PDHA1–acetylation signaling suppresses cuproptosis to attenuate anti-androgen effect in prostate cancer
PDHA1 乙酰化信号抑制铜死亡以减弱前列腺癌的抗雄激素作用

Ruilin Zhuang^{1, 2#}, Qianghua Zhou^3#, Bisheng Cheng^4#, Shirong Peng^{1, 2#}, Zhi Xiong^1，2, Zhaoxiang Xie^{1, 2}, Tong Su^{1, 2}, Weilong Lin^{1, 2}, Zean Li^{1, 2}, Zhiming Wu², Hai Huang^{1, 2, 5, 6}*, Kaiwen Li^{1, 2, 5}*
庄瑞林 ^{1, 2 #} ，周强华 ^{3 #} ，程碧生 ^{4 #} ，彭世荣 ^{1, 2 #} ，熊智 ^1，2 ，谢兆祥 ^{1, 2} ，苏通 ^{1, 2} ，林伟龙 ^{1, 2} ，李哲安 ^{1, 2} ，吴志明 ² ，黄海 ^{1, 2, 5 , 6} * ，李凯文 ^{1, 2, 5} *

¹Department of Urology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China.
¹ 儿科，中山大学孙逸仙纪念医院，中国广州 510120。

²Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.
² 广东省恶性肿瘤表观遗传与基因调控重点实验室，中山大学孙逸仙纪念医院，中国广州 510120。

³State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center, Guangzhou 510060, P. R. China
³ 中山大学肿瘤防治中心，广东省肿瘤临床研究中心，广东省人民医院泌尿外科，广东省人民医院泌尿外科，510060，中国

⁴Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
⁴ 中山大学南方医院泌尿外科，中国广州。

⁵Guangdong Provincial Clinical Research Center for Urological Diseases, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.
⁵ 广东省泌尿疾病临床研究中心，中山大学中山纪念医院，中山大学，510120，中国。

⁶Department of Urology, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, Qingyuan, 511518, Guangdong, China.
⁶ 广州医科大学附属第六医院泌尿外科，清远市人民医院，清远，511518，广东，中国。

# These authors contributed equally to this work.
# 这些作者对这项工作做出了同等贡献。

*Correspondence to Hai Huang (huangh9@mail.sysu.edu.cn) and Kaiwen Li (likw6@mail.sysu.edu.cn).
*通讯作者：黄海 (huangh9@mail.sysu.edu.cn) 和 李凯文 (likw6@mail.sysu.edu.cn)。

Abstract
摘要

Acquired resistance to enzalutamide (Enz) presents a significant challenge in castration-resistant prostate cancer (CRPC), and overcoming this resistance remains an unmet clinical need. Here, we identified cuproptosis, a copper-dependent mechanism of regulated cell death, as a key driver of Enz resistance. Both in vitro and in vivo models demonstrated that PDHA1 serves as a critical modulator of cuproptosis and Enz sensitivity. Mechanistically, PDHA1 increases intracellular acetyl-CoA, enhancing histone H3K27 acetylation and upregulating SLC7A11, which promotes cysteine uptake and glutathione (GSH) synthesis. Elevated GSH chelates intracellular copper, thereby suppressing cuproptosis and reducing Enz efficacy. Targeting PDHA1 significantly restores cuproptosis and sensitizes CRPC cells to Enz treatment. These findings underscore the potential of PDHA1 inhibition to counteract Enz resistance by reactivating cuproptosis, offering a promising therapeutic approach for treating refractory prostate cancer.
恩鲁达坦 (Enz) 获得性耐药是去势抵抗性前列腺癌 (CRPC) 的一个重要挑战，克服这种耐药性仍然是一个未满足的临床需求。在这里，我们确定了铜死亡，一种依赖铜的调节性细胞死亡机制，是恩鲁达坦耐药性的关键驱动因素。体外和体内模型均表明，PDHA1 作为铜死亡和恩鲁达坦敏感性的关键调节因子。从机制上讲，PDHA1 增加细胞内乙酰辅酶 A，增强组蛋白 H3K27 乙酰化，并上调 SLC7A11，从而促进半胱氨酸摄取和谷胱甘肽 (GSH) 合成。升高的 GSH 结合细胞内铜，从而抑制铜死亡并降低恩鲁达坦的疗效。靶向 PDHA1 显著恢复铜死亡，并使 CRPC 细胞对恩鲁达坦治疗敏感。这些发现强调了 PDHA1 抑制通过重新激活铜死亡来对抗恩鲁达坦耐药性的潜力，为治疗难治性前列腺癌提供了一种有前景的治疗方法。

Keywords：prostate cancer，enzalutamide，cuproptosis，epigenetic modification，GSH，SLC7A11
关键词：前列腺癌，恩鲁替尼，铜死亡，表观遗传修饰，GSH，SLC7A11

Graphical abstract
图示

Introduction
引言

Castration-resistant prostate cancer (CRPC) represents an advanced and aggressive stage of the disease, characterized by continued tumor growth despite androgen deprivation therapy (ADT), which targets testosterone production^1,2. The emergence of resistance to hormonal therapies and the associated poor prognosis underscore the urgent need for innovative therapeutic approaches. Enzalutamide (Enz), a second-generation androgen receptor inhibitor, initially provides clinical benefits by blocking androgen receptor signaling in CRPC patients³. However, resistance inevitably arises, driven by mechanisms such as androgen receptor mutations, alternative splicing, and activation of compensatory signaling pathways^4-6. As a result, current research is focused on uncovering the molecular mechanisms underlying this resistance and developing innovative strategies to overcome drug resistance in CRPC.
去势抵抗性前列腺癌（CRPC）是疾病的一种晚期和侵袭性阶段，其特征是在雄激素剥夺治疗（ADT）靶向睾酮产生后肿瘤持续生长 ^1,2 。对激素治疗的耐药性出现以及相关的预后不良突出了对创新治疗方法的迫切需求。恩鲁替尼（Enz）是一种第二代雄激素受体抑制剂，通过阻断 CRPC 患者中的雄激素受体信号传导最初提供临床获益 ³ 。然而，耐药性不可避免地出现，其机制包括雄激素受体突变、替代剪接和补偿性信号通路的激活 ^4-6 。因此，当前研究集中于揭示这种耐药性的分子机制，并开发克服 CRPC 耐药性的创新策略。

Cuproptosis, a recently identified copper-dependent form of regulated cell death (RCD), is marked by mitochondrial dysfunction and protein aggregation triggered by elevated intracellular copper levels⁷. This distinct pathway presents a promising therapeutic target in tumor with disrupted copper metabolism, including CRPC. In CRPC, elevated copper levels and androgen-mediated copper uptake have been observed, linking copper dysregulation to disease progression and resistance to treatments^8-10. Copper metabolism not only promotes tumor growth but also contributes to therapeutic failure^11-13. As Enz resistance is often driven by alterations in androgen receptor pathways, targeting cuproptosis offers a novel strategy to bypass these mechanisms³. Preclinical studies suggest that combining copper ionophores with Enz may selectively induce cell death in CRPC cells by exploiting copper accumulation, providing a new therapeutic approach for overcoming drug resistance in advanced PCa¹⁴.
铜死亡（Cuproptosis）是一种新发现的依赖铜的调节性细胞死亡（RCD）形式，其特征是线粒体功能障碍和蛋白质聚集，由细胞内铜水平升高触发。这一独特通路在铜代谢紊乱的肿瘤（包括去势抵抗性前列腺癌 CRPC）中呈现为有前景的治疗靶点。在 CRPC 中，观察到铜水平升高和雄激素介导的铜摄取，将铜失调与疾病进展和治疗耐药性联系起来。铜代谢不仅促进肿瘤生长，还导致治疗失败。由于 Enz 耐药性通常由雄激素受体通路改变驱动，靶向铜死亡为绕过这些机制提供了一种新策略。临床前研究表明，将铜离子载体与 Enz 结合可通过利用铜积累选择性地诱导 CRPC 细胞死亡，为克服晚期 PCa 耐药性提供了一种新的治疗途径。

Recent advances have identified PDHA1 as a critical regulator of cuproptosis through genome-wide CRISPR/Cas9 screening⁷. PDHA1 encodes the E1 α1 subunit of the pyruvate dehydrogenase complex (PDH), a key enzyme in the tricarboxylic acid (TCA) cycle responsible for converting pyruvate into acetyl-CoA¹⁵. Although the precise role of PDHA1 in cuproptosis remains incompletely understood, evidence suggests its involvement in epigenetic regulation through modulation of intracellular acetyl-CoA levels¹⁵. Our independent findings demonstrates that PDHA1 enhances histone H3K27 acetylation, facilitating the transcriptional upregulation of SLC7A11, which promotes cysteine uptake and glutathione (GSH) synthesis. This increase in GSH chelates intracellular copper ions, thereby inhibiting cuproptosis and diminishing cellular sensitivity to Enz. These findings establish a mechanistic connection between PDHA1, epigenetic regulation, and cuproptosis, suggesting that targeting PDHA1 could present a novel therapeutic strategy to overcome drug resistance by reactivating cuproptosis in CRPC. Through elucidating the molecular mechanisms involved and demonstrating efficacy in preclinical models, this study seeks to establish a novel therapeutic avenue for targeting CRPC, with potential applicability in overcoming Enz resistance.
近期研究进展已通过全基因组 CRISPR/Cas9 筛选鉴定出 PDHA1 是铜死亡的关键调控因子 ⁷ 。PDHA1 编码丙酮酸脱氢酶复合体（PDH）的 E1 α1 亚基，该复合体是三羧酸循环（TCA）中的关键酶，负责将丙酮酸转化为乙酰辅酶 A ¹⁵ 。尽管 PDHA1 在铜死亡中的确切作用尚未完全明了，但证据表明其通过调节细胞内乙酰辅酶 A 水平参与表观遗传调控 ¹⁵ 。我们的独立研究发现，PDHA1 增强组蛋白 H3K27 乙酰化，促进 SLC7A11 的转录上调，从而促进半胱氨酸摄取和谷胱甘肽（GSH）合成。GSH 的增加螯合细胞内铜离子，从而抑制铜死亡并降低细胞对 Enz 的敏感性。这些发现建立了 PDHA1、表观遗传调控和铜死亡之间的机制联系，表明靶向 PDHA1 可能通过重新激活铜死亡为克服 CRPC 的药物耐药性提供一种新的治疗策略。 通过阐明所涉及的分子机制并在临床前模型中展示其疗效，本研究旨在为靶向去势抵抗性前列腺癌（CRPC）建立一条新的治疗途径，并具有克服 Enz 耐药性的潜在应用价值。

Results
结果

Enz Induces Cuproptosis in PCa cells.
Enz 在前列腺癌细胞中诱导铜死亡。

Enz has been shown to induce ferroptosis in PCa cells via glutathione (GSH) depletion¹⁶. To investigate if Enz also triggers cuproptosis, PCa cells were pre-treated with regulated cell death (RCD) inhibitors, including ferroptosis (Fer-1), apoptosis (Z-VAD), and cuproptosis (TTM). Among these, TTM provided the most significant rescue of Enz-induced cell death, indicating cuproptosis as a primary mechanism (Fig. 1A). Enz treatment resulted in a dose-dependent increase in intracellular copper (II) levels, which was effectively blocked by TTM (Fig. 1B, Supplementary Fig. S1A-B). Using the copper (I) ion probe, Coppersensor-1 (CS1), we confirmed a significant rise in copper (I) levels following Enz treatment, which was reversed by TTM (Fig. 1C). These results underscore the role of copper accumulation in Enz-induced cytotoxicity.
研究表明，Enz 通过谷胱甘肽（GSH）耗竭在前列腺癌细胞中诱导铁死亡 ¹⁶ 。为探究 Enz 是否也触发铜死亡，前列腺癌细胞预先用调节性细胞死亡（RCD）抑制剂处理，包括铁死亡（Fer-1）、凋亡（Z-VAD）和铜死亡（TTM）。其中，TTM 提供了对 Enz 诱导细胞死亡的最为显著的挽救作用，表明铜死亡是主要机制（图 1A）。Enz 处理导致细胞内铜（II）水平呈剂量依赖性增加，这被 TTM 有效阻断（图 1B，补充图 S1A-B）。使用铜（I）离子探针 Coppersensor-1（CS1），我们确认 Enz 处理后铜（I）水平显著升高，这被 TTM 逆转（图 1C）。这些结果表明铜积累在 Enz 诱导的细胞毒性中起重要作用。

