NRF2 controls iron homeostasis and ferroptosis through HERC2 and VAMP8
NRF2 通过 HERC2 和 VAMP8 控制铁稳态和铁死亡

NFE2L2/NRF2 deletion sensitizes ovarian cancer cells to ferroptosis
NFE2L2/NRF2缺失使卵巢癌细胞对铁死亡敏感

One of the many cancers that have been shown to have high levels of NRF2 is epithelial ovarian cancer, the most lethal gynecological cancer due to its aggressiveness, propensity to develop drug resistance, recurrence, and metastatic potential. Although more than 80% of ovarian cancer patients respond to first-line chemotherapy, the majority will develop recurrent tumors that are drug resistant and fatal. Treatment relapses of ovarian cancer patients continue to represent a major obstacle for its clinical management (35, 36). To determine the role of NRF2 in mediating ovarian cancer cell resistance to ferroptosis, CRISPR-Cas9 gene editing was used to establish NFE2L2/NRF2 KO SKOV-3 ovarian carcinoma cells, and three different NFE2L2/NRF2 KO clones, which were established using two different single-guide RNAs (sgRNAs), were chosen for further study. Successful deletion of NFE2L2/NRF2 was confirmed, with NRF2 protein levels being undetectable in all three KO cell lines, as well as lower mRNA and protein levels of several NRF2 target genes (fig. S1, A and B). Compared to wild type (WT), NFE2L2/NRF2 KO cells treated with IKE, a ferroptosis inducer that inhibits system xc-, exhibited a notable increase in the number of cells undergoing the “ballooning” morphology indicative of ferroptotic cell death (representative images of 24-hour treatment are presented in Fig. 1A: left panel shows ferroptotic cell morphology; right panel is the percentage of ferroptotic cells). IKE induced 18.66% ferroptotic cells in NFE2L2/NRF2 WT cells versus 76% in NFE2L2/NRF2 KO cells (Table 1). Consistent with the pro-ferroptotic change in morphology (Fig. 1A), IKE treatment resulted in an ~30% decrease in cell viability in NFE2L2/NRF2 WT, as compared to an ~75% decrease in NFE2L2/NRF2 KO cells at 24 hours, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. 1B). Furthermore, IKE-induced ferroptotic cell death was completely abolished by deferoxamine (DFO; an iron chelator), ferrostatin 1 (Fer-1; an inhibitor of lipid peroxidation), but not Z-VAD-fmk (a pan-caspase inhibitor) (Fig. 1B), indicating the importance of free iron accumulation and lipid peroxidation in promoting ferroptosis. Consistent with a ferroptosis-dependent decrease in cell viability, higher levels of lipid peroxides were also observed in IKE-treated NFE2L2/NRF2 KO cells, which could be reversed when the cells were cotreated with DFO (Fig. 1C). Next, more ferroptotic cell death in IKE-treated NFE2L2/NRF2 KO cells compared to NFE2L2/NRF2 WT cells was further confirmed via assessment of key ferroptosis markers, including higher mRNA levels of prostaglandin endoperoxide synthase 2 (PTGS2) and its protein product cyclooxygenase 2 (COX2) (Fig. 1, D and E), as well as increased 4-hydroxynonenal (4-HNE) protein adduct formation (Fig. 1D). In addition, NFE2L2/NRF2 KO cells had decreased total GSH levels, higher levels of reactive oxygen species (ROS), and more malondialdehyde (MDA) formation under untreated conditions (Fig. 1, F to H); IKE treatment further exacerbated the differences in these measurements between NFE2L2/NRF2 WT and KO cells (Fig. 1, F to H). Similar results were obtained when a less specific ferroptosis inducer, SAS, was used (fig. S1, C to J).
上皮性卵巢癌是众多具有高水平 NRF2 的癌症之一，由于其侵袭性、产生耐药性、复发和转移潜力，它是最致命的妇科癌症。尽管超过 80% 的卵巢癌患者对一线化疗有反应，但大多数患者会出现耐药且致命的复发性肿瘤。卵巢癌患者的治疗复发仍然是其临床管理的主要障碍。 35 , 36 ）。为了确定NRF2在介导卵巢癌细胞对铁死亡的抵抗中的作用，使用CRISPR-Cas9基因编辑建立了NFE2L2/NRF2 KO SKOV-3卵巢癌细胞，以及使用两种不同的NFE2L2/NRF2 KO克隆建立了三个不同的NFE2L2/NRF2 KO克隆。选择单向导RNA（sgRNA）进行进一步研究。证实NFE2L2/NRF2成功删除，所有三种 KO 细胞系中均检测不到 NRF2 蛋白水平，并且多个 NRF2 靶基因的 mRNA 和蛋白水平较低（图 S1、A 和 B）。与野生型（WT）相比，用 IKE（一种抑制系统 xc- 的铁死亡诱导剂）处理的NFE2L2/NRF2 KO 细胞表现出经历“气球”形态的细胞数量显着增加，表明铁死亡细胞的数量（代表性图像24小时治疗介绍于 Fig. 1A ：左图​​显示铁死亡细胞形态；右图是铁死亡细胞的百分比）。 IKE 在NFE2L2/NRF2 WT 细胞中诱导 18.66% 的铁死亡细胞，而在NFE2L2/NRF2 KO 细胞中诱导 76%（ Table 1 ）。 与形态上的促铁死亡变化一致（ Fig. 1A ），通过3- (4,5-二甲基噻唑- 2-基）-2,5-二苯基溴化四唑（MTT）测定（ Fig. 1B ）。此外，去铁胺（DFO；一种铁螯合剂）、铁他汀 1（Fer-1；一种脂质过氧化抑制剂）可以完全消除 IKE 诱导的铁死亡细胞死亡，但不能被 Z-VAD-fmk（一种泛半胱天冬酶抑制剂）消除。 Fig. 1B ），表明游离铁积累和脂质过氧化在促进铁死亡中的重要性。与铁死亡依赖性细胞活力下降一致，在 IKE 处理的NFE2L2/NRF2 KO 细胞中也观察到较高水平的脂质过氧化物，当细胞与 DFO 共同处理时，这种情况可以逆转。 Fig. 1C ）。接下来，通过评估关键铁死亡标记物进一步证实，与NFE2L2/NRF2 WT 细胞相比，IKE 处理的NFE2L2/NRF2 KO 细胞中铁死亡更多，包括前列腺素内过氧化物合酶 2 ( PTGS2 ) 及其蛋白质产物环加氧酶 2 的 mRNA 水平较高(COX2) ( Fig. 1, D and E ），以及增加 4-羟基壬烯醛 (4-HNE) 蛋白质加合物的形成（ Fig. 1D ）。此外，在未经处理的条件下， NFE2L2/NRF2 KO细胞的总GSH水平降低，活性氧（ROS）水平升高，丙二醛（MDA）形成更多。 Fig. 1, F to H ）； IKE 处理进一步加剧了NFE2L2/NRF2 WT 和 KO 细胞之间这些测量结果的差异（ Fig. 1, F to H ）。 当使用特异性较低的铁死亡诱导剂 SAS 时，也获得了类似的结果（图 S1，C 至 J）。

