Targeting PKD2 aggravates ferritinophagy-mediated ferroptosis via promoting autophagosome-lysosome fusion and enhances efficacy of carboplatin in lung adenocarcinoma 靶向 PKD2 通过促进自噬体-溶酶体融合加重铁蛋白自噬介导的铁死亡，并增强卡铂在肺腺癌中的疗效
PKD2 promoted proliferation, migration and invasion of LUAD cells in vitro and vivo. PKD2在体外和体内促进LUAD细胞的增殖、迁移和侵袭。
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PKD2 inhibited ferritinophagy in LUAD via preventing autophagosome-lysosome fusion. PKD2 通过阻止自噬体-溶酶体融合来抑制 LUAD 中的铁蛋白自噬。
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Targeting PKD2 could enhance the sensitivity of LUAD cells to carboplatin via inducing ferroptosis and apoptosis. 靶向 PKD2 可以通过诱导铁死亡和细胞凋亡来增强 LUAD 细胞对卡铂的敏感性。
Abstract 抽象的
Ferroptosis is an iron-dependent cell death and affects efficacies of multiple antitumor regimens, showing a great potential in cancer therapy. Protein kinase D2 (PKD2) plays a crucial role in regulating necrosis and apoptosis. However, the relationship of PKD2 and ferroptosis is still elusive. In this study, we mainly analyzed the roles of PKD2 on ferroptosis and chemotherapy in lung adenocarcinoma (LUAD). We found PKD2 was highly expressed in LUAD and silencing PKD2 could promote erastin-induced reactive oxygen species (ROS), malondialdehyde (MDA) accumulation, intracellular iron content and LUAD cells death. Mechanistically, augmenting PKD2 could prevent autophagic degradation of ferritin, which could be impaired by bafilomycin A1. We further found that PKD2 overexpression would promote LC3B-II, p62/SQSTM1 accumulation and block autophagosome-lysosome fusion in a TFEB-independent manner, which could be impaired by bafilomycin A1. Bafilomycin A1 stimulation could weaken ferroptosis promotion by PKD2 abrogation. Silencing ferritin heavy chain-1 (FTH1) could reverse the resistance to ferroptosis by PKD2 overexpression. Additionally, in vitro and vivo experiments validated PKD2 promoted proliferation, migration and invasion of LUAD cells. PKD2 knockdown or pharmacological inhibition by CRT0066101 could enhance efficacy of carboplatin in LUAD via ferroptosis and apoptosis. Collectively, our study revealed that abrogation of PKD2 could aggravate ferritinophagy-mediated ferroptosis by promoting autophagosome-lysosome fusion and enhance efficacy of carboplatin in LUAD. Targeting PKD2 to induce ferroptosis may be a promising strategy for LUAD therapy. 铁死亡是一种铁依赖性细胞死亡，会影响多种抗肿瘤方案的疗效，在癌症治疗中显示出巨大的潜力。蛋白激酶D2 (PKD2) 在调节坏死和细胞凋亡中起着至关重要的作用。然而，PKD2 与铁死亡的关系仍不清楚。在本研究中，我们主要分析了PKD2在肺腺癌（LUAD）铁死亡和化疗中的作用。我们发现 PKD2 在 LUAD 中高表达，沉默 PKD2 可以促进erastin诱导的活性氧（ROS）、丙二醛（MDA）积累、细胞内铁含量和LUAD细胞死亡。从机制上讲，增强 PKD2 可以防止铁蛋白的自噬降解，而巴弗洛霉素 A1 可能会损害铁蛋白的自噬降解。我们进一步发现，PKD2 过表达会促进 LC3B-II、p62/SQSTM1 积累，并以不依赖 TFEB 的方式阻断自噬体-溶酶体融合，而巴弗洛霉素 A1 可能会损害这种融合。 Bafilomycin A1 刺激可减弱 PKD2 消除对铁死亡的促进作用。沉默铁蛋白重链 1 (FTH1) 可以逆转 PKD2 过表达对铁死亡的抵抗力。此外，体外和体内实验验证了 PKD2 促进 LUAD 细胞的增殖、迁移和侵袭。 PKD2 敲低或 CRT0066101 的药理学抑制可以通过铁死亡和细胞凋亡增强卡铂在 LUAD 中的疗效。总的来说，我们的研究表明，废除 PKD2 可以通过促进自噬体-溶酶体融合来加重铁蛋白自噬介导的铁死亡，并增强卡铂在 LUAD 中的疗效。靶向 PKD2 诱导铁死亡可能是 LUAD 治疗的一个有前途的策略。