Cuproptosis is characterized by DLAT oligomerization, which was markedly elevated by Enz but abolished with TTM co-treatment (Fig. 1D, Supplementary Fig. S1D). Additionally, Enz triggered the degradation of Fe-S cluster proteins and induced protein stress, both of which were alleviated by TTM (Fig. 1E). These findings suggest that copper-driven protein dysfunction is central to Enz-induced cell death. Immunohistochemical analysis of PCa tissues revealed reduced LIAS expression in patients treated with androgen receptor antagonists compared to untreated controls, implicating cuproptosis in clinical settings (Fig. 1F). Transmission electron microscopy (TEM) further confirmed Enz-induced mitochondrial damage, including disorganized cristae and mitochondrial loss, which were reversed by TTM (Fig. 1G). These data establish that Enz induces cuproptosis in PCa cells via copper accumulation, mitochondrial dysfunction, and protein aggregation. Targeting cuproptosis may enhance therapeutic efficacy in Enz-resistant PCa.
铜死亡的特征是 DLAT 寡聚化，Enz 显著提高了这种寡聚化，但 TTM 共处理则消除了这种现象（图 1D，补充图 S1D）。此外，Enz 触发了 Fe-S 簇蛋白的降解并诱导了蛋白质应激，而 TTM 则缓解了这些问题（图 1E）。这些发现表明，铜驱动的蛋白质功能障碍是 Enz 诱导细胞死亡的关键。对 PCa 组织的免疫组化分析显示，与未治疗的对照组相比，接受雄激素受体拮抗剂治疗的患者的 LIAS 表达减少，这表明铜死亡在临床环境中具有重要意义（图 1F）。透射电子显微镜（TEM）进一步证实了 Enz 诱导的线粒体损伤，包括嵴结构紊乱和线粒体丢失，而 TTM 则逆转了这些损伤（图 1G）。这些数据表明，Enz 通过铜积累、线粒体功能障碍和蛋白质聚集在 PCa 细胞中诱导铜死亡。靶向铜死亡可能增强对 Enz 耐药 PCa 的治疗效果。

Elevated PDHA1 Expression Correlates with Enz Resistance and Poor Prognosis in PCa.
PDHA1 表达升高与 PCa 的 Enz 耐药性和不良预后相关。

To identify factors contributing to Enz resistance, we cross-referenced three GEO datasets of Enz-resistant PCa with cuproptosis-related genes from CRISPR screening. PDHA1 emerged as the only gene consistently upregulated across these datasets (Fig. 2A-B). Immunohistochemical analysis of PCa tissues confirmed significantly higher PDHA1 expression in Enz-resistant patients compared to Enz-sensitive ones (Fig. 2C). Clinical relevance of PDHA1 was further supported by The Cancer Genome Atlas (TCGA) data, which showed that elevated PDHA1 levels were associated with decreased overall survival (OS) and disease-free survival (DFS) (Fig. 2D). Tissue microarray (TMA) analysis demonstrated that PDHA1 was significantly overexpressed in cancerous tissues compared to adjacent normal tissues, with higher PDHA1 expression correlating with worse overall survival (OS), disease-free survival (DFS), and advanced Gleason scores (Fig. 2E-H). At the cellular level, PDHA1 expression was markedly elevated in PCa cell lines relative to normal prostate cells, observed at both the mRNA and protein levels (Fig. 2I-J). Furthermore, Enz treatment dose-dependently increased PDHA1 expression, linking it directly to Enz resistance (Fig. 2K-M). In summary, elevated PDHA1 expression is strongly correlated with Enz resistance, adverse prognosis, and disease progression in PCa, establishing it as a potential therapeutic target.
为确定导致 Enz 耐药的因素，我们将三个 GEO 数据库中 Enz 耐药性前列腺癌（PCa）与铜死亡相关基因的 CRISPR 筛选结果进行交叉比对。PDHA1 是这些数据库中唯一持续上调的基因（图 2A-B）。对 PCa 组织的免疫组化分析证实，Enz 耐药患者的 PDHA1 表达显著高于 Enz 敏感患者（图 2C）。癌症基因组图谱（TCGA）数据进一步支持了 PDHA1 的临床相关性，显示 PDHA1 水平升高与总生存期（OS）和疾病无进展生存期（DFS）降低相关（图 2D）。组织微阵列（TMA）分析表明，与邻近正常组织相比，癌组织中 PDHA1 显著过表达，PDHA1 表达水平越高，总生存期（OS）、疾病无进展生存期（DFS）越差，且 Gleason 评分越高（图 2E-H）。在细胞水平上，与正常前列腺细胞相比，PCa 细胞系的 PDHA1 表达显著升高，这在 mRNA 和蛋白水平均可观察到（图 2I-J）。 此外，Enz 治疗剂量依赖性地增加了 PDHA1 表达，将其直接与 Enz 耐药性相关（图 2K-M）。总之，PDHA1 表达升高与 PCa 中的 Enz 耐药性、不良预后和疾病进展密切相关，确立其作为潜在治疗靶点的地位。

PDHA1 as a Key Regulator of Enz-Induced Cuproptosis in PCa.
PDHA1 作为 PCa 中 Enz 诱导的铜死亡的关键调节因子。

To elucidate the functional role of PDHA1 in Enz-induced cuproptosis, stable PDHA1 knockdown and overexpression models were developed in PCa cell lines (Supplementary Fig. S2A-D). Knockdown of PDHA1 significantly enhanced the cytotoxicity induced by Enz, whereas overexpression of PDHA1 markedly attenuated this effect (Fig. 3A-B). These results indicate that PDHA1 is a pivotal mediator of Enz resistance, conferring a protective effect on PCa cells. In parallel experiments, PDHA1 knockdown markedly enhanced the sensitivity of PCa cells to Elesclomol (ES), a potent inducer of cuproptosis, exerting a more pronounced cytotoxic effect than Enz. Conversely, PDHA1 overexpression diminished the cytotoxicity induced by ES (Fig. 3C-D), further supporting the notion that PDHA1 plays a pivotal role in regulating cuproptosis pathways in PCa.
为阐明 PDHA1 在 Enz 诱导的铜死亡中的作用，在 PCa 细胞系中建立了稳定的 PDHA1 敲低和过表达模型（补充图 S2A-D）。PDHA1 敲低显著增强了 Enz 诱导的细胞毒性，而 PDHA1 过表达则明显减弱了这种效应（图 3A-B）。这些结果表明 PDHA1 是 Enz 耐药性的关键介质，对 PCa 细胞具有保护作用。在平行实验中，PDHA1 敲低显著增强了 PCa 细胞对 Elesclomol（ES，一种强效铜死亡诱导剂）的敏感性，其产生的细胞毒性效应强于 Enz。相反，PDHA1 过表达减弱了 ES 诱导的细胞毒性（图 3C-D），进一步支持 PDHA1 在调节 PCa 铜死亡通路中发挥关键作用的观点。

PI staining confirmed that knockdown of PDHA1 led to a significant increase in cell death in response to Enz and ES treatments (Supplementary Fig. S2E-F). Additionally, colony formation assays showed that PDHA1 depletion greatly suppressed cell proliferation and enhanced the cytotoxic effects of Enz. (Supplementary Fig. S2G). Further mechanistic insights were provided by the observation that pretreatment with the copper chelator TTM reversed the enhanced cytotoxicity observed in PDHA1-knockdown cells exposed to Enz, effectively restoring cell viability (Fig. 3E), with similar effects noted in ES-treated cells (Fig. 3F). PDHA1 knockdown also induced a marked increase in intracellular copper (II) levels, degradation of Fe-S cluster proteins, and protein stress—all hallmarks of cuproptosis (Supplementary Fig. S2H-I). Notably, PDHA1 overexpression suppressed these cuproptosis markers (Fig. 3G-J), underscoring the regulatory role of PDHA1 in modulating copper-mediated cell death and resistance to Enz in PCa.
PI 染色证实 PDHA1 敲低导致细胞在 Enz 和 ES 处理下死亡显著增加（补充图 S2E-F）。此外，集落形成实验表明 PDHA1 耗竭显著抑制细胞增殖并增强 Enz 的细胞毒性（补充图 S2G）。进一步机制研究表明，铜螯合剂 TTM 预处理逆转了 PDHA1 敲低细胞在 Enz 暴露下增强的细胞毒性，有效恢复细胞活力（图 3E），在 ES 处理细胞中也观察到类似效果（图 3F）。PDHA1 敲低还诱导了细胞内铜（II）水平显著增加、Fe-S 簇蛋白降解和蛋白应激——这些都是铜死亡的特征标志（补充图 S2H-I）。值得注意的是，PDHA1 过表达抑制了这些铜死亡标志物（图 3G-J），突显了 PDHA1 在调节铜介导的细胞死亡和 PCa 对 Enz 的耐药性中的调控作用。

In Vivo Targeting of PDHA1 Enhances the Antitumor Effects of Enz in PCa.
体内 PDHA1 靶向增强 Enz 在 PCa 的抗肿瘤作用。

To investigate the impact of PDHA1 knockdown on Enz efficacy in vivo, 22Rv1 PCa cells with stable PDHA1 knockdown (shPDHA1) or control (shNC) were implanted into nude mice. Mice received Enz treatment at a dosage of 20 mg/kg via intraperitoneal injection every three days. PDHA1 knockdown significantly suppressed tumor growth and enhanced the therapeutic effects of Enz, as evidenced by decreased tumor volume and weight (Fig. 4A-D). Histological analyses indicated that Enz treatment alone resulted in partial tumor cell death, whereas the combination of PDHA1 knockdown and Enz induced extensive tumor cell mortality (Fig. 4E). Immunohistochemical staining confirmed a marked reduction in PDHA1 expression in the shPDHA1 group, with partial restoration upon Enz treatment (Fig. 4F). Additionally, Ki67 staining indicated significantly reduced tumor cell proliferation in the combined treatment group compared to controls (Fig. 4G).
为探究 PDHA1 敲低对 Enz 在体内的疗效影响，将稳定敲低 PDHA1（shPDHA1）或对照（shNC）的 22Rv1 前列腺癌细胞植入裸鼠体内。小鼠通过腹腔注射每三天给予 20 mg/kg 剂量的 Enz 治疗。PDHA1 敲低显著抑制肿瘤生长并增强 Enz 的治疗效果，表现为肿瘤体积和重量减少（图 4A-D）。组织学分析显示，单独 Enz 治疗导致部分肿瘤细胞死亡，而 PDHA1 敲低与 Enz 联合治疗则诱导广泛的肿瘤细胞死亡（图 4E）。免疫组化染色证实 shPDHA1 组 PDHA1 表达显著降低，Enz 治疗后部分恢复（图 4F）。此外，Ki67 染色显示联合治疗组肿瘤细胞增殖显著低于对照组（图 4G）。

Furthermore, both PDHA1 knockdown and Enz treatment led to a notable downregulation of LIAS expression, a marker associated with cuproptosis. This suggests increased copper-dependent cell death in tumors subjected to the combination of PDHA1 knockdown and Enz (Fig. 4H). In summary, targeting PDHA1 in vivo enhances the antitumor activity of Enz, leading to increased tumor cell death and reduced proliferation. These findings support PDHA1 as a promising therapeutic target to enhance Enz efficacy and overcome drug resistance in PCa.
此外，PDHA1 敲低和 Enz 处理均导致与铜死亡相关的 LIAS 表达显著下调。这表明在 PDHA1 敲低和 Enz 联合处理的肿瘤中铜依赖性细胞死亡增加（图 4H）。总之，体内靶向 PDHA1 增强了 Enz 的抗肿瘤活性，导致肿瘤细胞死亡增加和增殖减少。这些发现支持 PDHA1 作为增强 Enz 疗效和克服 PCa 耐药性的有前景的治疗靶点。