NFE2L2/NRF2 deletion impairs iron homeostasis and increases the LIP through depletion of its target gene HERC2
NFE2L2/NRF2缺失会损害铁稳态，并通过消除其靶基因HERC2来增加 LIP

As free iron (ferrous iron, Fe²⁺) is required to drive ferroptotic cell death, the intracellular Fe²⁺ concentration was determined using a Ferene-S–based colorimetric assay. Basal intracellular Fe²⁺ levels increased ~3- to 5-fold in NFE2L2/NRF2 KO compared to NFE2L2/NRF2 WT cells; IKE treatment further enhanced free Fe²⁺ levels (Fig. 2A). Increased free iron levels in NFE2L2/NRF2 KO cells under both untreated and IKE-treated conditions were verified by immunofluorescence using the Fe²⁺-sensitive probe FerroOrange (Fig. 2B). Please note that IKE treatment led to an increase in free Fe²⁺ levels even in NFE2L2/NRF2 WT control cells, which is consistent with previous reports when cells were treated with erastin (22, 37). Next, expression of genes critical in maintaining iron homeostasis was compared in NFE2L2/NRF2 WT versus KO cells. Transferrin receptor 1 (TFRC/TFR1), the primary receptor controlling iron uptake, was moderately induced by IKE treatment at the protein but not mRNA level in all the cell lines, whereas there was a slight reduction in TFRC/TFR1 mRNA levels, but not protein, in all NFE2L2/NRF2 KO cells compared to NFE2L2/NRF2 WT cells (Fig. 2, C and D). In contrast, NFE2L2/NRF2 KO altered the expression of many proteins involved in ferritin synthesis and degradation [HERC2, F-box and leucine-rich repeat protein 5 (FBXL5), iron regulatory protein 2 (IRP2), and NCOA4] (Fig. 2, C and D), which in turn would affect the LIP. First, NFE2L2/NRF2 KO affected ferritin synthesis as evidenced by the following findings: NFE2L2/NRF2 KO cells exhibited altered levels of key iron sensor proteins constituting the HERC2-FBXL5-IRP1/2-FTH1/FTL pathway, including decreased HERC2, increased FBXL5, decreased IRP2, and increased ferritin heavy/light chains (FTH1 and FTL) (Fig. 2D). As shown in the model (Fig. 2E), HERC2 is an E3 ubiquitin ligase for both FBXL5 and NCOA4; however, HERC2 binds FBXL5 in the absence of free iron (Fe²⁺), whereas HERC2 binds NCOA4 only in the presence of free iron (38–41). In addition, IRP2 binds to the iron-responsive element in the 5′ untranslated region of FTH1 and FTL mRNA to repress the synthesis of ferritin during low free iron conditions. Thus, this NFE2L2/NRF2 deletion–mediated decrease in IRP2 should ultimately result in enhanced synthesis of ferritin in NFE2L2/NRF2-KO SKOV-3 cells. Consistent with this notion, increased protein levels of both FTH1 and FTL were observed (Fig. 2D) although their mRNA expression decreased (Fig. 2C), which was in accordance with FTH1/FTL being NRF2 target genes (42). Second, NFE2L2/NRF2 KO also affected ferritin degradation. NCOA4 is a critical cargo receptor that specifically recruits ferritin to the autophagosome for degradation, a process termed ferritinophagy (43). Both the mRNA and protein levels of NCOA4 were increased in NFE2L2/NRF2 KO cells (Fig. 2, C and D). The increased NCOA4 protein level seen in NFE2L2/NRF2 KO cells was partly due to decreased expression of HERC2, resulting in its stabilization (Fig. 2, D and E). Collectively, these results indicate that NFE2L2/NRF2 deletion impairs iron homeostasis by altering expression of proteins controlling ferritin synthesis and degradation, resulting in an increased LIP and enhanced sensitivity of cancer cells to ferroptosis.
由于需要游离铁（二价铁，Fe ²⁺ ）来驱动铁死亡细胞死亡，因此使用基于 Ferene-S 的比色测定法测定细胞内 Fe ²⁺浓度。与NFE2L2/NRF2 WT 细胞相比， NFE2L2/NRF2 KO 细胞中基础细胞内 Fe ²⁺水平增加约 3 至 5 倍； IKE 处理进一步提高了游离 Fe ²⁺水平（ Fig. 2A ）。使用 Fe ²⁺敏感探针 FerroOrange 通过免疫荧光验证了未经处理和 IKE 处理条件下NFE2L2/NRF2 KO 细胞中游离铁水平的增加（ Fig. 2B ）。请注意，即使在NFE2L2/NRF2 WT 对照细胞中，IKE 处理也会导致游离 Fe ²⁺水平增加，这与之前用erastin 处理细胞时的报告一致（ 22 , 37 ）。接下来，比较了NFE2L2/NRF2 WT 和 KO 细胞中维持铁稳态的关键基因的表达。转铁蛋白受体 1 ( TFRC /TFR1) 是控制铁摄取的主要受体，在所有细胞系中，IKE 处理在蛋白质水平（而非 mRNA 水平）中度诱导，而TFRC /TFR1 mRNA 水平略有降低，但没有降低。与NFE2L2/NRF2 WT 细胞相比，所有NFE2L2/NRF2 KO 细胞中的蛋白质（ Fig. 2, C and D ）。相比之下， NFE2L2/NRF2 KO 改变了许多参与铁蛋白合成和降解的蛋白质的表达 [HERC2、F-box 和富含亮氨酸的重复蛋白 5 (FBXL5)、铁调节蛋白 2 (IRP2) 和 NCOA4]。 Fig. 2, C and D ），这反过来又会影响 LIP。 首先， NFE2L2/NRF2 KO影响铁蛋白合成，如下结果所证明： NFE2L2 / NRF2 KO细胞表现出构成HERC2-FBXL5-IRP1/2-FTH1/FTL途径的关键铁传感器蛋白水平改变，包括HERC2减少、FBXL5增加，IRP2 减少，铁蛋白重链/轻链（FTH1 和 FTL）增加（ Fig. 2D ）。如模型所示（ Fig. 2E )，HERC2 是 FBXL5 和 NCOA4 的 E3 泛素连接酶；然而，HERC2 在没有游离铁 (Fe ²⁺ ) 的情况下结合 FBXL5，而 HERC2 仅在存在游离铁的情况下结合 NCOA4 ( 38 – 41 ）。此外，IRP2 与FTH1和FTL mRNA 5' 非翻译区的铁响应元件结合，在低游离铁条件下抑制铁蛋白的合成。因此，这种NFE2L2/NRF2缺失介导的 IRP2 减少最终应导致NFE2L2/NRF2 -KO SKOV-3 细胞中铁蛋白的合成增强。与这一观点一致，观察到 FTH1 和 FTL 的蛋白质水平增加（ Fig. 2D ）尽管它们的 mRNA 表达降低了（ Fig. 2C ），这与FTH1 / FTL是 NRF2 靶基因一致（ 42 ）。其次， NFE2L2/NRF2 KO 也影响铁蛋白降解。 NCOA4 是一种关键的货物受体，专门将铁蛋白招募到自噬体进行降解，这一过程称为铁蛋白自噬。 43 ）。 NFE2L2/NRF2 KO 细胞中 NCOA4 的 mRNA 和蛋白水平均增加（ Fig. 2, C and D ）。 NFE2L2/NRF2 KO 细胞中 NCOA4 蛋白水平增加的部分原因是 HERC2 表达减少，从而导致其稳定化。 Fig. 2, D and E ）。总的来说，这些结果表明， NFE2L2/NRF2缺失通过改变控制铁蛋白合成和降解的蛋白质表达来损害铁稳态，导致 LIP 增加并增强癌细胞对铁死亡的敏感性。