Lung cancer is the leading cause of cancer death worldwide and causes a great burden for human public health [1]. Lung adenocarcinoma (LUAD) is the most common pathological subtype, accounting for about 40% of all lung cancer cases [2]. Platinum-based chemotherapy still plays a pivotal role in the first-line treatment of advanced LUAD patients. However, the development of resistance to chemotherapy remains a major challenge. Extensive evidence has demonstrated that platinum combined with other antitumor regimens such as immunotherapy, radiotherapy can enhance chemotherapy sensitivity and improve outcomes of patients [3,4]. Combination administration for refractory LUAD has become a trend in clinical practice. 肺癌是全球癌症死亡的主要原因，给人类公共卫生造成巨大负担[1]。肺腺癌(LUAD)是最常见的病理亚型，约占所有肺癌病例的40%[2]。铂类化疗在晚期 LUAD 患者的一线治疗中仍然发挥着关键作用。然而，化疗耐药性的发展仍然是一个重大挑战。大量证据表明，铂类联合其他抗肿瘤方案如免疫治疗、放疗等可增强化疗敏感性，改善患者预后[3 , 4 ]。难治性LUAD的联合用药已成为临床实践的趋势。
Ferroptosis emerges as an iron-dependent and reactive oxygen species (ROS)-reliant cell death that differs from other forms of programmed cell death like apoptosis, pyroptosis. It has been reported that ferroptosis can be modulated by selective autophagy via degradation of ferritin, lipid droplets, circadian proteins, and GPX4 [5,6]. Ferritin is an iron storage protein complex comprising 24 subunits, including two basic subunits: a heavy subunit (FTH1) and a light subunit (FTL). Ferritin prevents Fe2+ from being oxidized by reactive oxygen species (ROS). NCOA4, a selective autophagy receptor, mediates the degradation of ferritin and release of irons through autophagosome-lysosome pathway [7]. This process is called ferritinophagy. The studies have revealed that multiple mechanisms are involved in inhibiting ferritinophagy of tumor cells to resist ferroptosis. PCBP1 represses ferritinophagy-mediated ferroptosis in head and neck cancer via repressing BECN1 and ALOX15 mRNAs [8]. TRIM11 is overexpressed and suppressed ferritinophagy through UBE2N-TAX1BP1 signaling in pancreatic ductal adenocarcinomas [9]. Intriguingly, some studies have reported that activation of ferroptosis by manipulating ferritinophagy can exert strong antitumor effects. Dihydroartemisinin can induce ferroptosis and suppress proliferation of leukemia cells by promoting ferritinophagy [10]. TMEM164 plays a key role in ATG5-dependent autophagosome formation during ferroptosis and mediates autophagic degradation of ferritin, GPX4, and lipid droplets to increase iron content and lipid peroxidation, thereby promoting ferroptosis. Abrogation of TMEM164 limits antitumor effects of ferroptosis-mediated cytotoxicity [11]. Additionally, numerous studies have suggested that inducing ferroptosis can reverse resistance of multiple antitumor drugs including chemotherapy, targeted therapy, immunotherapy etc. [12]. Consequently, inducing ferritinophagy-dependent ferroptosis is a promising therapeutic strategy for cancer. 