Mechanistic Elucidation of PDHA1-Regulated Cuproptosis through Metabolic Pathways and SLC7A11-Mediated GSH Synthesis
通过代谢途径和 SLC7A11 介导的 GSH 合成阐明 PDHA1 调控的铜死亡机制

To explore the molecular mechanisms underlying PDHA1's role in regulating cuproptosis, untargeted metabolomic analysis was conducted on PDHA1-knockdown PCa cells. Principal component analysis (PCA) revealed significant metabolic reprogramming, with 278 and 146 differentially expressed metabolites identified in C4-2 and 22Rv1 PCa cell lines, respectively (Supplementary Fig. S3A-B). Enrichment analysis indicated dysregulation in key metabolic pathways, particularly those related to glutamate, cysteine, and glutathione (GSH) (Supplementary Fig. S3C). Downregulation of intermediates in these pathways (Fig. 5A) suggests a diminished capacity for GSH synthesis, which is crucial for chelating intracellular copper ions—a key player in cuproptosis induction¹⁷. Consistently, PDHA1 knockdown resulted in a significant decrease in intracellular GSH levels, as confirmed by flow cytometry analysis (Fig. 5C-D). This decrease correlated with a marked decline in cysteine, the rate-limiting precursor for GSH synthesis (Fig. 5B), whereas PDHA1 overexpression elevated both cysteine and GSH levels (Supplementary Fig. S3D-F)¹⁸. Addition of exogenous GSH significantly reversed the increase in intracellular copper (II) ions observed in PDHA1-knockdown cells (Fig. 5E), along with a reduction in the degradation of Fe-S cluster proteins and protein stress associated with cuproptosis (Fig. 5F).
为探究 PDHA1 在调控铜死亡中的作用机制，对 PDHA1 敲降的 PCa 细胞进行了非靶向代谢组学分析。主成分分析（PCA）揭示出显著的代谢重编程，在 C4-2 和 22Rv1 PCa 细胞系中分别鉴定出 278 和 146 种差异表达代谢物（补充图 S3A-B）。富集分析表明关键代谢通路失调，特别是与谷氨酸、半胱氨酸和谷胱甘肽（GSH）相关的通路（补充图 S3C）。这些通路中间体的下调（图 5A）提示 GSH 合成能力减弱，而 GSH 对于螯合细胞内铜离子——铜死亡诱导的关键因素至关重要 ¹⁷ 。一致地，PDHA1 敲降导致细胞内 GSH 水平显著下降，流式细胞术分析证实了这一结果（图 5C-D）。这种下降与半胱氨酸——GSH 合成限速前体的显著减少相关（图 5B），而 PDHA1 过表达则提高了半胱氨酸和 GSH 水平（补充图 S3D-F） ¹⁸ 。 外源性 GSH 的添加显著逆转了 PDHA1 敲低细胞中观察到的细胞内铜(II)离子增加（图 5E），同时降低了 Fe-S 簇蛋白的降解和与铜死亡相关的蛋白质应激（图 5F）。

Further investigation into cysteine transport revealed a significant downregulation of several cysteine transporters in PDHA1-knockdown cells, with SLC7A11 showing the most pronounced decrease (Fig. 5G-H). Overexpression of PDHA1 restored SLC7A11 expression, suggesting that SLC7A11 plays a crucial role in mediating PDHA1-regulated cuproptosis resistance (Supplementary Fig. S3G-H). Moreover, overexpression of SLC7A11 in PCa cells rescued the alterations in cuproptosis-associated proteins induced by Enz treatment (Fig. 5I), and exogenous GSH supplementation attenuated the cytotoxic effects of Enz (Fig. 5J). In summary, PDHA1 regulates cuproptosis by modulating the SLC7A11-GSH metabolic axis. It enhances GSH synthesis, which chelates copper (II) ions, thereby mitigating copper-induced protein stress and preventing cuproptosis. These findings position PDHA1 as a critical regulator of copper metabolism and cuproptosis in PCa.
对半胱氨酸转运的进一步研究揭示了 PDHA1 敲低细胞中多个半胱氨酸转运蛋白显著下调，其中 SLC7A11 下降最为明显（图 5G-H）。PDHA1 过表达恢复了 SLC7A11 的表达，表明 SLC7A11 在介导 PDHA1 调控的铜死亡抗性中起着关键作用（补充图 S3G-H）。此外，在 PCa 细胞中过表达 SLC7A11 挽救了 Enz 处理诱导的铜死亡相关蛋白的改变（图 5I），而外源性 GSH 补充减轻了 Enz 的细胞毒性作用（图 5J）。总之，PDHA1 通过调节 SLC7A11-GSH 代谢轴调控铜死亡。它增强 GSH 的合成，GSH 螯合铜(II)离子，从而减轻铜诱导的蛋白质应激并预防铜死亡。这些发现将 PDHA1 定位为 PCa 中铜代谢和铜死亡的关键调节因子。

PDHA1 Regulates Epigenetic Modifications and SLC7A11 Expression through Acetyl-CoA-Mediated Histone Acetylation in PCa
PDHA1 通过乙酰辅酶 A 介导的组蛋白乙酰化调控 PCa 中的表观遗传修饰和 SLC7A11 表达

PDHA1, an essential subunit of the pyruvate dehydrogenase (PDH) complex, catalyzes the conversion of pyruvate to acetyl-CoA, a pivotal metabolite in epigenetic regulation, particularly in histone acetylation¹⁹. Given the role of acetyl-CoA in modulating histone acetylation, we hypothesized that PDHA1 influences histone acetylation and thereby regulates the expression of genes such as SLC7A11. To evaluate this hypothesis, we initially measured intracellular acetyl-CoA levels in PCa cells. PDHA1 knockdown significantly reduced acetyl-CoA levels (Fig. 6A), whereas PDHA1 overexpression elevated acetyl-CoA levels (Fig. 6B). This confirms that PDHA1 activity directly affects acetyl-CoA production in PCa cells. Since acetyl-CoA is essential for histone acetylation, we next analyzed global histone acetylation²⁰. Western blotting revealed that PDHA1 overexpression significantly increased histone acetylation levels, particularly at H3K27ac sites, a key marker of active transcription (Fig. 6C). Treatment with the histone acetylation inhibitor JQAD1 reversed these effects, reducing H3K27ac levels and downregulating SLC7A11 expression (Fig. 6D), suggesting that histone acetylation is critical for PDHA1-mediated transcriptional regulation of SLC7A11.
PDHA1 是丙酮酸脱氢酶（PDH）复合物的一个必需亚基，催化丙酮酸转化为乙酰辅酶 A，乙酰辅酶 A 是表观遗传调控中的一个关键代谢物，特别是在组蛋白乙酰化中 ¹⁹ 。鉴于乙酰辅酶 A 在调节组蛋白乙酰化中的作用，我们假设 PDHA1 影响组蛋白乙酰化，从而调节 SLC7A11 等基因的表达。为了验证这一假设，我们最初测量了 PCa 细胞内的乙酰辅酶 A 水平。PDHA1 敲低显著降低了乙酰辅酶 A 水平（图 6A），而 PDHA1 过表达则提高了乙酰辅酶 A 水平（图 6B）。这证实了 PDHA1 活性直接影响了 PCa 细胞中乙酰辅酶 A 的产生。由于乙酰辅酶 A 对组蛋白乙酰化至关重要，我们接下来分析了全基因组组蛋白乙酰化 ²⁰ 。Western blotting 显示，PDHA1 过表达显著增加了组蛋白乙酰化水平，特别是在 H3K27ac 位点，这是活性转录的关键标志物（图 6C）。使用组蛋白乙酰化抑制剂 JQAD1 处理逆转了这些效应，降低了 H3K27ac 水平并下调了 SLC7A11 的表达（图。 6D），表明组蛋白乙酰化对于 PDHA1 介导的 SLC7A11 转录调控至关重要。

To further explore the epigenetic role of PDHA1, we performed chromatin immunoprecipitation sequencing (ChIP-seq). PDHA1 overexpression resulted in a notable enrichment of H3K27ac around transcription start sites (TSS), with significant increases in promoter regions linked to genes involved in cellular stress responses (Fig. 6E, Supplementary Fig. S4A-B). Furthermore, the ROSE algorithm identified an increase in super-enhancer formation in PDHA1-overexpressing cells (Fig. 6F), which is known to drive robust gene expression under oncogenic stress. Using the Integrative Genomics Viewer (IGV), we observed a substantial increase in H3K27ac peaks at the SLC7A11 promoter in PDHA1-overexpressing cells (Fig. 6G), linking PDHA1-driven acetylation directly to the transcriptional upregulation of SLC7A11. ChIP-qPCR data further confirmed that PDHA1 enhances H3K27ac levels at the SLC7A11 promoter (Fig. 6H), solidifying the link between PDHA1 and SLC7A11 transcriptional control via histone acetylation. In conclusion, PDHA1 modulates epigenetic modifications by increasing intracellular acetyl-CoA levels, which, in turn, enhances H3K27ac acetylation and promotes the expression of SLC7A11. This regulatory axis supports resistance to cuproptosis and contributes to the progression of PCa, particularly under anti-androgen therapies like Enz.
为进一步探索 PDHA1 的表观遗传作用，我们进行了染色质免疫沉淀测序（ChIP-seq）。PDHA1 过表达导致转录起始位点（TSS）周围的 H3K27ac 显著富集，与参与细胞应激反应的基因启动子区域相关（图 6E，补充图 S4A-B）。此外，ROSE 算法识别出 PDHA1 过表达细胞中超级增强子形成的增加（图 6F），已知其在致癌应激下驱动基因表达的增强。使用整合基因组浏览器（IGV），我们观察到 PDHA1 过表达细胞中 SLC7A11 启动子处的 H3K27ac 峰显著增加（图 6G），将 PDHA1 驱动的乙酰化直接与 SLC7A11 转录上调联系起来。ChIP-qPCR 数据进一步证实 PDHA1 增强 SLC7A11 启动子处的 H3K27ac 水平（图 6H），通过组蛋白乙酰化巩固了 PDHA1 与 SLC7A11 转录调控之间的联系。总之，PDHA1 通过增加细胞内乙酰辅酶 A 水平来调节表观遗传修饰，进而增强 H3K27ac 乙酰化并促进 SLC7A11 的表达。 该调控轴支持抗铜死亡，并促进前列腺癌（PCa）的进展，尤其是在使用恩度等抗雄激素治疗时。

Synergistic Efficacy of CPI 613 Combined with Enz in PCa Treatment
CPI 613 联合恩度治疗前列腺癌的协同疗效

CPI 613, a novel PDHA1 inhibitor, has demonstrated antitumor activity by disrupting mitochondrial metabolism through phosphorylation and inactivation of PDHA1, which plays a key role in PCa cell survival²¹. Given that PDHA1 is implicated in Enz resistance in PCa, we explored the potential therapeutic synergy of combining CPI 613 with Enz. We initially treated PCa cells with CPI 613 and observed a significant increase in intracellular copper (I) and (II) ion levels, which could be mitigated by tetrathiomolybdate (TTM) (Supplementary Fig. S5A-B). The treatment also elevated PDHA1 phosphorylation and induced cytotoxic protein stress, as evidenced by Fe-S protein degradation (Supplementary Fig. S5C). The combination of CPI 613 with Enz significantly reduced PCa cell viability, and combination index (CI) analysis revealed a synergistic interaction between the two drugs (CI < 1) (Fig. 7A-B). Further, in organoid models derived from P10P53 knockout mice, the combination treatment led to extensive cell death and disintegration, with markedly higher propidium iodide (PI) staining compared to monotherapies (Fig. 7C).
CPI 613 是一种新型 PDHA1 抑制剂，通过磷酸化和失活 PDHA1 来破坏线粒体代谢，从而显示出抗肿瘤活性，而 PDHA1 在 PCa 细胞存活中起着关键作用 ²¹ 。鉴于 PDHA1 与 PCa 中的 Enz 耐药性相关，我们探索了将 CPI 613 与 Enz 联合使用的潜在治疗协同作用。我们最初用 CPI 613 处理 PCa 细胞，观察到细胞内铜(I)和(II)离子水平显著增加，这可以通过四硫代钼酸盐(TTM)减轻（补充图 S5A-B）。该处理还提高了 PDHA1 的磷酸化水平，并诱导了细胞毒性蛋白应激，这通过 Fe-S 蛋白降解得到证实（补充图 S5C）。CPI 613 与 Enz 的联合使用显著降低了 PCa 细胞活力，组合指数(CI)分析显示两种药物之间存在协同作用（CI < 1）（图 7A-B）。此外，在源自 P10P53 敲除小鼠的类器官模型中，联合治疗导致了广泛的细胞死亡和崩解，与单药治疗相比，碘化丙啶(PI)染色明显更高（图 7C）。