Since both FBXL5-IRP2–mediated ferritin synthesis and NCOA4-dependent ferritin degradation through the autophagy-lysosome pathway can be controlled by the key E3 ubiquitin ligase HERC2 (Fig. 2E) (41, 43), coupled with the result described in Fig. 2D indicating a decrease in HERC2 protein levels in NFE2L2/NRF2 KO cells, the possibility of HERC2 being an NRF2 target gene was explored. Consistent with the observed positive correlation between the protein levels of NRF2 and HERC2 (Fig. 2D), a higher level of HERC2 mRNA in NFE2L2/NRF2 WT cells was observed in both untreated and IKE-treated conditions, compared to NFE2L2/NRF2 KO cells (Fig. 2C). The NRF2 activators sulforaphane (SF), arsenic (As), and tert-butylhydroquinone (tBHQ) or the NRF2 inhibitor brusatol (BRU) was able to increase or decrease both the mRNA and protein level of HERC2, respectively (Fig. 2F). In silico analysis identified 12 putative antioxidant response elements (AREs) in HERC2, which were individually cloned into the promoter region of a firefly luciferase vector, followed by measurement of firefly luciferase activity using a dual luciferase reporter assay (renilla was used as an internal control). Three functional AREs (ARE-1, ARE-2, and ARE-8) were identified as their luciferase activity was enhanced by SF treatment; one nonfunctional ARE (ARE-11) was also included for comparison (Fig. 2G). Endogenous binding of NRF2 to these three AREs was further verified to occur in WT, but not NFE2L2/NRF2 KO, cells by chromatin immunoprecipitation (ChIP)–polymerase chain reaction (PCR) (Fig. 2H). Last, using biotinylated 41–base pair (bp) DNA probes containing the WT ARE-1, ARE-2, ARE-8, or their mutated ARE sequences (mt-AREs), NRF2 was confirmed to specifically bind to ARE-1, ARE-2, and ARE-8, but not their mutant counterparts (Fig. 2I). These results demonstrate that the HERC2 promoter, intron1, and exon1 contain functional AREs that are regulated by NRF2. In addition, NRF2 activators were able to induce NRF2 and HERC2 protein levels in two additional ovarian cell lines with low basal levels of NRF2 (Fig. 2J). More broadly, a positive correlation between NRF2 and HERC2 was also observed in most ovarian cell lines tested where NFE2L2/NRF2 expression was reduced by transient knockdown using two different NFE2L2/NRF2 small interfering RNAs (siRNAs) (fig. S2).
由于 FBXL5-IRP2 介导的铁蛋白合成和通过自噬-溶酶体途径的 NCOA4 依赖性铁蛋白降解都可以由关键的 E3 泛素连接酶 HERC2 控制（ Fig. 2E ) ( 41 , 43 ），加上中描述的结果 Fig. 2D 表明NFE2L2/NRF2 KO 细胞中 HERC2 蛋白水平降低，探讨了HERC2作为 NRF2 靶基因的可能性。与观察到的 NRF2 和 HERC2 蛋白水平之间的正相关性一致（ Fig. 2D ），与NFE2L2/NRF2 KO 细胞相比，在未经处理和 IKE 处理的条件下， NFE2L2/NRF2 WT 细胞中的HERC2 mRNA 水平较高（ Fig. 2C ）。 NRF2 激活剂萝卜硫素 (SF)、砷 (As) 和叔丁基氢醌 (tBHQ) 或 NRF2 抑制剂布鲁萨醇 (BRU) 能够分别增加或减少 HERC2 的 mRNA 和蛋白质水平。 Fig. 2F ）。计算机分析确定了HERC2中的 12 个假定的抗氧化反应元件 (ARE)，将其单独克隆到萤火虫荧光素酶载体的启动子区域，然后使用双荧光素酶报告基因测定（使用海肾作为内部对照）测量萤火虫荧光素酶活性）。确定了三种功能性 ARE（ARE-1、ARE-2 和 ARE-8），因为 SF 处理增强了它们的荧光素酶活性；还包括一种非功能性 ARE (ARE-11) 进行比较（ Fig. 2G ）。通过染色质免疫沉淀 (ChIP)-聚合酶链式反应 (PCR) 进一步证实 NRF2 与这三种 ARE 的内源性结合发生在 WT 细胞中，但在NFE2L2/NRF2 KO 细胞中则不然。 Fig. 2H ）。 最后，使用含有 WT ARE-1、ARE-2、ARE-8 或其突变 ARE 序列 (mt-ARE) 的生物素化 41 碱基对 (bp) DNA 探针，证实 NRF2 与 ARE-1 特异性结合， ARE-2 和 ARE-8，但不是它们的突变对应物（ Fig. 2I ）。这些结果表明， HERC2启动子、内含子 1 和外显子 1 含有受 NRF2 调控的功能性 ARE。此外，NRF2 激活剂能够在另外两种 NRF2 基础水平较低的卵巢细胞系中诱导 NRF2 和 HERC2 蛋白水平（ Fig. 2J ）。更广泛地说，在大多数测试的卵巢细胞系中也观察到 NRF2 和 HERC2 之间的正相关性，其中使用两种不同的NFE2L2/NRF2小干扰 RNA (siRNA) 进行瞬时敲低， NFE2L2/NRF2表达降低（图 S2）。

