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Meta-Analysis
. 2023 Oct 2;83(19):3192-3204.
doi: 10.1158/0008-5472.CAN-23-0285.

Androgen Receptor Variants Confer Castration Resistance in Prostate Cancer by Counteracting Antiandrogen-Induced Ferroptosis

Rui Sun #  1   2   3 , Binyuan Yan #  3   4 , Hao Li  3 , Donglin Ding  3 , Liguo Wang  5 , Jun Pang  4 , Dingwei Ye  1   2 , Haojie Huang  3   6   7
Affiliations
Meta-Analysis

Androgen Receptor Variants Confer Castration Resistance in Prostate Cancer by Counteracting Antiandrogen-Induced Ferroptosis

Rui Sun et al. Cancer Res. .

Abstract

Androgen receptor (AR) inhibition by androgen deprivation and/or antiandrogen administration is the mainstay therapy for advanced prostate cancer. However, most prostate cancers ultimately become resistant to these therapies, indicating the importance of identifying mechanisms driving resistance to improve patient outcomes. Here we demonstrated that acute treatment with the antiandrogen enzalutamide (ENZ) decreased glutathione (GSH) production, increased lipid peroxidation, and induced ferroptosis in prostate cancer cells. Consistently, meta-analysis of transcriptomic data linked the androgen-AR axis to metabolism-related biological processes, including lipid metabolism. The cystine transporter gene SLC7A11 was a key AR target, and full-length AR (AR-FL) transactivated SLC7A11 transcription by directly occupying the SLC7A11 promoter and putative enhancer regions. AR variants (AR-V) preferentially bound the SLC7A11 enhancer and upregulated SLC7A11 expression, thereby conferring resistance to ferroptosis induced by ENZ treatment. However, this effect was abolished following downregulation of AR-Vs using the dual CBP/p300 and BET inhibitor NEO2734. These findings reveal ferroptosis induction as an anticancer mechanism of antiandrogens and SLC7A11 as a direct target gene of AR-FL and AR-Vs. AR-V-mediated SLC7A11 expression represents a mechanism coupling ferroptosis resistance to prostate cancer progression.

Significance: Upregulation of SLC7A11 can be induced by androgen receptor variants to inhibit antiandrogen-induced prostate cancer cell ferroptosis and to drive castration resistance in prostate cancer.

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Figures

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 μmol/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.
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 μmol/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.
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 μmol/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 = 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 = 20) and metastatic CRPC samples (n = 20).
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 μmol/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 = 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 = 20) and metastatic CRPC samples (n = 20).
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.
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.
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.
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.
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 μmol/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 μm; 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.
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 μmol/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 μm; 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.
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).
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).
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 = 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.
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 = 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.
Figure 8. Hypothetical working model. AR-FL transactivates SLC7A11 gene transcription by directly occupying at the promoter and enhancer regions of SLC7A11 gene. Antiandrogen 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 expression, 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.
Figure 8.
Hypothetical working model. AR-FL transactivates SLC7A11 gene transcription by directly occupying at the promoter and enhancer regions of SLC7A11 gene. Antiandrogen 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 expression, 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.
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