In vivo experiments using a subcutaneous xenograft mouse model demonstrated that the combination of CPI 613 and Enz produced significantly greater tumor inhibition compared to either agent alone (Fig. 7D-G). Hematoxylin and eosin (H&E) staining revealed larger necrotic areas in tumors treated with the combination, accompanied by a significant reduction in Ki67-positive proliferative cells and downregulation of LIAS, a marker of cuproptosis (Fig. 7H-I). To assess the translational relevance of this combination, patient-derived xenograft (PDX) models from CRPC patients were treated with CPI 613 and Enz. Consistent with earlier findings, the combination therapy outperformed monotherapies, leading to greater tumor growth inhibition, extensive necrosis, and reduced vascularity, as confirmed by H&E and Ki67 staining (Fig. 7J-O). These findings present strong evidence that combining CPI 613 with Enz enhances antitumor efficacy by inducing cuproptosis, offering a promising approach to overcoming Enz resistance in PCa.
体内实验采用皮下异种移植小鼠模型表明，CPI 613 与 Enz 联合使用比单独使用任一药物具有显著更强的肿瘤抑制作用（图 7D-G）。苏木精和伊红（H&E）染色显示，联合治疗组肿瘤的坏死区域更大，Ki67 阳性增殖细胞显著减少，且 LIAS（铜死亡标志物）下调（图 7H-I）。为评估该联合用药的临床转化意义，对来自去势抵抗性前列腺癌（CRPC）患者的异种移植（PDX）模型使用 CPI 613 和 Enz 进行治疗。与早期研究结果一致，联合治疗优于单药治疗，导致更强的肿瘤生长抑制、广泛的坏死和血管密度降低，通过 H&E 和 Ki67 染色得到证实（图 7J-O）。这些研究结果有力证明，将 CPI 613 与 Enz 联合使用可通过诱导铜死亡增强抗肿瘤疗效，为克服前列腺癌（PCa）中 Enz 耐药性提供了一种有前景的策略。

Discussion
讨论

Prostate cancer (PCa), especially in its castration-resistant form CRPC, often develops resistance to treatments like Enz, leading to poor patient outcomes. A key emerging area of research is the role of copper metabolism and its contribution to tumor cell survival. Cuproptosis, a copper-dependent form of regulated cell death, has gained attention as a novel pathway that could be exploited for therapeutic purposes^7,22-24. In several cancers, including renal clear cell carcinoma, cuproptosis inhibition has been linked to chemoresistance, while induction of cuproptosis has been shown to sensitize tumors to treatment^25-27 . Given that copper metabolism is dysregulated in PCa, and elevated copper levels have been associated with tumor progression, targeting cuproptosis presents a promising therapeutic strategy .
前列腺癌（PCa），尤其是去势抵抗型前列腺癌（CRPC），常对依那西普（Enz）等治疗产生耐药性，导致患者预后不良。铜代谢及其对肿瘤细胞存活的作用是当前研究的一个关键领域。铜死亡（Cuproptosis）作为一种依赖铜的调节性细胞死亡形式，已被关注作为一种可能用于治疗的新途径 ^7,22-24 。在多种癌症中，包括肾透明细胞癌，铜死亡抑制与化疗耐药性相关，而铜死亡的诱导则显示出提高肿瘤对治疗的敏感性 ^25-27 。鉴于前列腺癌中铜代谢失调，且铜水平升高与肿瘤进展相关，靶向铜死亡为一种有前景的治疗策略。

Our study builds on recent findings that GSH, a key antioxidant, not only plays a critical role in ferroptosis but also regulates cuproptosis by chelating copper ions and mitigating their cytotoxic effects ^16,28,29. Previous research has shown that depletion of GSH enhances cell sensitivity to various forms of regulated cell death, including ferroptosis, and that the SLC7A11 transporter is a key player in this process by facilitating cysteine uptake for GSH synthesis^30,31​. Our metabolomics data confirm that PDHA1, a regulator of mitochondrial metabolism, influences GSH synthesis by modulating SLC7A11 expression. The upregulation of SLC7A11 in PCa cells with high PDHA1 expression highlights a previously unrecognized link between PDHA1 and the modulation of GSH levels, which in turn affects copper-induced cell death .
我们的研究基于近期发现，谷胱甘肽（GSH）作为一种关键的抗氧化剂，不仅对铁死亡起着至关重要的作用，还通过螯合铜离子并减轻其细胞毒性效应来调节铜死亡 ^16,28,29 。先前研究表明，GSH 的耗竭增强了细胞对多种调节性细胞死亡的敏感性，包括铁死亡，并且 SLC7A11 转运蛋白通过促进半胱氨酸摄取以合成 GSH，在这一过程中起着关键作用 ^30,31 。我们的代谢组学数据证实，线粒体代谢调节因子 PDHA1 通过调节 SLC7A11 的表达来影响 GSH 的合成。高 PDHA1 表达的 PCa 细胞中 SLC7A11 的上调突显了 PDHA1 与 GSH 水平调节之间先前未被认识到的联系，进而影响铜诱导的细胞死亡。

While GSH has traditionally been associated with protecting cells from oxidative stress, recent studies suggest that it also plays a pivotal role in preventing copper-induced cytotoxicity by chelating excess copper ions³¹ . In our experiments, PDHA1 knockdown reduced intracellular GSH levels and sensitized PCa cells to copper-induced cuproptosis, suggesting that targeting PDHA1 could help overcome Enz resistance. Conversely, overexpression of PDHA1 led to increased GSH synthesis, protecting PCa cells from Enz-induced cuproptosis . This finding is consistent with studies in other cancers where copper metabolism and GSH synthesis were shown to play critical roles in resistance mechanisms .
虽然谷胱甘肽（GSH）传统上被认为可以保护细胞免受氧化应激，但最近的研究表明，它还可以通过螯合过量的铜离子来预防铜诱导的细胞毒性 ³¹ 。在我们的实验中，PDHA1 敲低降低了细胞内 GSH 水平，并使前列腺癌细胞对铜诱导的铜死亡更加敏感，这表明靶向 PDHA1 可以帮助克服 Enz 耐药性。相反，PDHA1 过表达导致 GSH 合成增加，保护前列腺癌细胞免受 Enz 诱导的铜死亡。这一发现与其他癌症研究中铜代谢和 GSH 合成在耐药机制中起关键作用的研究结果一致。

Interestingly, PDHA1’s role extends beyond metabolic regulation. As a critical enzyme in the pyruvate dehydrogenase complex, PDHA1 is also involved in epigenetic modifications. Recent studies have shown that PDHA1 can increase nuclear acetyl-CoA levels, thereby promoting histone acetylation and gene expression¹⁵ . Our study expands on these findings by demonstrating that PDHA1 upregulates histone H3K27 acetylation at the SLC7A11 promoter, enhancing SLC7A11 expression and boosting GSH synthesis. This epigenetic mechanism likely contributes to the observed resistance to Enz in PCa cells, as increased GSH levels help neutralize copper ions, preventing the activation of cuproptosis​.
有趣的是，PDHA1 的作用不仅限于代谢调节。作为丙酮酸脱氢酶复合体中的关键酶，PDHA1 还参与表观遗传修饰。近期研究表明，PDHA1 可以增加细胞核中乙酰辅酶 A 的水平，从而促进组蛋白乙酰化及基因表达 ¹⁵ 。我们的研究在此基础上进一步证实，PDHA1 上调 SLC7A11 启动子处的组蛋白 H3K27 乙酰化，增强 SLC7A11 表达并促进 GSH 合成。这种表观遗传机制可能有助于解释 PCa 细胞对 Enz 的耐药性，因为 GSH 水平升高有助于中和铜离子，从而防止铜依赖性细胞死亡的发生。

Another significant finding from our study is the potential therapeutic synergy between PDHA1 inhibition and Enz treatment. The combination of the PDHA1 inhibitor CPI 613 with Enz significantly enhanced antitumor efficacy in both in vitro and in vivo models²¹. CPI 613 disrupted mitochondrial metabolism, sensitizing PCa cells to Enz-induced copper toxicity, thereby promoting cuproptosis . This combination was more effective than either treatment alone, and our results suggest that CPI 613 may represent a promising addition to the therapeutic arsenal for overcoming Enz resistance in CRPC .
我们研究的另一个重要发现是 PDHA1 抑制和 Enz 治疗之间的潜在治疗协同作用。PDHA1 抑制剂 CPI 613 与 Enz 联合使用，在体外和体内模型中均显著增强了抗肿瘤疗效 ²¹ 。CPI 613 破坏了线粒体代谢，使 PCa 细胞对 Enz 诱导的铜毒性敏感，从而促进铜死亡。这种联合治疗的效果优于单独治疗，我们的结果表明 CPI 613 可能代表一种有前景的治疗手段，用于克服 CRPC 中的 Enz 耐药性。

In conclusion, our study provides strong evidence that PDHA1 plays a dual role in PCa, regulating both metabolism and epigenetics to modulate copper-induced cell death and Enz resistance. By targeting the PDHA1-SLC7A11-GSH axis, we can enhance the efficacy of Enz and potentially overcome therapeutic resistance. The combination of CPI 613 with Enz offers a novel therapeutic approach for CRPC, and future studies should explore its potential in clinical settings .
总之，我们的研究提供了强有力的证据，表明 PDHA1 在 PCa 中发挥双重作用，调节代谢和表观遗传学，以调节铜诱导的细胞死亡和 Enz 耐药性。通过靶向 PDHA1-SLC7A11-GSH 轴，我们可以增强 Enz 的疗效，并有可能克服治疗耐药性。CPI 613 与 Enz 的联合为 CRPC 提供了一种新的治疗策略，未来的研究应探索其在临床环境中的潜力。

Materials and Methods
材料和方法的

Materials
材料

Enzalutamide (MDV3100, catalog no. S1250, Selleck Chemicals), Ferrostatin-1 (Fer-1, catalog no. S7243, Selleck Chemicals), Z-VAD-FMK (ZVAD, catalog no. S7023, Selleck Chemicals), Elesclomol (ES, catalog no. S1052, Selleck Chemicals), CPI 613 (catalog no. A4333, APExBIO), JQAD1 (catalog no. HY-145765, MedChemExpress), and Coppersensor-1 (CS1, catalog no. HY-141511, MedChemExpress) were purchased from the specified suppliers. Tetrathiomolybdate (TTM) (catalog no. 323446, Sigma-Aldrich) and Copper (Cu) (catalog no. 349216, Sigma-Aldrich) were also used in the experiments.
恩唑鲁胺（MDV3100，货号 S1250，Selleck Chemicals）、铁稳态素-1（Fer-1，货号 S7243，Selleck Chemicals）、Z-VAD-FMK（ZVAD，货号 S7023，Selleck Chemicals）、伊莱斯科莫尔（ES，货号 S1052，Selleck Chemicals）、CPI 613（货号 A4333，APExBIO）、JQAD1（货号 HY-145765，MedChemExpress）和铜传感器-1（CS1，货号 HY-141511，MedChemExpress）均从指定供应商处购买。实验中还使用了四硫钼酸钠（TTM）（货号 323446，Sigma-Aldrich）和铜（Cu）（货号 349216，Sigma-Aldrich）。