VAMP8 down-regulation in NFE2L2/NRF2 KO cells results in a defect of ferritinophagy and autophagosomal accumulation of apoferritin/NCOA4
NFE2L2 / NRF2 KO细胞中VAMP8下调导致铁蛋白自噬缺陷和脱铁铁蛋白/NCOA4自噬体积累

As shown in Fig. 2D, the protein levels of FTH1, FTL, and NCOA4 were increased in NFE2L2/NRF2 KO cells, indicative of a defect in ferritin degradation. Since ferritin is degraded by the autophagy-lysosome pathway, a process termed ferritinophagy (see a model illustration for ferritinophagy in Fig. 3A), this prompted us to examine the possibility that the observed increase in these proteins was a result of blockage of autophagic flux. NFE2L2/NRF2 KO cells exhibited a larger number of microtubule-associated protein 1A/1B-light chain 3 (LC3)–positive autophagosomes, but not autolysosomes, as assessed by monomeric–red fluorescent protein (mRFP)–green fluorescent protein (GFP)–LC3 tandem–based immunofluorescence (Fig. 3B). This result indicates that there was no defect in autophagosome formation, but instead a blockage at the later stage of the pathway in NFE2L2/NRF2 KO cells. Transmission electron microscopy (TEM) also indicated an accumulation of protein aggregates and double-membrane autophagosomes in NFE2L2/NRF2 KO cells (Fig. 3C). Next, the snap receptor (SNARE) complex [VAMP8, synaptosome-associated protein 29 (SNAP29), and syntaxin (STX17)], which plays a critical role in mediating autophagosome-lysosome fusion (44), was examined. Unexpectedly, the protein level of VAMP8, a lysosomal SNARE protein, was markedly reduced, while there were no obvious changes in STX17 or SNAP29 protein levels in NFE2L2/NRF2 KO cells (Fig. 3D). Furthermore, NFE2L2/NRF2 KO cells had ~30 to 40% lower VAMP8 mRNA levels compared to WT cells (Fig. 3E). Transcription of lysosomal genes has been shown to be regulated by transcription factor EB (TFEB), whose activity is controlled by serine phosphorylation. Previous studies indicate that phosphorylation at S142/S138 inactivates TFEB by enhancing its nuclear export, whereas S211 phosphorylation mediates cytosolic retention. Thus, the net effect of TFEB phosphorylation at these sites is less nuclear localization and reduced transcription of lysosomal genes (45–49). Therefore, the phosphorylation status of TFEB and mammalian target of rapamycin (mTOR), one of the upstream kinases for TFEB, were assessed. Basal phosphorylation of mTOR at S2448 was higher in NFE2L2/NRF2 KO cells, while the total mTOR level remained unchanged; TFEB phosphorylation was also enhanced at S211 and S142 with no changes in its total protein expression (Fig. 3F). Consistently, when cells with nuclear localization of TFEB were counted, significantly more cells with nuclear TFEB localization in NFE2L2/NRF2 WT cells than NFE2L2/NRF2 KO cells were identified (Fig. 3G). TFEB binds to DNA sequences containing an EBOX motif (CANNTG), and in silico analysis identified six putative EBOX motifs in the promoter region of VAMP8. Using a dual luciferase assay, one functional EBOX (⁻¹⁹³²CACTTG⁻¹⁹²⁷) was identified, as demonstrated by increased EBOX-driven firefly luciferase activity in the presence of mTOR inhibitors (torin and rapamycin) and adenosine monophosphate–activated protein kinase (AMPK) activator (AICAR), as well as a decreased luciferase activity following mTOR activation (amino acids) (Fig. 3H). These results indicate that NFE2L2/NRF2 KO leads to ferritinophagy blockage through TFEB phosphorylation and nuclear exclusion, which decreases the transcription of VAMP8, although the precise mechanism by which deletion of NFE2L2/NRF2 leads to activation of mTOR, or possibly other kinases that may regulate TFEB phosphorylation and nuclear localization, requires further investigation. The notion that mTOR was activated in NFE2L2/NRF2 KO cells, resulting in decreased VAMP8 transcription, was further supported by the results in fig. S3. Rapamycin treatment reduced phosphorylated mTOR levels in NFE2L2/NRF2 KO cells without affecting the total mTOR level, indicating inactivation of mTOR by rapamycin in NFE2L2/NRF2 KO cells. Rapamycin increased VAMP8 expression in NFE2L2/NRF2 KO cells, which partially rescued the blockage of ferritinophagy flux as indicated by the reduced level of NCOA4, p62, and LC3-II in rapamycin-treated NFE2L2/NRF2 KO cells (fig. S3C). Similar to DFO, rapamycin also reduced the number of ferroptotic cells (fig. S3A) and improved cell survival in response to SAS in NFE2L2/NRF2 KO cells (fig. S3B). Similar to HERC2, the positive correlation between NRF2 and VAMP8 in a variety of ovarian cell lines was also observed when NFE2L2/NRF2 expression was reduced by NFE2L2/NRF2 siRNAs (fig. S2). Immortalized normal ovarian surface epithelial (OSE) (50) and fallopian tube (FT-246) (51) cell lines had a low basal protein level of NRF2 and an undetectable level of VAMP8, while NFE2L2/NRF2 knockdown in the ovarian cancer cell lines tested all resulted in a decrease in VAMP8 (fig. S2).
如图所示 Fig. 2D ， NFE2L2/NRF2 KO 细胞中 FTH1、FTL 和 NCOA4 的蛋白水平增加，表明铁蛋白降解存在缺陷。由于铁蛋白通过自噬-溶酶体途径降解，这一过程称为铁蛋白自噬（参见铁蛋白自噬的模型说明） Fig. 3A ），这促使我们研究观察到的这些蛋白质的增加是否是自噬流受阻的结果。通过单体-红色荧光蛋白 (mRFP)-绿色荧光蛋白 (GFP) 评估， NFE2L2/NRF2 KO 细胞表现出大量微管相关蛋白 1A/1B-轻链 3 (LC3) 阳性自噬体，但没有自溶酶体–LC3 串联免疫荧光（ Fig. 3B ）。这一结果表明， NFE2L2/NRF2 KO细胞中自噬体形成并没有缺陷，而是在该通路的后期被阻断。透射电子显微镜 (TEM) 还表明NFE2L2/NRF2 KO 细胞中蛋白质聚集体和双膜自噬体的积累。 Fig. 3C ）。接下来是 snap 受体 (SNARE) 复合物 [VAMP8、突触体相关蛋白 29 (SNAP29) 和突触蛋白 (STX17)]，它在介导自噬体 - 溶酶体融合中发挥着关键作用。 44 ），进行了检查。出乎意料的是， NFE2L2/NRF2 KO细胞中溶酶体SNARE蛋白VAMP8的蛋白水平显着降低，而STX17或SNAP29蛋白水平没有明显变化。 Fig. 3D ）。此外，与 WT 细胞相比， NFE2L2/NRF2 KO 细胞的VAMP8 mRNA 水平降低约 30% 至 40%（ Fig. 3E ）。 溶酶体基因的转录已被证明受到转录因子 EB (TFEB) 的调节，其活性由丝氨酸磷酸化控制。先前的研究表明，S142/S138 的磷酸化通过增强 TFEB 的核输出而使其失活，而 S211 磷酸化则介导细胞质保留。因此，这些位点 TFEB 磷酸化的净效应是核定位减少和溶酶体基因转录减少。 45 – 49 ）。因此，评估了 TFEB 和哺乳动物雷帕霉素靶蛋白 (mTOR)（TFEB 上游激酶之一）的磷酸化状态。 NFE2L2/NRF2 KO 细胞中 mTOR 在 S2448 的基础磷酸化较高，而总 mTOR 水平保持不变； TFEB 磷酸化也在 S211 和 S142 处增强，但其总蛋白表达没有变化（ Fig. 3F ）。一致地，当对具有 TFEB 核定位的细胞进行计数时，发现NFE2L2/NRF2 WT 细胞中具有核 TFEB 定位的细胞明显多于NFE2L2/NRF2 KO 细胞（ Fig. 3G ）。 TFEB 与含有 EBOX 基序 (CANNTG) 的 DNA 序列结合，并通过计算机分析在VAMP8启动子区域鉴定出六个假定的 EBOX 基序。使用双荧光素酶测定，鉴定出一个功能性 EBOX ( ^-1932 CACTTG ^-1927 )，这通过在 mTOR 抑制剂（torin 和雷帕霉素）和腺苷单磷酸激活蛋白激酶 (AMPK) 存在的情况下 EBOX 驱动的萤火虫荧光素酶活性增加来证明。激活剂 (AICAR)，以及 mTOR 激活（氨基酸）后荧光素酶活性降低（ Fig. 3H ）。 这些结果表明， NFE2L2/NRF2 KO 通过 TFEB 磷酸化和核排斥导致铁蛋白自噬阻断，从而降低VAMP8的转录，尽管删除NFE2L2/NRF2导致 mTOR 或可能调节的其他激酶激活的精确机制TFEB磷酸化和核定位，需要进一步研究。 mTOR 在NFE2L2/NRF2 KO 细胞中被激活，导致 VAMP8 转录减少，这一观点得到了图 2 中结果的进一步支持。 S3。雷帕霉素处理降低了NFE2L2/NRF2 KO 细胞中的磷酸化 mTOR 水平，但不影响总 mTOR 水平，表明雷帕霉素在NFE2L2/NRF2 KO 细胞中使 mTOR 失活。雷帕霉素增加了NFE2L2 / NRF2 KO 细胞中 VAMP8 的表达，这部分缓解了铁蛋白自噬流的阻断，如雷帕霉素处理的NFE2L2/NRF2 KO 细胞中 NCOA4、p62 和 LC3-II 水平降低所示（图 S3C）。与 DFO 类似，雷帕霉素还减少了NFE2L2/NRF2 KO 细胞中铁死亡细胞的数量（图 S3A），并提高了 SAS 对 SAS 的细胞存活率（图 S3B）。与HERC2类似，当NFE2L2/NRF2 siRNA减少NFE2L2/NRF2表达时，在多种卵巢细胞系中也观察到NRF2和VAMP8之间的正相关性（图S2）。 永生化正常卵巢表面上皮（OSE）（ 50 ）和输卵管（FT-246）（ 51 ）细胞系的 NRF2 基础蛋白水平较低，VAMP8 水平不可检测，而测试的卵巢癌细胞系中NFE2L2/NRF2敲低均导致 VAMP8 减少（图 S2）。