铁死亡是一种铁依赖性和活性氧(ROS) 依赖性细胞死亡，与细胞凋亡、细胞焦亡等其他形式的程序性细胞死亡不同。据报道，铁死亡可以通过选择性自噬通过铁蛋白、脂滴、昼夜节律蛋白和GPX4的降解来调节[5 , 6]。铁蛋白是一种铁储存蛋白复合物，包含 24 个亚基，其中包括两个基本亚基：重亚基 (FTH1) 和轻亚基 (FTL)。铁蛋白可防止 Fe2+被活性氧(ROS) 氧化。 NCOA4是一种选择性自噬受体，通过自噬体-溶酶体途径介导铁蛋白的降解和铁的释放[7]。这个过程称为铁蛋白自噬。研究表明，抑制肿瘤细胞的铁蛋白自噬以抵抗铁死亡涉及多种机制。 PCBP1通过抑制BECN1和 ALOX15 mRNA来抑制头颈癌中铁蛋白自噬介导的铁死亡 [8]。 TRIM11 在胰腺导管腺癌中过表达并通过 UBE2N-TAX1BP1 信号传导抑制铁蛋白自噬 [ 9]。有趣的是，一些研究报道，通过操纵铁蛋白自噬来激活铁死亡可以发挥强大的抗肿瘤作用。双氢青蒿素可通过促进铁蛋白自噬来诱导铁死亡并抑制白血病细胞的增殖[10]。 TMEM164在铁死亡过程中ATG5依赖性自噬体形成中发挥关键作用，并介导铁蛋白、 GPX4和脂滴的自噬降解，以增加铁含量和脂质过氧化，从而促进铁死亡。 TMEM164 的废除限制了铁死亡介导的细胞毒性的抗肿瘤作用[11]。此外，大量研究表明，诱导铁死亡可以逆转多种抗肿瘤药物的耐药性，包括化疗、靶向治疗、免疫治疗等[12 ]。因此，诱导铁蛋白自噬依赖性铁死亡是一种有前途的癌症治疗策略。
Protein kinase D2 (PKD2) belongs to a family of serine/threonine protein kinases operating in the signaling network of the second messenger diacylglycerol (DAG) [13]. PKD2 can be activated by various cellular stimuli, such as phorbol esters, G protein-coupled receptor (GPCR) agonists, growth factors, hormones, cellular stress, and cytokines/chemokines. It has now emerged as a key signaling node in many pathological conditions, including cancer, metabolic disorders, cardiac diseases, central nervous system disorders, inflammatory diseases, and immune dysregulation. PKD2 has been reported to be upregulated in most tumors and induce angiogenesis, inhibit apoptosis of prostate, pancreas, gastric and glioblastoma cancer by activating NF-kB, MMP signaling pathways [[14], [15], [16], [17]]. Elevated PKD2 also positively regulates epithelial-to-mesenchymal transition (EMT) to promote metastasis of lung adenocarcinoma and indicates poor prognosis [18]. In cardiomyocytes, PKD depletion will promote autophagy and inhibit hypertrophy via AKT/mTOR/S6K pathway [19]. However, 2,3′,4,4′,5-Pentachlorobiphenyl (PCB118) has been shown to induce autophagy of thyrocytes via DAPK2/PKD/VPS34 pathway [20]. The effects of PKD on autophagy are controversial. As a result, whether the effects of PKD on autophagy are context-dependent and whether PKD regulate ferritinophagy-dependent ferroptosis still need further exploration. 蛋白激酶D2 (PKD2) 属于丝氨酸/苏氨酸蛋白激酶家族，在第二信使二酰甘油(DAG) 信号网络中发挥作用 [13 ]。 PKD2 可以被各种细胞刺激激活，例如佛波酯、G 蛋白偶联受体 (GPCR) 激动剂、生长因子、激素、细胞应激和细胞因子/趋化因子。它现在已成为许多病理状况的关键信号节点，包括癌症、代谢紊乱、心脏病、中枢神经系统疾病、炎症性疾病和免疫失调。据报道，PKD2 在大多数肿瘤中上调，并通过激活 NF-kB、MMP 信号通路诱导血管生成、抑制前列腺癌、胰腺癌、胃癌和胶质母细胞瘤细胞凋亡 [ [14] 、 [15] 、 [16] 、 [17]]。 PKD2升高还积极调节上皮间质转化（EMT），促进肺腺癌转移并提示不良预后[18 ]。在心肌细胞中，PKD 耗竭将通过 AKT/mTOR/S6K 通路促进自噬并抑制肥大 [ 19 ]。然而，2,3',4,4',5-五氯联苯 (PCB118) 已被证明可通过 DAPK2/PKD/VPS34 途径诱导甲状腺细胞自噬 [ 20 ]。 PKD 对自噬的影响存在争议。 因此，PKD 对自噬的影响是否具有背景依赖性以及 PKD 是否调节铁蛋白自噬依赖性铁死亡仍需要进一步探索。