The following antibodies were used: Lipoic Acid (Abcam, catalog no. ab58724), HSP70 (Proteintech, catalog no. 10995-1-AP), LIAS (Proteintech, catalog no. 11577-1-AP), FDX1 (Proteintech, catalog no. 12592-1-AP), DLAT (Proteintech, catalog no. 13426-1-AP), PDHA1 (Proteintech, catalog no. 18068-1-AP), p-PDHA1 (Proteintech, catalog no. 29582-1-AP), α-Tubulin (Proteintech, catalog no. 11224-1-AP), H3 (Proteintech, catalog no. 17168-1-AP), and H3K27ac (Abclonal, catalog no. A7253).
以下抗体被使用：硫辛酸（Abcam，货号 ab58724）、热休克蛋白 70（HSP70）（Proteintech，货号 10995-1-AP）、乳酸脱氢酶激酶 A（LIAS）（Proteintech，货号 11577-1-AP）、醛缩酶 1（FDX1）（Proteintech，货号 12592-1-AP）、二氢辅酶 A 硫激酶（DLAT）（Proteintech，货号 13426-1-AP）、丙酮酸脱氢酶 E1α（PDHA1）（Proteintech，货号 18068-1-AP）、磷酸化丙酮酸脱氢酶 E1α（p-PDHA1）（Proteintech，货号 29582-1-AP）、α-微管蛋白（α-Tubulin）（Proteintech，货号 11224-1-AP）、组蛋白 H3（H3）（Proteintech，货号 17168-1-AP）和组蛋白 H3K27ac（Abclonal，货号 A7253）。

Cell Culture
细胞培养

Human PCa cell lines, including 22RV1, C4-2, and IE8, were obtained from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (GIBCO, catalog no. C11875500BT), supplemented with 10% fetal bovine serum (FBS) (ExCell Bio, catalog no. FSP500) and 1% penicillin-streptomycin (GIBCO, catalog no. 15140-122). All cell lines were regularly tested for mycoplasma contamination using PCR analysis to ensure a sterile environment.
人前列腺癌细胞系，包括 22RV1、C4-2 和 IE8，均由中国科学院分子细胞科学卓越中心（上海，中国）获取。细胞在 RPMI-1640 培养基（GIBCO，目录号 C11875500BT）中培养，补充 10%胎牛血清（FBS）（ExCell Bio，目录号 FSP500）和 1%青霉素-链霉素（GIBCO，目录号 15140-122）。所有细胞系均定期使用 PCR 分析检测支原体污染，以确保无菌环境。

Xenograft Mouse Model
异种移植小鼠模型

All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (approval no. SYSU-IACUC-2021-000041). Male BALB/c nude mice were purchased from GemPharmatech LLC. For the animal studies involving PDHA1 knockdown and Enz, 3×10⁶ stable shNC or shPDHA1-transfected 22Rv1 cells were subcutaneously injected into the flanks of each mouse. When the average tumor volume reached 50-60 mm³, the mice bearing shPDHA1 or shNC xenografts were randomly divided into the Control group (10% DMSO + 40% PEG300 + 5% Tween80 + 45% ddH2O) and the Enz group (20 mg/kg, DMSO:PEG300=10%:40%:45%). For the animal studies involving CPI 613 and Enz, 3×10^6 wild-type 22Rv1 cells were injected into the flanks of each mouse, and the mice bearing xenografts were randomly divided into four groups: the control group (10% DMSO + 40% PEG300 + 5% Tween80 + 45% ddH2O), the Enz group (20 mg/kg, DMSO:PEG300=10%:40%:45%), the CPI 613 group (20 mg/kg, DMSO:PEG300=10%:40%:45%), and the combination group (Enz and CPI 613 co-treatment). The treatments were administered via intraperitoneal injection every three days for a total of 21 days. On the 22nd day, the mice were euthanized, and tumors were collected. Tumor volume was calculated using the formula: a × b² × 0.5 (“b” is the shortest diameter and “a” is the diameter perpendicular to “b”).
所有动物实验均获得中山大学实验动物管理委员会批准（批准号：SYSU-IACUC-2021-000041）。雄性 BALB/c 无胸腺裸鼠购自 GemPharmatech LLC 公司。在涉及 PDHA1 敲低和 Enz 的动物研究中，将 3×10^6 稳定 shNC 或 shPDHA1 转染的 22Rv1 细胞皮下注射到每只小鼠的腹股沟。当平均肿瘤体积达到 50-60 mm³时，携带 shPDHA1 或 shNC 异种移植物的小鼠随机分为对照组（10% DMSO + 40% PEG300 + 5% Tween80 + 45% ddH2O）和 Enz 组（20 mg/kg，DMSO:PEG300=10%:40%:45%）。在涉及 CPI 613 和 Enz 的动物研究中，将 3×10^6 野生型 22Rv1 细胞注射到每只小鼠的腹股沟，携带异种移植物的小鼠随机分为四组：对照组（10% DMSO + 40% PEG300 + 5% Tween80 + 45% ddH2O）、Enz 组（20 mg/kg，DMSO:PEG300=10%:40%:45%）、CPI 613 组（20 mg/kg，DMSO:PEG300=10%:40%:45%）和联合组（Enz 和 CPI 613 联合治疗）。治疗通过每三天一次的腹腔注射进行，共 21 天。 第 22 天，将小鼠安乐死，并收集肿瘤。使用公式计算肿瘤体积：a × b² × 0.5（"b"为最短直径，"a"为垂直于"b"的直径）。

Cell Viability Assay
细胞活力测定

Cell viability was assessed using the CCK8 assay (APExBIO, K1018) following the manufacturer's instructions. Briefly, 5 × 10³ PCa cells were treated with DMSO (control), or various concentrations of Enz, ES (supplemented with an equal dose of Cu), or CPI 613 for specified durations. One to two hours before the end of the treatment, 10 μL of CCK8 reagent mixed with 90 μL of RPMI 1640 medium was added to each well. Absorbance at 450 nm (OD450) was measured using a microplate reader to determine cell viability.
按照制造商说明使用 CCK8 试剂盒（APExBIO，K1018）评估细胞活力。简而言之，5 × 10⁴ PCa 细胞用 DMSO（对照组）或不同浓度的 Enz、ES（补充等量的 Cu）或 CPI 613 处理指定时间。在治疗结束前 1-2 小时，向每个孔中加入 10 μL CCK8 试剂与 90 μL RPMI 1640 培养基的混合物。使用微孔板读取器测量 450 nm 处的吸光度（OD450）以确定细胞活力。

The combination index (CI) was calculated using CalcuSyn software version 2.0 to analyze the synergistic, additive, or antagonistic effects of drug combinations. A CI<1 indicates synergism, CI=1 indicates additivity, and CI>1 indicates antagonism.
使用 CalcuSyn 软件版本 2.0 计算组合指数（CI），以分析药物组合的协同、相加或拮抗作用。CI<1 表示协同作用，CI=1 表示相加作用，CI>1 表示拮抗作用。

Cell Death Assay
细胞死亡检测

PCa cells were treated with the specified drugs for 24 hours. After washing, 2 μg/ml DAPI (Sevicebio, G1012) and 10 μg/ml PI (Sevicebio, G1021) were added. The cells were incubated in the dark for 10 minutes, and fluorescent images were captured using an inverted fluorescence microscope (Olympus IX71).
PCa 细胞用指定药物处理 24 小时。清洗后，加入 2 μg/ml DAPI（Sevicebio，G1012）和 10 μg/ml PI（Sevicebio，G1021）。细胞在避光条件下孵育 10 分钟，使用倒置荧光显微镜（Olympus IX71）捕获荧光图像。

Colony Formation Assay
集落形成检测

Approximately 1,000 PCa cells were seeded in each well of a six-well plate and cultured for 14 days. Colonies were then fixed with 4% paraformaldehyde and stained with crystal violet for about 20 minutes. Imaging and colony counting were performed using Quantity One 1-D analysis software (Bio-Rad, Hercules, CA, USA).
约 1000 个 PCa 细胞接种在每个六孔板的孔中，培养 14 天。然后使用 4%多聚甲醛固定集落，并用结晶紫染色约 20 分钟。使用 Quantity One 1-D 分析软件（Bio-Rad，Hercules，CA，USA）进行成像和集落计数。

Western Blot

Cell lysates were prepared using RIPA lysis buffer (Beyotime, P0013B) supplemented with 1% protease inhibitor cocktail (Cwbiotech, CW2200S) and 1% phosphatase inhibitor cocktail (Cwbiotech, CW2383S). Protein concentrations were determined using a BCA protein assay kit (Beyotime, P0011). Proteins were mixed with 5× SDS-PAGE protein loading buffer (Beyotime, P0286) and boiled at 95°C for 10 minutes. Boiled protein samples were stored at -80°C until further use. Extracted proteins were separated on 8%-12% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, ISEQ00010). Membranes were blocked with TBST (TBS containing 0.05% Tween-20) containing 5% non-fat milk or 5% BSA and incubated with primary antibodies at 4°C overnight. After washing three times with 1× TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG at room temperature for 1 hour, washed three times with 1× TBST, and visualized using enhanced chemiluminescence (ECL).
使用 RIPA 裂解缓冲液（碧云天，P0013B）制备细胞裂解物，并添加 1%蛋白酶抑制剂混合物（Cwbiotech，CW2200S）和 1%磷酸酶抑制剂混合物（Cwbiotech，CW2383S）。蛋白质浓度通过 BCA 蛋白质测定试剂盒（碧云天，P0011）测定。蛋白质与 5× SDS-PAGE 蛋白上样缓冲液（碧云天，P0286）混合，并在 95°C 煮沸 10 分钟。煮沸后的蛋白质样品储存在-80°C 直至进一步使用。提取的蛋白质在 8%-12% SDS-PAGE 凝胶上进行分离，并转移到 PVDF 膜（Millipore，ISEQ00010）上。膜用含 0.05%吐温-20 的 TBST（TBS）封闭，加入 5%脱脂奶粉或 5% BSA，并在 4°C 孵育过夜。用一抗在 4°C 孵育过夜后，用 1× TBST 洗涤三次，然后在室温下用 HRP 标记的山羊抗兔或抗鼠 IgG 孵育 1 小时，再用 1× TBST 洗涤三次，最后通过增强化学发光（ECL）进行可视化。

RNA Isolation and qRT-PCR
RNA 提取和 qRT-PCR

Total RNA was extracted and purified using the RNA-Quick Purification Kit (ESscience, #RN001) according to the manufacturer’s instructions. RT-PCR was performed using the qPCR SYBR Green Master Mix (Vazyme, Q711) and the cDNA SuperMix Kit (Vazyme, R333). The mRNA expression levels of the genes were measured using an ABI Quanstudio DX real-time PCR system. The detailed list of all primers used in this study is provided in Supplementary Table 1.
总 RNA 的提取和纯化按照 RNA-Quick 纯化试剂盒（ESscience，#RN001）的说明书进行。使用 qPCR SYBR Green Master Mix（Vazyme，Q711）和 cDNA SuperMix 试剂盒（Vazyme，R333）进行 RT-PCR。使用 ABI Quanstudio DX 实时 PCR 系统测量基因的 mRNA 表达水平。本研究中使用的所有引物详细列表见补充表 1。

siRNA Transient Transfection
siRNA 瞬时转染

RNA interference was conducted using specific siRNA and a non-targeting negative control siRNA. The siRNA oligonucleotides were purchased from GenePharma (Suzhou, China). Transient transfection of siRNA into cells was performed using Lipofectamine RNAiMAX (Invitrogen, 13778075). The medium was replaced with fresh culture medium 24 hours post-transfection. The efficiency of gene silencing was evaluated 48 hours post-transfection using RT-PCR. The target sequences of the siRNAs used in the experiments are listed in Supplementary Table 2.
RNA 干扰采用特异性 siRNA 和非靶向阴性对照 siRNA 进行。siRNA 寡核苷酸购自 GenePharma（中国苏州）。使用 Lipofectamine RNAiMAX（Invitrogen，13778075）将 siRNA 瞬时转染到细胞中。转染后 24 小时更换新鲜培养基。转染后 48 小时使用 RT-PCR 评估基因沉默效率。实验中使用的 siRNA 靶序列见补充表 2。

Establishment of PDHA1 Knockdown and Overexpression Cell Lines
PDHA1 敲低和过表达细胞系的建立