We have shown that decreased HERC2 expression led to enhanced protein synthesis of FTH1/FTL and enhanced NCOA4 protein stability, and that decreased VAMP8 expression resulted in ferritinophagy blockage in NFE2L2/NRF2 KO cells. Next, the effect of NFE2L2/NRF2 deletion on autophagic ferritin turnover and intracellular free iron was assessed. Immunofluorescence analysis revealed that while WT cells had very little FTH1/FTL signal (less ferritin aggregates shown as puncta), NFE2L2/NRF2 KO cells had significantly more FTH1/FTL aggregates, most of which colocalized with LC3-positive puncta (Fig. 3I, Table 2, and fig. S4A). However, while FTH1/FTL colocalized with LC3, indicating recruitment of ferritin to the autophagosome, FTH1/FTL failed to be delivered to the lysosome, as very few FTH1/FTL aggregates colocalized with LAMP1-positive lysosomes in NFE2L2/NRF2 KO cells (Fig. 3J, Table 2, and fig. S4B). These results support a model whereby FTH1/FTL was recruited by NCOA4 into autophagosomes, which then accumulated due to the blockage of VAMP8-mediated autophagosome-lysosome fusion in NFE2L2/NRF2 KO cells. In addition, accumulation of FTH1 aggregates was observed in other ovarian cell lines where NFE2L2/NRF2 was knocked down via siRNA (fig. S4C). Paradoxically, enhanced ferritin synthesis and blockage of ferritin degradation (inhibition of ferritinophagy) in NFE2L2/NRF2 KO cells should have resulted in a decrease, rather than a significant increase, of the LIP (Fig. 2, A and B). We hypothesized that the availability of higher than normal NCOA4, a critical cargo receptor that recruits ferritin to the autophagosome, would overwhelm the other proteins, such as poly(RC) binding protein 1 (PCBP1), needed to deliver iron to ferritin (52), resulting in NCOA4-mediated recruitment of apoferritin into the autophagosome. As shown in Fig. 3K, NFE2L2/NRF2 WT cells showed an increase in Perls staining (dark dots in the cytosol) after ferric ammonium citrate (FAC) treatment, indicative of ferric iron stored in ferritin cages, while the NFE2L2/NRF2 KO cells had very limited Perls staining after FAC treatment, although these cells contained many ferritin aggregates colocalized with LC3 (Fig. 3K), indicating recruitment of apoferritin into autophagosomes in NFE2L2/NRF2 KO cells. This result suggests that the excessive amount of NCOA4 resulted in the rapid recruitment of apoferritin into autophagosomes before iron incorporation into the ferritin complex. Next, the effect of siRNA-mediated knockdown of NCOA4 on alleviating the increased LIP and ferroptosis observed in NFE2L2/NRF2 KO cells was tested. NCOA4-siRNA had a slight effect on the protein levels of FTH1/FTL (increased) and LC3-I/LC3-II (decreased) in both the WT and KO cell lines (fig. S4D). Knockdown of NCOA4 alleviated 4-HNE–adducted protein formation in IKE-treated NFE2L2/NRF2 KO cells (fig. S4, D and E). The protection afforded by NCOA4 knockdown in NFE2L2/NRF2 KO cells was also visualized by a reduction in FTH1 puncta formation, decreased Fe²⁺-sensitive FerroOrange fluorescence intensity, and fewer cells undergoing ferroptotic death (fig. S4F). Consistent with these results, NCOA4 knockdown significantly enhanced the survival of NFE2L2/NRF2 KO cells under IKE treatment (fig. S4G). In addition, the mRNA levels of ferroportin (SLC40A1) were significantly higher in the NFE2L2/NRF2 KO cells compared to WT (fig. S4H), demonstrating that the accumulation of free labile iron observed in the NFE2L2/NRF2 KO cells is most likely not due to changes in ferroportin expression, despite previous reports (53).
我们发现， HERC2表达减少导致 FTH1/FTL 蛋白合成增强，NCOA4 蛋白稳定性增强， VAMP8表达减少导致NFE2L2/NRF2 KO 细胞中铁蛋白自噬阻断。接下来，评估了NFE2L2/NRF2缺失对自噬铁蛋白周转和细胞内游离铁的影响。免疫荧光分析显示，虽然 WT 细胞具有非常少的 FTH1/FTL 信号（较少的铁蛋白聚集体，显示为斑点），但NFE2L2/NRF2 KO 细胞具有明显更多的 FTH1/FTL 聚集体，其中大多数与 LC3 阳性斑点共定位。 Fig. 3I , Table 2 ，和图。 S4A）。然而，虽然 FTH1/FTL 与 LC3 共定位，表明铁蛋白被招募到自噬体，但 FTH1/FTL 未能被递送至溶酶体，因为在NFE2L2/NRF2 KO 细胞中，很少有 FTH1/FTL 聚集体与 LAMP1 阳性溶酶体共定位。 Fig. 3J , Table 2 ，和图。 S4B）。这些结果支持这样的模型：FTH1/FTL被NCOA4招募到自噬体中，然后由于VAMP8介导的自噬体-溶酶体融合在NFE2L2/NRF2 KO细胞中被阻断而积累。此外，在通过 siRNA 敲除NFE2L2/NRF2 的其他卵巢细胞系中观察到 FTH1 聚集体的积累（图 S4C）。矛盾的是， NFE2L2/NRF2 KO 细胞中铁蛋白合成的增强和铁蛋白降解的阻断（铁蛋白自噬的抑制）应该导致 LIP 的减少，而不是显着增加。 Fig. 2, A and B ）。 我们假设，NCOA4（一种将铁蛋白招募到自噬体的关键货物受体）的可用性高于正常水平，将压倒其他蛋白质，例如将铁传递到铁蛋白所需的聚（RC）结合蛋白 1 (PCBP1)。 52 ），导致 NCOA4 介导的脱铁铁蛋白募集到自噬体中。如图所示 Fig. 3K ， NFE2L2/NRF2 WT 细胞在柠檬酸铁铵 (FAC) 处理后显示 Perls 染色增加（细胞质中的黑点），表明三价铁储存在铁蛋白笼中，而NFE2L2/NRF2 KO 细胞在柠檬酸铁铵 (FAC) 处理后 Perls 染色非常有限FAC 处理，尽管这些细胞含有许多与 LC3 共定位的铁蛋白聚集体（ Fig. 3K ），表明脱铁铁蛋白被招募到NFE2L2/NRF2 KO 细胞的自噬体中。这一结果表明，过量的 NCOA4 导致脱铁铁蛋白在铁掺入铁蛋白复合物之前快速招募到自噬体中。接下来，测试了 siRNA 介导的NCOA4敲低对缓解NFE2L2/NRF2 KO 细胞中观察到的 LIP 增加和铁死亡的影响。 NCOA4 -siRNA对WT和KO细胞系中FTH1/FTL（增加）和LC3-I/LC3-II（减少）的蛋白质水平有轻微影响（图S4D）。 NCOA4的敲低减轻了 IKE 处理的NFE2L2/NRF2 KO 细胞中 4-HNE 加合蛋白的形成（图 S4、D 和 E）。 NFE2L2/NRF2 KO 细胞中NCOA4敲低所提供的保护还通过 FTH1 斑点形成的减少、Fe ²⁺敏感性铁橙荧光强度的降低以及经历铁死亡的细胞减少来显现（图 S4F）。与这些结果一致， NCOA4敲低显着增强了 IKE 处理下NFE2L2/NRF2 KO 细胞的存活率（图 S4G）。此外，与 WT 相比， NFE2L2/NRF2 KO 细胞中铁转运蛋白 ( SLC40A1 ) 的 mRNA 水平显着较高（图 S4H），这表明在NFE2L2/NRF2 KO 细胞中观察到的游离不稳定铁的积累很可能不是由于铁转运蛋白表达的变化，尽管之前有报道（ 53 ）。

