORC1 couples cyst
development of androgen independence. J Natl Cancer Inst 1999;91:1869–76. (e)ine availability with GPX4 protein synthesis and ferroptosis regulation.
19. Heinlein CA, Chang C. Androgen receptor in prostate cancer. Endocr Rev 2004; Nat Commun 2021;12:1589.
25:276–308. 35. Beltran H, Prandi D, Mosquera JM, Benelli M, Puca L, Cyrta J, et al. Divergent
20. Dehm SM, Tindall DJ. Alternatively spliced androgen receptor variants. clonal evolution of castration-resistant neuroendocrine prostate cancer.
Endocr Relat Cancer 2011;18:R183–96. Nat Med 2016;22:298–305.
21. Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, et al. AR-V7 36. Robinson D, Van Allen EM, Wu YM, Schultz N, Lonigro RJ, Mosquera JM, et al.
and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med Integrative clinical genomics of advanced prostate cancer. Cell 2015;161:
2014;371:1028–38. 1215–28.
22. Sun R, Wei T, Ding D, Zhang J, Chen S, He HH, et al. CYCLIN K down- 37. Cai C, Wang H, He HH, Chen S, He L, Ma F, et al. ERG induces androgen
regulation induces androgen receptor gene intronic polyadenylation, variant receptor-mediated regulation of SOX9 in prostate cancer. J Clin Invest 2013;123:
expression and PARP inhibitor vulnerability in castration-resistant prostate 1109–22.
cancer. Proc Natl Acad Sci USA 2022;119:e2205509119. 38. Zhao Y, Wang L, Ren S, Wang L, Blackburn PR, McNulty MS, et al. Activation of
23. Van Etten JL, Nyquist M, Li Y, Yang R, Ho Y, Johnson R, et al. Targeting a P-TEFb by androgen receptor-regulated enhancer RNAs in castration-resistant
single alternative polyadenylation site coordinately blocks expression of prostate cancer. Cell Rep 2016;15:599–610.

Downloaded from http://aacrjournals.org/cancerres/article-pdf/83/19/3192/3367220/3192.pdf by guest on 13 January 2024

androgen receptor mRNA splice variants in prostate cancer. Cancer Res 39. Dehm SM, Schmidt LJ, Heemers HV, Vessella RL, Tindall DJ. Splicing of a novel
2017;77:5228–35. androgen receptor exon generates a constitutively active androgen receptor that
24. Watson PA, Chen YF, Balbas MD, Wongvipat J, Socci ND, Viale A, et al. mediates prostate cancer therapy resistance. Cancer Res 2008;68:5469–77.
Constitutively active androgen receptor splice variants expressed in castration- 40. He Y, Lu J, Ye Z, Hao S, Wang L, Kohli M, et al. Androgen receptor splice variants
resistant prostate cancer require full-length androgen receptor. Proc Natl Acad bind to constitutively open chromatin and promote abiraterone-resistant growth
Sci USA 2010;107:16759–65. of prostate cancer. Nucleic Acids Res 2018;46:1895–911.
25. Bohrer LR, Liu P, Zhong J, Pan Y, Angstman J, Brand LJ, et al. FOXO1 binds to 41. Li Y, Chan SC, Brand LJ, Hwang TH, Silverstein KA, Dehm SM. Androgen
the TAU5 motif and inhibits constitutively active androgen receptor splice receptor splice variants mediate enzalutamide resistance in castration-resistant
variants. Prostate 2013;73:1017–27. prostate cancer cell lines. Cancer Res 2013;73:483–9.
26. He Y, Wei T, Ye Z, Orme JJ, Lin D, Sheng H, et al. A noncanonical AR addiction 42. Denmeade SR, Lin XS, Isaacs JT. Role of programmed (apoptotic) cell death
drives enzalutamide resistance in prostate cancer. Nat Commun 2021;12:1521. during the progression and therapy for prostate cancer. Prostate 1996;28:251–65.
27. Yan Y, Ma J, Wang D, Lin D, Pang X, Wang S, et al. The novel BET-CBP/p300 43. Ohlson N, Bergh A, Nygren K, Stattin P, Wikstrom P. The magnitude of early
dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer. castration-induced primary tumour regression in prostate cancer does not
EMBO Mol Med 2019;11:e10659. predict clinical outcome. Eur Urol 2006;49:675–83.
28. Zhang Y, Shi J, Liu X, Feng L, Gong Z, Koppula P, et al. BAP1 links metabolic 44. Yuan F, Hankey W, Wu D, Wang H, Somarelli J, Armstrong AJ, et al. Molecular
regulation of ferroptosis to tumour suppression. Nat Cell Biol 2018;20:1181–92. determinants for enzalutamide-induced transcription in prostate cancer.
29. Sun R, Xie HY, Qian JX, Huang YN, Yang F, Zhang FL, et al. FBXO22 possesses Nucleic Acids Res 2019;47:10104–14.
both protumorigenic and antimetastatic roles in breast cancer progression. 45. Murillo H, Huang H, Schmidt LJ, Smith DI, Tindall DJ. Role of PI3K signaling in
Cancer Res 2018;78:5274–86. survival and progression of LNCaP prostate cancer cells to the androgen
30. Kyprianou N, Isaacs JT. Activation of programmed cell death in the rat ventral refractory state. Endocrinology 2001;142:4795–805.
prostate after castration. Endocrinology 1988;122:552–62. 46. Leung JK, Tam T, Wang J, Sadar MD. Isolation and characterization of
31. Baron A, Migita T, Tang D, Loda M. Fatty acid synthase: a metabolic oncogene in castration-resistant prostate cancer LNCaP95 clones. Hum Cell 2021;34:
prostate cancer? J Cell Biochem 2004;91:47–53. 211–8.

3204 Cancer Res; 83(19) October 1, 2023 CANCER RESEARCH