To stably knock down PDHA1 expression, shPDHA1 and PDHA1 overexpression vectors were cloned into the pLVX-puro vector (purchased from Youbio). The shRNA target sequences were 5'-GGAUUGCUCUAGCCUGUAATT-3' (#1) and 5'-GCUGCUGCCUAUUGUAGAUTT-3' (#2). Cells were infected with the lentiviral particles for 3 days, followed by selection with puromycin (10 mg/mL) for 14 days to establish stable cell lines.
为了稳定敲低 PDHA1 表达，将 shPDHA1 和 PDHA1 过表达载体克隆到 pLVX-puro 载体（购自 Youbio）中。shRNA 靶向序列为 5'-GGAUUGCUCUAGCCUGUAATT-3' (#1)和 5'-GCUGCUGCCUAUUGUAGAUTT-3' (#2)。细胞用慢病毒颗粒感染 3 天，然后用 puromycin (10 mg/mL) 筛选 14 天以建立稳定细胞系。

Copper Ion Detection
铜离子检测

The intracellular copper ion levels were measured using the Cell Copper (Cu) Colorimetric Assay Kit (Complexing Method) (Elabscience, E-BC-K775-M). Briefly, 2×10^6 cells were treated with the specified drugs for 24 hours, and the copper ion levels were determined according to the manufacturer's instructions. For the detection of copper ion levels in tumor tissues, the Copper (Cu) Colorimetric Assay Kit (Elabscience, E-BC-K300-M) was used. A 10% tissue homogenate was prepared from 0.1g of tumor tissue, and the copper ion levels were measured following the manufacturer’s protocol. To detect intracellular copper ions using fluorescent dye, cells were treated with the specified drugs for 24 hours, followed by incubation with 2 μg/ml DAPI (Sevicebio, G1012) and 5 μM Coppersensor 1 (CS1, MedChemExpress, HY-141511) for 10 minutes in the dark. Images were captured using a Leica SP8 STED 3X confocal microscope.
细胞内铜离子水平采用细胞铜（Cu）比色测定试剂盒（络合法）（Elabscience，E-BC-K775-M）进行检测。简而言之，2×10^6 个细胞用指定药物处理 24 小时，根据制造商说明测定铜离子水平。为检测肿瘤组织中的铜离子水平，使用铜（Cu）比色测定试剂盒（Elabscience，E-BC-K300-M）。取 0.1g 肿瘤组织制备 10%组织匀浆，按照制造商方案测定铜离子水平。为使用荧光染料检测细胞内铜离子，细胞用指定药物处理 24 小时，随后在避光条件下用 2 μg/ml DAPI（Sevicebio，G1012）和 5 μM Coppersensor 1（CS1，MedChemExpress，HY-141511）孵育 10 分钟。使用 Leica SP8 STED 3X 共聚焦显微镜捕获图像。

Cysteine Detection
半胱氨酸检测

The levels of cysteine in cells were assessed using the CheKine™ Micro Cysteine (Cys) Assay Kit (Abbkine, KTB1450). In brief, 2×10^6 cells were treated with the specified drugs for 24 hours, and cysteine levels were measured according to the manufacturer's instructions.
细胞内半胱氨酸水平采用 CheKine™ Micro 半胱氨酸（Cys）检测试剂盒（Abbkine，KTB1450）进行检测。简而言之，2×10^6 个细胞用指定药物处理 24 小时，根据说明书测量半胱氨酸水平。

Glutathione (GSH) Detection
谷胱甘肽（GSH）检测

Intracellular GSH levels were detected using the fluorescent dye Monochlorobimane (MedChemExpress, HY-101899). In summary, cells were treated with the specified drugs for 24 hours, collected, resuspended in PBS, and incubated with 20 μM Monochlorobimane for 2 hours at 25°C in the dark. The cells were then collected, washed, and resuspended, and GSH levels were analyzed using a Beckman flow cytometer and FlowJo v10 software.
细胞内 GSH 水平采用荧光染料单氯生物碱（MedChemExpress，HY-101899）进行检测。总之，细胞用指定药物处理 24 小时，收集，重悬于 PBS 中，在 25°C 避光条件下与 20 μM 单氯生物碱孵育 2 小时。然后收集细胞，洗涤，重悬，并使用贝克曼流式细胞仪和 FlowJo v10 软件分析 GSH 水平。

Untargeted Metabolomics analysis
非靶向代谢组学分析

Untargeted metabolomics profiling was performed by Metabo-Profile (Shanghai, China) following these steps: centrifugation, extraction, evaporation, lyophilization, derivatization, and injection. Separation, measurement, and analysis were conducted using a time-of-flight mass spectrometry system (Pegasus HT, Leco Corp., St. Joseph, MO, USA) coupled with gas chromatography (Agilent 7890B, Santa Clara, CA, USA) and a Gerstel MPS2 MultiPurpose Sampler (Gerstel, Mülheim, Germany). Quality control procedures included the use of test mixtures, retention indices, internal standards, and pooled biological QC samples on a comprehensive metabolomics platform. For metabolite annotation, retention indices and mass spectral data were compared against reference standards in the JiaLib metabolite database (Metabo-Profile, Shanghai, China) using the iMAP software.
靶向代谢组学分析由上海中国的 Metabo-Profile 公司按照以下步骤进行：离心、提取、蒸发、冷冻干燥、衍生化和进样。分离、测量和分析使用飞行时间质谱系统（Pegasus HT，Leco 公司，密苏里州圣约瑟夫，美国）与气相色谱（Agilent 7890B，加利福尼亚州圣克拉拉，美国）和 Gerstel MPS2 多功能采样器（Gerstel，德国米尔海姆）联用。质量控制程序包括使用测试混合物、保留指数、内标和综合代谢组学平台上的混合生物 QC 样本。对于代谢物鉴定，保留指数和质谱数据通过与 JiaLib 代谢物数据库（Metabo-Profile，上海中国）中的参考标准进行比较，使用 iMAP 软件进行。

ChIP Assay and ChIP-sequence Analysis
染色质免疫沉淀（ChIP）实验和 ChIP 测序分析

Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer’s instructions (Thermo Fisher Scientific, USA). Briefly, wild-type and PDHA1-overexpressing cells were crosslinked with 1% formaldehyde and neutralized with glycine. Cells were then lysed, sonicated, and incubated overnight at 4°C with the specified antibodies (anti-H3K27ac and IgG). The chromatin-antibody complexes were subsequently incubated with Protein A/G magnetic beads on ice for 2 hours and collected. The beads were washed three times, and DNA was purified through phenol-chloroform extraction and ethanol precipitation. ChIP-qPCR primers are listed in Supplementary Supplementary Table 3. ChIP libraries were prepared and sequenced using Illumina instruments according to the manufacturer’s instructions.
染色质免疫沉淀（ChIP）实验按照制造商说明书进行（Thermo Fisher Scientific，美国）。简而言之，野生型和 PDHA1 过表达的细胞用 1%甲醛交联，然后用甘氨酸中和。细胞随后被裂解、超声处理，并在 4℃与指定抗体（抗 H3K27ac 和 IgG）孵育过夜。染色质-抗体复合物随后在冰上与蛋白 A/G 磁珠孵育 2 小时并收集。磁珠洗涤三次，通过酚-氯仿萃取和乙醇沉淀纯化 DNA。ChIP-qPCR 引物列于补充补充表 3。ChIP 文库的制备和测序按照制造商说明书使用 Illumina 仪器进行。

Immunohistochemistry (IHC) and Scoring
免疫组化（IHC）和评分

Xenograft tumor samples were fixed in formalin and embedded in paraffin. Four-micron-thick sections were cut from these samples and mounted on slides. Antigen retrieval and immunostaining were performed as previously described³². Staining intensity was categorized as negative (0), weak (1), moderate (2), or strong (3). To evaluate PDHA1 and Ki67 expression, the extent of staining was scored based on the percentage of immunoreactive tumor cells. The H-Score, ranging from 0 to 300, was calculated by multiplying the staining intensity by the corresponding extent of staining.Human PCa tissue microarrays were obtained from Shanghai OUTDO BIOTECH Co., Ltd. Immunohistochemistry and scoring were conducted as described above.
异种移植肿瘤样本用福尔马林固定并包埋于石蜡中。从这些样本中切取 4 微米厚的切片并置于载玻片上。抗原修复和免疫染色按先前描述的方法进行 ³² 。染色强度分为阴性（0）、弱（1）、中（2）或强（3）四级。为评估 PDHA1 和 Ki67 的表达，根据免疫反应性肿瘤细胞的百分比对染色范围进行评分。通过将染色强度乘以相应的染色范围计算 H-Score，H-Score 范围为 0 至 300。人前列腺癌组织微阵列由上海 OUTDO BIOTECH 有限公司提供。免疫组化和评分按上述方法进行。

Data Collection
数据收集

Transcriptomic data and clinical information of 494 PCa patients from The Cancer Genome Atlas (TCGA, PanCancer Atlas, https://www.cbioportal.org/) were collected from the cBioPortal database for survival analysis. Enz resistance datasets (GSE150809, GSE163240, GSE169305) were retrieved from the GEO database to identify genes associated with cuproptosis and Enz resistance. The cuproptosis gene set was based on findings by Tsvetkov et al.⁷ . PCa patient transcriptomic data (GSE3325) were also obtained from the GEO database to validate PDHA1 expression.
TCGA（癌症基因组图谱，PanCancer 图谱，https://www.cbioportal.org/）中 494 例前列腺癌（PCa）患者的转录组数据和临床信息从 cBioPortal 数据库收集，用于生存分析。从 GEO 数据库检索了 Enz 耐药数据集（GSE150809、GSE163240、GSE169305），以识别与铜死亡和 Enz 耐药相关的基因。铜死亡基因集基于 Tsvetkov 等人的研究 ⁷ 。同时从 GEO 数据库获取了 PCa 患者转录组数据（GSE3325），以验证 PDHA1 的表达。

Statistical Methods
统计方法

Two-tailed unpaired Student's t-test and one-way analysis of variance (ANOVA) were used to determine statistical significance. A p-value < 0.05 was considered statistically significant. Statistical analyses were conducted using GraphPad Prism 9.0 software. Quantitative values are presented as mean ± standard deviation. Statistical significance was denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
采用双尾非配对 Student's t 检验和单因素方差分析（ANOVA）确定统计学显著性。p 值<0.05 被认为具有统计学显著性。统计分析使用 GraphPad Prism 9.0 软件进行。定量值以均值±标准差表示。统计学显著性表示如下：*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001。

Acknowledgements
致谢

This study was supported by the National Natural Science Foundation of China (#82173088), the Natural Science Foundation of Guangdong (#2022A1515012383), Fundamental Research Funds for the Central Universities, Sun Yat-sen University (23ptpy168), and Baiqiuen Fund to K.L..
本研究得到了中国国家自然科学基金（#82173088）、广东省自然科学基金（#2022A1515012383）、中央高校基本科研业务费专项基金（中山大学 23ptpy168）以及白求恩基金对 K.L.的支持。

Competing Interests
利益冲突

The authors declare no competing interests.
作者声明无利益冲突。

Author contributions
作者贡献

R.Z., Q.Z., and S.P. conceived and designed the research. R.Z., Q.Z., and S.P. coordinated the acquisition, distribution, and quality assessment of PCa tumor and adjacent tissue samples. B.C., Z.X., and S.Y. directed and performed mass spectrometry data analysis and quality control. Z.X., T.S., W.L., and B.L. analyzed genomic and transcriptomic data. J.X., Y.O., P.C., and Z.W. conducted tissue microarray immunohistochemistry experiments. R.Z., Q.Z., and S.P. performed the cell experiments. K.L. and H.H. interpreted the data within the context of PCa biology and provided guidance. R.Z., Q.Z., and S.P. drafted the manuscript, while K.L. and H.H. revised the manuscript and supervised the study.
R.Z.、Q.Z.和 S.P.构思并设计了研究。R.Z.、Q.Z.和 S.P.协调了 PCa 肿瘤和邻近组织样本的获取、分配和质量评估。B.C.、Z.X.和 S.Y.指导并执行了质谱数据分析和质量控制。Z.X.、T.S.、W.L.和 B.L.分析了基因组和转录组数据。J.X.、Y.O.、P.C.和 Z.W.进行了组织微阵列免疫组化实验。R.Z.、Q.Z.和 S.P.进行了细胞实验。K.L.和 H.H.在 PCa 生物学背景下解释了数据，并提供了指导。R.Z.、Q.Z.和 S.P.起草了稿件，而 K.L.和 H.H.修订了稿件并监督了研究。