NRF2 regulates iron homeostasis through HERC2, VAMP8, and NCOA4
NRF2 通过 HERC2、VAMP8 和 NCOA4 调节铁稳态

Collectively, our results have revealed a previously undiscovered function of NRF2, i.e., NRF2 regulates iron homeostasis and ferroptosis through HERC2, VAMP8, and NCOA4 (illustration in Fig. 4). By controlling their protein levels, NRF2 regulates both ferritin synthesis and degradation. First, HERC2, an E3 ubiquitin ligase for FBXL5 and NCOA4, is an NRF2 target gene; deletion of NRF2 results in reduced HERC2 expression, increased stability of FBXL5 and NCOA4, decreased IRP2 protein stability, and enhanced FTH synthesis. Second, NRF2 indirectly controls VAMP8 through the mTOR-TFEB axis. In NFE2L2/NRF2 KO cells, decreased TFEB-dependent transcription of VAMP8 results in blockage of autophagosome-lysosome fusion and inhibition of ferritinophagy. Third, excessive accumulation of NCOA4 in NFE2L2/NRF2 KO cells causes recruitment of apoferritin into autophagosomes, leading to autophagosomal accumulation of apoferritin/NCOA4, increased LIP, and enhanced sensitivity to ferroptotic cell death.
总的来说，我们的结果揭示了 NRF2 以前未被发现的功能，即 NRF2 通过 HERC2、VAMP8 和 NCOA4 调节铁稳态和铁死亡（图 1） Fig. 4 ）。通过控制蛋白质水平，NRF2 调节铁蛋白的合成和降解。首先， HERC2是FBXL5和NCOA4的E3泛素连接酶，是NRF2靶基因； NRF2 的缺失导致 HERC2 表达减少，FBXL5 和 NCOA4 稳定性增加，IRP2 蛋白稳定性降低，FTH 合成增强。其次，NRF2通过mTOR-TFEB轴间接控制VAMP8 。在NFE2L2/NRF2 KO 细胞中， VAMP8的 TFEB 依赖性转录减少导致自噬体-溶酶体融合受阻并抑制铁蛋白自噬。第三， NFE2L2/NRF2 KO细胞中NCOA4的过度积累导致脱铁铁蛋白募集到自噬体中，导致脱铁铁蛋白/NCOA4在自噬体中积累，LIP增加，并增强对铁死亡细胞死亡的敏感性。