In this study, we investigated the roles of PKD2 on autophagy and ferroptosis in LUAD. Abrogation of PKD2 could promote autophagy and ferroptosis, which could be impaired by inhibitors of autophagosome-lysosome fusion (bafilomycin A1, chloroquine (CQ)). Mechanistically, PKD2 inhibited degradation of ferritin through blocking autophagosome-lysosome fusion in a TFEB-independent manner. In addition, we validated that PKD2 could promote the proliferation, migration, and invasion of LUAD cells. PKD2 knockdown or inhibitor in combination with carboplatin could further enhance antitumor efficiency of carboplatin via inducing ferroptosis and apoptosis in LUAD. These findings suggested PKD2 could alleviate ferritinophagy-mediated ferroptosis via preventing autophagosome-lysosome fusion in LUAD and might represent a promising candidate to sensitize LUAD patients to carboplatin. 在本研究中，我们研究了 PKD2 对 LUAD 中自噬和铁死亡的作用。废除 PKD2 可能会促进自噬和铁死亡，而自噬体-溶酶体融合抑制剂（巴弗洛霉素 A1、氯喹(CQ)）可能会损害自噬和铁死亡。从机制上讲，PKD2 通过以不依赖 TFEB 的方式阻断自噬体-溶酶体融合来抑制铁蛋白的降解。此外，我们验证了PKD2可以促进LUAD细胞的增殖、迁移和侵袭。 PKD2敲低或抑制剂与卡铂联用可以通过诱导LUAD中铁死亡和细胞凋亡进一步增强卡铂的抗肿瘤效率。这些发现表明 PKD2 可以通过阻止 LUAD 中的自噬体-溶酶体融合来减轻铁蛋白自噬介导的铁死亡，并且可能是使 LUAD 患者对卡铂敏感的有希望的候选者。
2. Materials and methods 2 材料与方法
2.1. Cell culture and transfection 2.1.细胞培养和转染
All cell lines (A549, PC9 and HEK) were obtained from the Procell, Wuhan, China. A549, PC9, and HEK cells were cultured in F12K (HyClone, USA), RPMI H1640 (HyClone, USA), and dulbecco's modified eagle medium (DMEM) (HyClone, USA), respectively, supplemented with 10% fetal bovine serum (BI, Israel) in a humidified atmosphere of 5% CO2 and 37 °C according to protocol. All of the cell culture media contained 100 U/mL penicillin and 100 mg/mL streptomycin. 所有细胞系（A549、PC9 和 HEK）均获自 Procell，武汉，中国。 A549、PC9和HEK细胞分别在F12K（HyClone，美国）、RPMI H1640（HyClone，美国）和杜尔贝科改良鹰培养基（DMEM）（HyClone，美国）中培养，并补充有10％胎牛血清（BI） ，以色列）根据协议在 5% CO 2和 37 °C的潮湿气氛中进行。所有细胞培养基均含有 100 U/mL青霉素和 100 mg/mL链霉素。
GFP-PKD2 plasmids (from Peter Storz, Department of Cancer Biology, Mayo Clinic), HA-PKD2 plasmids (from MiaoLingPlasmid, Wuhan, China) and siRNA duplexes against PKD2 or FTH1 were transfected into cells using jetPRIME (Polyplus-transfection, Illkirch, France) for transient transfection according to the manufacturer's instructions. The siRNA duplex sense sequences were as follows: 使用jetPRIME (Polyplus -transfection , Illkirch,法国）根据制造商的说明进行瞬时转染。 siRNA双链有义序列如下：
Lentiviruses were produced using HEK293T cells co-transfected with PKD2shRNA plasmid (from Derek C. Radisky, Department of Cancer Biology, Mayo Clinic), pSPAX2 (Addgene, USA), 2 μg pMD2.G (Addgene, USA) and EZ Trans (Life iLAB Biotech, Shanghai, China). 使用与 PKD2 shRNA质粒（来自 Mayo Clinic 癌症生物学系 Derek C. Radisky）、pSPAX2（Addgene，美国）、2 μg pMD2.G（Addgene，美国）和 EZ Trans（来自 Derek C. Radisky）共转染的 HEK293T 细胞产生慢病毒。 Life iLAB Biotech，上海，中国）。