Ethics approval and consent to participate
伦理批准和参与同意

All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (SYSU-IACUC-2021-000041) and conducted in accordance with the university's guidelines for Animal Care and Use. The use of human tissues was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital (SYSEC-KY-KS-2020-201).
所有动物实验均获得中山大学动物伦理委员会批准（SYSU-IACUC-2021-000041），并依照大学动物护理和使用指南进行。人体组织的使用获得中山大学纪念医院伦理委员会批准（SYSEC-KY-KS-2020-201）。

Data availability
数据可用性

Sequencing data are available by contacting the corresponding author on reasonable request.
测序数据可在合理请求下通过联系通讯作者获得。

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Fig1. Enz Induces Cuproptosis in PCa cells.
图 1. Enz 诱导 PCa 细胞发生铜死亡。

A. PCa cells were pre-treated overnight with TTM (5 μM), Fer-1 (5 μM), and Z-VAD (10 μM), followed by 48 hours of treatment with Enz (40 μM). Cell viability was then assessed. B. The intracellular copper (II) ion levels were measured after PCa cells were treated with TTM (5 μM) and Enz (20 μM), either alone or in combination. C. After treatment of PCa cells with TTM (5 μM) and Enz (20 μM), either alone or in combination, CS1 (green fluorescence) and DAPI (blue fluorescence) were used to stain copper (I) ions and cell nuclei, respectively (scale bar: 20 μM). D. After treatment of PCa cells with TTM (5 μM) and Enz (20 μM), either alone or in combination, DLAT (red fluorescence) and DAPI (blue fluorescence) were used to stain DLAT and cell nuclei, respectively (scale bar: 20 μM). E. The protein levels of HSP70, LIAS, and FDX1 were evaluated after treating PCa cells with TTM (5 μM) and Enz (20 μM), either alone or in combination. Tubulin was used as a loading control. F. Representative IHC images of LIAS in PCa tissues treated with AR antagonists and untreated PCa tissues (scale bar: 100 μm). G. Transmission electron microscopy (TEM) images were acquired after PCa cells were treated with TTM (5 μM) and Enz (20 μM), either alone or in combination (scale bar: 1 μM).
A. PCa 细胞用 TTM（5 μM）、Fer-1（5 μM）和 Z-VAD（10 μM）预处理过夜，然后用 Enz（40 μM）处理 48 小时。随后评估细胞活力。B. 在单独或联合使用 TTM（5 μM）和 Enz（20 μM）处理 PCa 细胞后，测量细胞内铜（II）离子水平。C. 在单独或联合使用 TTM（5 μM）和 Enz（20 μM）处理 PCa 细胞后，分别用 CS1（绿色荧光）和 DAPI（蓝色荧光）染色铜（I）离子和细胞核（标尺：20 μM）。D. 在单独或联合使用 TTM（5 μM）和 Enz（20 μM）处理 PCa 细胞后，分别用 DLAT（红色荧光）和 DAPI（蓝色荧光）染色 DLAT 和细胞核（标尺：20 μM）。E. 在单独或联合使用 TTM（5 μM）和 Enz（20 μM）处理 PCa 细胞后，评估 HSP70、LIAS 和 FDX1 的蛋白水平。微管蛋白用作加载对照。F. AR 拮抗剂处理和未处理 PCa 组织的 LIAS 代表性免疫组化图像（标尺：100 μm）。G. 透射电子显微镜（TEM）图像在 PCa 细胞用 TTM（5 μM）和 Enz（20 μM）单独或联合处理后被获取（标尺：1 μM）。

Fig2. Elevated PDHA1 Expression Correlates with Enz Resistance and Poor Prognosis in PCa.
图 2. PDHA1 表达升高与 PCa 的 Enz 耐药性和不良预后相关。

A. Venn diagram of copper-induced cell death-related gene sets from Enz-resistant prostate cancer datasets (GSE150809, GSE163240, GSE169305). B. PDHA1 expression levels in Enz-resistant prostate cancer datasets (GSE150809, GSE163240, GSE169305). C. Representative IHC images of PDHA1 in Enz-resistant and Enz-sensitive PCa tissues (scale bar: 100 μm). D. Kaplan-Meier survival curves showing overall survival (OS) and recurrence-free survival (RFS) of prostate cancer patients with high versus low PDHA1 expression in the TCGA cohort. E. Images showing PDHA1 expression levels in a prostate cancer tissue microarray (TMA). F. H-score of PDHA1 expression in cancerous versus adjacent normal tissues from the prostate cancer TMA. G. Kaplan-Meier survival curves showing overall survival (OS) and disease-free survival (DFS) in prostate cancer patients with high versus low PDHA1 expression in the TMA cohort. H. H-score of PDHA1 expression in prostate cancer tissues with different Gleason scores from the TMA. I-J. PDHA1 mRNA (I) and protein (J) levels in normal prostate cells and PCa cell lines. K-M. Changes in PDHA1 mRNA (K-L) and protein (M) levels in PCa cells after treatment with varying concentrations of Enz. (*P < 0.05, **P < 0.01, ***P < 0.001).
A. 来自恩诺沙星耐药性前列腺癌数据集（GSE150809、GSE163240、GSE169305）的铜诱导细胞死亡相关基因集的维恩图。B. 恩诺沙星耐药性前列腺癌数据集（GSE150809、GSE163240、GSE169305）中 PDHA1 的表达水平。C. 恩诺沙星耐药性和恩诺沙星敏感性 PCa 组织中 PDHA1 的代表性免疫组化图像（比例尺：100 μm）。D. TCGA 队列中 PDHA1 高表达与低表达的前列腺癌患者总生存期（OS）和无复发生存期（RFS）的 Kaplan-Meier 生存曲线。E. 显示前列腺癌组织微阵列（TMA）中 PDHA1 表达水平的图像。F. 前列腺癌 TMA 中癌组织与邻近正常组织中 PDHA1 表达的 H 评分。G. TMA 队列中 PDHA1 高表达与低表达的前列腺癌患者总生存期（OS）和无病生存期（DFS）的 Kaplan-Meier 生存曲线。H. TMA 中不同 Gleason 评分的前列腺癌组织中 PDHA1 表达的 H 评分。I-J. 正常前列腺细胞和 PCa 细胞系中 PDHA1 mRNA（I）和蛋白（J）水平。K-M. PDHA1 mRNA（K-L）和蛋白（M）水平在治疗后不同浓度 Enz 处理 PCa 细胞中的变化（*P < 0.05, **P < 0.01, ***P < 0.001）。

Fig3. Targeting PDHA1 Enhances Enz-Induced Cytotoxicity in PCa.
图 3. 靶向 PDHA1 增强 Enz 诱导的 PCa 细胞毒性。

B. CCK8 assay was used to assess cell viability in control and PDHA1 knockdown PCa cells (A), as well as wild-type (WT) and PDHA1 overexpression PCa cells (B) after treatment with Enz at the specified concentrations. C-D. CCK8 assay was used to assess cell viability in control and PDHA1 knockdown PCa cells (C), as well as WT and PDHA1 overexpression PCa cells (D) after treatment with ES at the specified concentrations. E. CCK8 assay was used to assess cell viability in control and PDHA1 knockdown PCa cells after treatment with TTM (5 μM) and Enz (20 μM), either alone or in combination. F. CCK8 assay was used to assess cell viability in control and PDHA1 knockdown PCa cells after treatment with TTM (5 μM) and ES (2 nM), either alone or in combination. H. The intracellular copper (II) ion concentration was measured after PCa cells were treated with TTM (5 μM) and ES (2 nM), either alone or in combination. I-J. Protein levels of HSP70, LIAS, and FDX1 were evaluated in WT and PDHA1 overexpression PCa cells after treatment with Enz (20 μM) (I) or ES (2 nM) (J). Tubulin was used as a loading control.
B. 采用 CCK8 法检测对照组和 PDHA1 敲低 PCa 细胞（A）以及野生型（WT）和 PDHA1 过表达 PCa 细胞（B）在用 Enz 以指定浓度处理后的细胞活力。C-D. 采用 CCK8 法检测对照组和 PDHA1 敲低 PCa 细胞（C）以及 WT 和 PDHA1 过表达 PCa 细胞（D）在用 ES 以指定浓度处理后的细胞活力。E. 采用 CCK8 法检测对照组和 PDHA1 敲低 PCa 细胞在用 TTM（5 μM）和 Enz（20 μM）单独或联合处理后的细胞活力。F. 采用 CCK8 法检测对照组和 PDHA1 敲低 PCa 细胞在用 TTM（5 μM）和 ES（2 nM）单独或联合处理后的细胞活力。H. 测量 PCa 细胞在用 TTM（5 μM）和 ES（2 nM）单独或联合处理后的细胞内铜（II）离子浓度。I-J. 评估 WT 和 PDHA1 过表达 PCa 细胞在用 Enz（20 μM）（I）或 ES（2 nM）（J）处理后 HSP70、LIAS 和 FDX1 蛋白水平。微管蛋白用作加载对照。

Fig4. In Vivo Targeting of PDHA1 Enhances the Antitumor Effects of Enz in PCa.
图 4. 体内靶向 PDHA1 增强 Enz 在 PCa 中的抗肿瘤作用。

A. Schematic illustration of the 22Rv1 xenograft model with control and PDHA1 knockdown groups treated with Enz (10 mg/kg, intraperitoneally, every 3 days), created using Biorender. B. PDHA1 knockdown in nude mice sensitized PCa cells to Enz, as shown by the representative tumor images . C. Tumor growth curves were recorded every three days (mean ± SD, n = 5 per group). D. Tumor tissues were weighed, and data were summarized. E. Representative H&E and IHC staining of indicated proteins in tumor tissues from each group. Scale bar, 100 μm. H-score for PDHA1 (F), Ki67 (G), and LIAS (H) in the specified groups.
A. 使用 Biorender 创建的 22Rv1 异种移植模型示意图，包括对照组和 PDHA1 敲低组，均用 Enz（10 mg/kg，腹腔注射，每 3 天一次）处理。B. PDHA1 敲低使裸鼠中的 PCa 细胞对 Enz 敏感，如图所示为代表性肿瘤图像。C. 每 3 天记录肿瘤生长曲线（均值±标准差，每组 n=5）。D. 称重肿瘤组织，并汇总数据。E. 每组肿瘤组织中指示蛋白的代表性 H&E 和 IHC 染色。标尺，100 μm。F. 指定组中 PDHA1 的 H-score，G. Ki67 的 H-score，H. LIAS 的 H-score。

Heatmap showing Z-score normalized analysis of metabolites, highlighting shared changes in glutamine and cysteine metabolism intermediates. B. Cysteine levels in control and PDHA1 knockdown cells were measured using a cysteine detection kit. C-D. Intracellular GSH levels in control and PDHA1 knockdown cells were analyzed using flow cytometry. E. Intracellular copper (II) ion levels in control and PDHA1 knockdown cells were measured with or without the presence of GSH (10 μM). F. Changes in HSP70, LIAS, and FDX1 protein levels in control and PDHA1 knockdown cells were evaluated with or without GSH (10 μM). Tubulin was used as a loading control. G. qRT-PCR analysis of mRNA expression of cysteine transporters (SLC1A1, SLC1A5, SLC7A5, SLC7A11) in control and PDHA1 knockdown cells. H. Western blot analysis of SLC7A11 protein expression in control and PDHA1 knockdown cells. I. Changes in HSP70, LIAS, and FDX1 protein levels in wild-type (WT) and SLC7A11-overexpressing cells, with or without Enz (20 μM). Tubulin was used as a loading control. J. Cell viability after 48-hour treatment with Enz (40 μM) with or without GSH (10 μM).
热图显示代谢物的 Z 分数标准化分析，突出谷氨酰胺和半胱氨酸代谢中间体的共同变化。B. 使用半胱氨酸检测试剂盒测量对照组和 PDHA1 敲低细胞的半胱氨酸水平。C-D. 使用流式细胞术分析对照组和 PDHA1 敲低细胞的细胞内 GSH 水平。E. 测量对照组和 PDHA1 敲低细胞的细胞内铜(II)离子水平，有或无 GSH (10 μM)的存在。F. 评估对照组和 PDHA1 敲低细胞中 HSP70、LIAS 和 FDX1 蛋白水平的变化，有或无 GSH (10 μM)。微管蛋白用作加载对照。G. 对照组和 PDHA1 敲低细胞中半胱氨酸转运蛋白（SLC1A1、SLC1A5、SLC7A5、SLC7A11）mRNA 表达的 qRT-PCR 分析。H. 对照组和 PDHA1 敲低细胞中 SLC7A11 蛋白表达的 Western blot 分析。I. 野生型（WT）和 SLC7A11 过表达细胞中 HSP70、LIAS 和 FDX1 蛋白水平的变化，有或无 Enz (20 μM)的存在。微管蛋白用作加载对照。J. 使用 Enz (40 μM)或无 GSH (10 μM)处理 48 小时后的细胞活力。