Contribution of the NRF2-HERC2 and NRF2-VAMP8/NCOA4 axes in controlling iron homeostasis and dictating ferroptosis sensitivity
NRF2-HERC2 和 NRF2-VAMP8/NCOA4 轴在控制铁稳态和决定铁死亡敏感性中的贡献

To further validate the contribution of the newly identified function of NRF2 in iron homeostasis, and to ensure the anti-ferroptotic function of NRF2 extends beyond just its mediation of redox balance, HERC2 and VAMP8 single or double KO cells were generated. Like what was observed in NFE2L2/NRF2 KO cells, KO of HERC2 or VAMP8 resulted in increased protein levels of FTH1 and NCOA4, as well as the ferroptosis markers COX2 and 4-HNE, with double- KO of HERC2/VAMP8 exhibiting a more pronounced phenotype than single KO (Fig. 5A). Also, KO of HERC2 or double KO significantly increased free iron levels as measured by both FerroOrange (Fig. 5B, FerroOrange panel) and Ferene-S colorimetric assay (Fig. 5B, bar graph, bottom), but decreased ferric iron–loaded ferritin cages (Fig. 5B, Perls staining). Consistently, KO enhanced ferroptotic cell death (Fig. 5, C and D) and reduced cell survival (Fig. 5E). The percentage of ferroptotic cells was ~20% in IKE-treated WT cells versus ~75% in the HERC2/VAMP8 double KO cells (Fig. 5D), with an ~30% decrease in cell viability in NFE2L2/NRF2 WT compared to an ~80% decrease in HERC2/VAMP8 double KO cells at 24 hours (Fig. 5E). A comparison of the percentage of ferroptotic cells in NFE2L2/NRF2 WT and the different KO cell lines following IKE treatment shows that cells with double KO of HERC2/VAMP8 exhibit an equivalent ferroptosis sensitivity as NFE2L2/NRF2 KO cells (Table 1). These results demonstrated that HERC2/VAMP8 double KO cells behaved like NFE2L2/NRF2 KO cells, as indicated by a similar accumulation of apoferritin/NCOA4, increased LIP, enhanced ferroptosis markers (COX2 and 4-HNE), and increased sensitivity to ferroptotic cell death.
为了进一步验证新发现的 NRF2 功能在铁稳态中的贡献，并确保 NRF2 的抗铁死亡功能超出其介导氧化还原平衡的范围，生成了HERC2和VAMP8单或双 KO 细胞。与在NFE2L2/NRF2 KO 细胞中观察到的情况一样， HERC2或VAMP8的 KO 导致 FTH1 和 NCOA4 以及铁死亡标记物 COX2 和 4-HNE 的蛋白水平增加，其中HERC2 / VAMP8的双 KO 表现出更明显的表型比单一 KO ( Fig. 5A ）。此外，通过 FerroOrange 测量， HERC2的 KO 或双重 KO 显着增加了游离铁水平（ Fig. 5B ，FerroOrange 面板）和 Ferene-S 比色测定（ Fig. 5B ，条形图，底部），但铁蛋白笼中的三价铁含量减少（ Fig. 5B ，Perls 染色）。一致地，KO 增强了铁死亡细胞死亡（ Fig. 5, C and D ）和细胞存活率降低（ Fig. 5E ）。 IKE 处理的 WT 细胞中铁死亡细胞的百分比约为 20%，而HERC2 / VAMP8双 KO 细胞中的铁死亡细胞百分比约为 75%（ Fig. 5D ），24 小时时， NFE2L2/NRF2 WT 细胞活力下降约 30%，而HERC2 / VAMP8双 KO 细胞活力下降约 80%（ Fig. 5E ）。对 IKE 处理后NFE2L2/NRF2 WT 和不同 KO 细胞系中铁死亡细胞的百分比进行比较，结果显示， HERC2 / VAMP8双 KO 的细胞表现出与NFE2L2/NRF2 KO 细胞相当的铁死亡敏感性。 Table 1 ）。 这些结果表明， HERC2 / VAMP8双 KO 细胞的行为与NFE2L2/NRF2 KO 细胞相似，脱铁铁蛋白/NCOA4 的相似积累、LIP 增加、铁死亡标记物（COX2 和 4-HNE）增强以及对铁死亡细胞死亡的敏感性增加表明了这一点。 。