Measurement of acetyl-CoA levels in control and PDHA1 knockdown cells using an acetyl-CoA detection kit. B. Measurement of acetyl-CoA levels in wild-type and PDHA1-overexpressing cells using the same detection kit. C. Analysis of global acetylation levels in wild-type and PDHA1-overexpressing cells using a pan-acetylation antibody. D. Western blot analysis of target protein levels in wild-type and PDHA1-overexpressing cells after treatment with or without JQAD1 (1 μM) for 24 hours. E. ChIP-seq analysis using H3K27ac antibody to assess acetylation at transcription start sites in wild-type and PDHA1-overexpressing cells. The plot shows normalized ChIP H3K27ac signal enrichment. F. Analysis of enhancers and super-enhancers in wild-type and PDHA1-overexpressing cells using the modified ROSE algorithm. G. UCSC Genome Browser screenshot showing H3K27ac ChIP-seq peaks at the SLC7A11 locus in wild-type and PDHA1-overexpressing cells. H. ChIP-qPCR analysis of wild-type and PDHA1-overexpressing cells using IgG and H3K27ac antibodies.
A. 使用乙酰辅酶 A 检测试剂盒检测对照组和 PDHA1 敲低细胞的乙酰辅酶 A 水平。B. 使用相同检测试剂盒检测野生型和 PDHA1 过表达的细胞的乙酰辅酶 A 水平。C. 使用全乙酰化抗体分析野生型和 PDHA1 过表达细胞的全球乙酰化水平。D. 在野生型和 PDHA1 过表达细胞中，经或不经 JQAD1（1 μM）处理 24 小时后，进行靶蛋白水平的 Western blot 分析。E. 使用 H3K27ac 抗体进行 ChIP-seq 分析，评估野生型和 PDHA1 过表达细胞转录起始位点的乙酰化情况。图示为标准化 ChIP H3K27ac 信号富集。F. 使用改进的 ROSE 算法分析野生型和 PDHA1 过表达细胞中的增强子和超级增强子。G. UCSC 基因组浏览器截图显示野生型和 PDHA1 过表达细胞中 SLC7A11 基因座处的 H3K27ac ChIP-seq 峰。H. 使用 IgG 和 H3K27ac 抗体对野生型和 PDHA1 过表达细胞进行 ChIP-qPCR 分析。

B. PCa cells were treated for 48 hours with Enz and CPI 613, either alone or in combination, at the specified concentrations. Cell viability was measured, and the combination index (CI) was calculated using CalcuSyn software. CI value of <1, =1, and >1 indicates synergistic, additive, and antagonistic effects, respectively. C. Organoids were treated for 48 hours with Enz (40 μM) and CPI 613 (160 μM), either alone or in combination, at the specified concentrations. Representative images of PI-stained dead cells were obtained. In nude mice, the combination of Enz and CPI 613 more effectively inhibited tumor growth. At the end of the experiment, mice were sacrificed, and (D) representative tumor images were shown. F. Tumor growth curves were recorded every three days (mean ± SD, n = 5/group). G. Tumor tissues were weighed and summarized. E. Representative H&E and IHC staining of indicated proteins in tumor tissues from each group. Scale bar, 100 μm. H-score analysis for (H) Ki67 and (I) LIAS in the specified groups. In NSG mice, the combination of Enz and CPI 613 showed a stronger inhibitory effect on PDX model growth. J. Representative tumor images were shown. L. Tumor growth curves were recorded every three days (mean ± SD, n = 3/group). M. Tumor tissues were weighed and summarized. K. Representative H&E and IHC staining of indicated proteins in the tumor tissues. Scale bar, 100 μm. H-score analysis for (N) Ki67 and (O) LIAS in the specified groups.
B. PCa 细胞用 Enz 和 CPI 613 分别单独或联合处理 48 小时，在指定浓度下。细胞活力被测量，并使用 CalcuSyn 软件计算组合指数（CI）。CI 值<1、=1 和>1 分别表示协同、相加和拮抗作用。C.类器官用 Enz（40 μM）和 CPI 613（160 μM）分别单独或联合处理 48 小时，在指定浓度下。获得 PI 染色的死细胞代表性图像。在小鼠中，Enz 和 CPI 613 的组合更有效地抑制肿瘤生长。实验结束时，小鼠被处死，(D)显示代表性肿瘤图像。F.每三天记录肿瘤生长曲线（均值±标准差，每组 n=5）。G.肿瘤组织称重并汇总。E.每组肿瘤组织中指示蛋白的代表性 H&E 和 IHC 染色。标尺，100 μm。H.指定组中 Ki67 的 H-score 分析，(I) LIAS 的 H-score 分析。在小鼠中，Enz 和 CPI 613 的组合对 PDX 模型生长显示出更强的抑制作用。J.显示代表性肿瘤图像。L. 肿瘤生长曲线每三天记录一次（均值±标准差，每组 n=3）。M. 肿瘤组织称重并汇总。K. 肿瘤组织中指定蛋白的代表性 H&E 和 IHC 染色。标尺，100 μm。指定组中(N) Ki67 和(O) LIAS 的 H 评分分析。

A-B. Intracellular copper (II) ion concentrations in PCa cells after treatment with Enz at the specified concentrations. C. Changes in the protein levels of HSP70, LIAS, and FDX1 in PCa cells after treatment with Enz at the specified concentrations. Tubulin was used as a loading control. D. DLAT oligomerization in PCa cells after treatment with Enz at the specified concentrations. Tubulin was used as a loading control.
A-B. 处理后 PCa 细胞内铜（II）离子浓度。C. 处理后 PCa 细胞中 HSP70、LIAS 和 FDX1 蛋白水平的改变。使用微管蛋白作为加载对照。D. 处理后 PCa 细胞中 DLAT 寡聚化。使用微管蛋白作为加载对照。

B. Validation of PDHA1 knockdown in PCa cells by measuring PDHA1 mRNA (A) or protein levels (B) using qPCR and Western blot, respectively. C-D. Validation of PDHA1 overexpression in PCa cells by measuring PDHA1 mRNA (C) or protein levels (D) using qPCR and Western blot, respectively. E-F. Representative images of dead cell staining with PI dye in control and PDHA1 knockdown PCa cells after treatment with Enz (40 μM). G. Colony formation assay images of control and PDHA1 knockdown PCa cells after treatment with Enz (40 μM). H. Changes in HSP70, LIAS, and FDX1 protein levels in control and PDHA1 knockdown PCa cells. Tubulin was used as a loading control. I. Intracellular copper (II) ion levels in control and PDHA1 knockdown PCa cells. J. Changes in HSP70, LIAS, and FDX1 protein levels in control and PDHA1 knockdown PCa cells, with or without TTM (5 μM) treatment. Tubulin was used as a loading control.
B. 通过 qPCR 和 Western blot 分别检测 PDHA1 mRNA (A)或蛋白水平 (B) 验证 PCa 细胞中 PDHA1 敲低。C-D. 通过 qPCR 和 Western blot 分别检测 PDHA1 mRNA (C)或蛋白水平 (D) 验证 PCa 细胞中 PDHA1 过表达。E-F. 治疗 Enz (40 μM)后，在对照组和 PDHA1 敲低 PCa 细胞中 PI 染料死细胞染色的代表性图像。G. 治疗 Enz (40 μM)后，对照组和 PDHA1 敲低 PCa 细胞的集落形成实验图像。H. 对照组和 PDHA1 敲低 PCa 细胞中 HSP70、LIAS 和 FDX1 蛋白水平的变化。使用微管蛋白作为加载对照。I. 对照组和 PDHA1 敲低 PCa 细胞中细胞内铜(II)离子水平。J. 对照组和 PDHA1 敲低 PCa 细胞中 HSP70、LIAS 和 FDX1 蛋白水平的变化，有或无 TTM (5 μM)处理。使用微管蛋白作为加载对照。

A. 2D principal component analysis (PCA) of metabolomic sequencing data from PDHA1 knockdown cells. B. Volcano plot showing differential metabolite expression in PDHA1 knockdown cells, with thresholds set at P < 0.05 and |log2FC| ≥ 0. C. Pathway enrichment analysis of differential metabolites using the SMPDB (Small Molecule Pathway Database) following untargeted metabolomics sequencing. D. Cysteine levels in wild-type and PDHA1-overexpressing cells, measured using a cysteine assay kit. E-F. Intracellular GSH levels in wild-type and PDHA1-overexpressing cells, measured using flow cytometry. G. Protein level changes of SLC7A11 in PDHA1-overexpressing cells. H. Protein level changes of SLC7A11 in SLC7A11-overexpressing cells.
A. PDHA1 敲低细胞的代谢组测序数据的 2D 主成分分析（PCA）。B. 显示 PDHA1 敲低细胞中差异代谢物表达的火山图，阈值设置为 P < 0.05 和|log2FC| ≥ 0。C. 使用 SMPDB（小分子通路数据库）对差异代谢物进行通路富集分析，基于非靶向代谢组测序。D. 野生型和 PDHA1 过表达细胞中半胱氨酸水平的测定，使用半胱氨酸测定试剂盒。E-F. 野生型和 PDHA1 过表达细胞中细胞内 GSH 水平的测定，使用流式细胞术。G. PDHA1 过表达细胞中 SLC7A11 蛋白水平的改变。H. SLC7A11 过表达细胞中 SLC7A11 蛋白水平的改变。

A-B. ADistribution of ChIP-seq peaks across various functional genomic regions, analyzed using the ChIPseeker software. C-D. KEGG enrichment scatter plots of peak-associated genes.
A-B. 使用 ChIPseeker 软件分析 ChIP-seq 峰在各个功能基因组区域的分布。C-D. 峰相关基因的 KEGG 富集散点图。

Supplementary Fig5. CPI 613 Induces Cuproptosis.
补充图 5. CPI 613 诱导铜死亡。

Intracellular copper ion levels in control and PDHA1 knockdown PCa cells, measured using a copper ion assay kit after treatment with DMSO or CPI 613 (100 μM) for 24 hours. B. CS1 (green fluorescence) and DAPI (blue fluorescence) staining of copper (I) ions and cell nuclei, respectively, in PCa cells treated with DMSO or CPI 613 (100 μM) (scale bar: 20 μM). H. Changes in protein levels of PDHA1, pPDHA1, HSP70, LIAS, and FDX1 in PCa cells after treatment with DMSO or CPI 613 (100 μM). Tubulin was used as a loading control.
使用 DMSO 或 CPI 613（100 μM）处理 24 小时后，通过铜离子测定试剂盒检测对照组和 PDHA1 敲低 PCa 细胞内的铜离子水平。B. CS1（绿色荧光）和 DAPI（蓝色荧光）分别对处理 DMSO 或 CPI 613（100 μM）的 PCa 细胞中的铜(I)离子和细胞核进行染色（标尺：20 μM）。H. 处理 DMSO 或 CPI 613（100 μM）后 PCa 细胞中 PDHA1、pPDHA1、HSP70、LIAS 和 FDX1 蛋白水平的改变。微管蛋白用作加载对照。