As shown in our model (Fig. 4), NFE2L2/NRF2 KO cells exhibit excessive accumulation of NCOA4, which is a critical factor in mediating the recruitment of apoferritin into autophagosomes, leading to the observed increase in the LIP and enhanced sensitivity to ferroptotic cell death. To fully confirm this mechanism, and also validate the NCOA4 siRNA knockdown results presented in fig. S4 (D to G), CRISPR NCOA4 KO NFE2L2/NRF2 WT and NFE2L2/NRF2 KO cell lines were generated. NCOA4 KO in NFE2L2/NRF2 WT cells enhanced ferroptosis as indicated by increased COX2 and 4-HNE (Fig. 5F), which was accompanied by increased ferroptosis and decreased survival (Fig. 5, H, I, and J). A marked contrast was observed when NCOA4 was knocked out in NFE2L2/NRF2 KO cells, as NCOA4 deletion reduced FTH1 protein levels, reduced ferroptosis markers COX2 and 4-HNE (Fig. 5F), decreased LIP as measured by both FerroOrange (Fig. 5G, FerroOrange panel) and Ferene-S colorimetic assay (Fig. 5G, bar graph, bottom), increased ferric iron–loaded ferritin cages (Fig. 5G, Perls staining), decreased percentage of ferroptotic cells (Fig. 5, H and I), and restored NFE2L2/NRF2 KO cell survival following IKE treatment to that of NFE2L2/NRF2 WT cells (Fig. 5J). In addition, overexpression of VAMP8 in NFE2L2/NRF2 KO SKOV3 cells alleviated autophagy blockage, as indicated by reduced protein levels of FTH1, NCOA4, and LC3-I/II (Fig. 5K), as well as reduced ferroptosis sensitivity in IKE-treated NFE2L2/NRF2 KO but not NFE2L2/NRF2 WT cells (Fig. 5L). Together, these results demonstrate the significance of NRF2-mediated iron sensing, by controlling ferritin synthesis/degradation, in determining a cancer cell’s response to ferroptosis inducers.
如我们的模型所示（ Fig. 4 ）， NFE2L2/NRF2 KO细胞表现出NCOA4的过度积累，这是介导脱铁铁蛋白募集到自噬体中的关键因素，导致观察到的LIP增加和对铁死亡细胞死亡的敏感性增强。为了充分证实这一机制，并验证图 1 所示的NCOA4 siRNA 敲低结果。生成了 S4（D 至 G）、CRISPR NCOA4 KO NFE2L2/NRF2 WT 和NFE2L2/NRF2 KO 细胞系。 COX2 和 4-HNE 增加表明NFE2L2/NRF2 WT 细胞中的NCOA4 KO 增强了铁死亡（ Fig. 5F ），伴随着铁死亡增加和存活率下降（ Fig. 5, H, I, and J ）。当在NFE2L2/NRF2 KO 细胞中敲除NCOA4时，观察到显着的对比，因为NCOA4缺失降低了 FTH1 蛋白水平，降低了铁死亡标记物 COX2 和 4-HNE（ Fig. 5F ），通过 FerroOrange 测量，LIP 降低（ Fig. 5G ，FerroOrange 面板）和 Ferene-S 比色测定（ Fig. 5G ，条形图，底部），增加三价铁负载铁蛋白笼（ Fig. 5G ，Perls 染色），铁死亡细胞百分比下降（ Fig. 5, H and I ），并将 IKE 处理后的NFE2L2/NRF2 KO 细胞存活率恢复至NFE2L2/NRF2 WT 细胞的存活率（ Fig. 5J ）。此外， NFE2L2/NRF2 KO SKOV3 细胞中VAMP8的过表达减轻了自噬阻断，FTH1、NCOA4 和 LC3-I/II 的蛋白水平降低表明了这一点。 Fig. 5K ），以及 IKE 处理的NFE2L2 / NRF2 KO 细胞中铁死亡敏感性降低，但NFE2L2 / NRF2 WT 细胞没有降低（ Fig. 5L ）。 总之，这些结果证明了 NRF2 介导的铁感应通过控制铁蛋白合成/降解在确定癌细胞对铁死亡诱导剂的反应中的重要性。

NRF2 expression correlates with HERC2 and VAMP8 levels in human cancer tissues, as well as ferroptosis resistance of cancer cell lines
NRF2 表达与人类癌症组织中的 HERC2 和 VAMP8 水平以及癌细胞系的铁死亡抗性相关

The translational relevance of this study was evaluated using human ovarian patient tissues to test whether there is a correlation between the expression of HERC2, VAMP8, and NRF2. NRF2 protein levels correlated strongly with HERC2 and VAMP8 (SLC7A11, a known NRF2 target gene, was used as a positive control) (Fig. 6A and fig. S5A). Notably, NRF2 expression was low in normal tissues but up-regulated in tumors (Fig. 6A, red versus black dots). Similarly, there was a positive correlation between NRF2 and HERC2, as well as NRF2 and VAMP8 across a wide variety of different ovarian cancer cell lines (Fig. 6B). Normal ovarian cell lines FT-246 and OSE were excluded in the correlation curve because of the low levels of HERC2 and VAMP8 (Fig. 6B). In support of our model, most cancer cell lines showed a negative correlation between the levels of NRF2 and either FTH1 or NCOA4 (Fig. 6B). Next, the response of these cell lines to ferroptosis and other mode of cell death inducers was measured (fig. S5B) and the 50% growth inhibition concentration (GI₅₀) for each individual compound was calculated (Table 3). As expected, a positive correlation between NRF2 expression and GI₅₀ values of the different ferroptosis inducers among these cell lines was observed (Fig. 6C, top two rows). In contrast, there was no correlation between NRF2 and GI₅₀ values of the other cytotoxic compounds that do not induce ferroptosis, including chemotherapeutic drugs (cisplatin and mitomycin C) and the apoptosis inducer staurosporine (Fig. 6C, bottom row). The finding that NRF2 determines the resistance of ovarian cancer cell lines to ferroptosis inducers was further illustrated in a heatmap indicating the GI₅₀ profiles of these cell lines in response to ferroptotic inducers, as the three NFE2L2/NRF2 KO SKOV-3 cell lines were clustered as the most sensitive to all ferroptotic inducers, far apart from WT SKOV-3 (Fig. 6D, top). In contrast, the sensitivity of the three NFE2L2/NRF2 KO SKOV-3 cell lines to other compounds in relation to the other cell lines tested was only increased slightly (Fig. 6D, bottom).
本研究的翻译相关性使用人类卵巢患者组织进行评估，以测试 HERC2、VAMP8 和 NRF2 的表达之间是否存在相关性。 NRF2 蛋白水平与 HERC2 和 VAMP8 密切相关（已知 NRF2 靶基因 SLC7A11 用作阳性对照）（ Fig. 6A 和图。 S5A）。值得注意的是，NRF2 表达在正常组织中较低，但在肿瘤中表达上调。 Fig. 6A ，红点与黑点）。同样，在多种不同的卵巢癌细胞系中，NRF2 和 HERC2 以及 NRF2 和 VAMP8 之间存在正相关性。 Fig. 6B ）。由于 HERC2 和 VAMP8 水平较低，正常卵巢细胞系 FT-246 和 OSE 被排除在相关曲线中（ Fig. 6B ）。为了支持我们的模型，大多数癌细胞系显示 NRF2 水平与 FTH1 或 NCOA4 水平呈负相关（ Fig. 6B ）。接下来，测量这些细胞系对铁死亡和其他细胞死亡诱导剂模式的反应（图 S5B），并计算每种化合物的 50% 生长抑制浓度（GI ₅₀ ）（ Table 3 ）。正如预期的那样，在这些细胞系中观察到 NRF2 表达与不同铁死亡诱导剂的 GI ₅₀值呈正相关（ Fig. 6C ，前两行）。相比之下，其他不诱导铁死亡的细胞毒性化合物的 NRF2 和 GI ₅₀值之间没有相关性，包括化疗药物（顺铂和丝裂霉素 C）和凋亡诱导剂星形孢菌素（staurosporine）。 Fig. 6C ，底行）。 NRF2 决定卵巢癌细胞系对铁死亡诱导剂的抗性这一发现在热图中得到了进一步说明，该热图显示了这些细胞系响应铁死亡诱导剂的 GI ₅₀概况，因为三个NFE2L2/NRF2 KO SKOV-3 细胞系聚集在一起与 WT SKOV-3 相比，它是对所有铁死亡诱导剂最敏感的（ Fig. 6D ， 顶部）。相比之下，与测试的其他细胞系相比，三种NFE2L2/NRF2 KO SKOV-3 细胞系对其他化合物的敏感性仅略有增加（ Fig. 6D ， 底部）。