Medium containing lentivirus was collected 48 h after transfection. For lentiviral transduction in a 12-well plate, 500 μl regular medium, 500 μl viral supernatant and 1 μl polybrene were used. After 24 h the virus-containing medium was renewed by the fresh virus-containing medium. Then transduced tumor cells were selected with puromycin. 转染后48小时收集含有慢病毒的培养基。对于 12 孔板中的慢病毒转导，使用 500 μl 常规培养基、500 μl 病毒上清液和 1 μl Polybrene 。 24小时后，用新鲜的含病毒培养基更新含病毒培养基。然后用嘌呤霉素选择转导的肿瘤细胞。
2.2. Chemicals 2.2.化学品
Autophagy inducer, rapamycin and Earle's Balanced Salt Solution (EBSS) were purchased from MedChemExpress and Beyotime, respectively. Ferroptosis inducer, erastin, and autophagic flux blocker, Bafilomycin A1 and chloroquine (CQ) were also purchased from MedChemExpress. 自噬诱导剂、雷帕霉素和厄尔平衡盐溶液(EBSS)分别购自MedChemExpress和Beyotime。铁死亡诱导剂、erastin 和自噬流阻滞剂、巴弗洛霉素A1 和氯喹(CQ) 也购自 MedChemExpress。
2.3. Bioinformatic analyses 2.3.生物信息学分析
The expression of PKD2 in pan-cancer was obtained from TIMER 2.0 (http://timer.cistrome.org/). The analyses of drug sensitivity between high and low PKD2 expression groups were performed by R package ‘pRRophetic’. TCGALUAD samples were ranked by PKD2 expression and the top and the bottom 100 samples were selected for Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and Gene Ontology (GO) pathway analyses performed by GSEA software (version 4.2.3). The results of GO pathway analyses were visualized by Bioinformatics website (http://www.bioinformatics.com.cn/). PKD2在泛癌中的表达从TIMER 2.0获得（http://timer.cistrome.org/）。通过 R 包“pRRophetic”进行高 PKD2 表达组和低 PKD2 表达组之间的药物敏感性分析。 TCGALUAD样本按照 PKD2 表达量进行排序，并选择前 100 个样本进行京都基因与基因组百科全书 (KEGG) 富集分析和通过 GSEA 软件（版本 4.2.3）进行基因本体(GO) 通路分析。 GO通路分析结果通过生物信息学网站（http://www.bioinformatics.com.cn/ ）可视化）。
2.4. Specimens collection 2.4.标本采集
Eight LUAD and the corresponding paracarcinoma tissues were collected from the Department of Thoracic Surgery, Shandong Provincial Hospital. In addition, LUAD slides were obtained for immunohistochemical (IHC) staining from the Department of Pathology, Shandong Provincial Hospital. A IHC microarray including 61 LUAD cases, 5 paracarcinoma samples and 9 normal samples was purchased from Bioaitech (Xian, China). The project has been approved by the Ethics Committee of Shandong Province Hospital (approval number: SWYX: NO. 2022-558). The requirement for written informed consent was waived.
2.5. RNA extraction and quantitative real-time PCR
Briefly, total RNA was extracted using AG RNAex Pro Reagent (Accurate Biotechnology, Hunan, China) and was reverse-transcribed by Evo M-MLV RT kit (Accurate Biotechnology, Hunan, China) according to the protocol. Gene expression levels were detected by qRT-PCR using the Roche LightCycler ® 480 system and SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China). All primers used were listed in Supplementary Table 1.
2.6. Western blot
Total proteins were extracted from cells by RIPA lysis buffer and the concentration was measured by BCA protein assay kit (Beyotime, Shanghai, China). Nuclear and cytoplasmic proteins were extracted and collected according to the manufacturer's protocol (Beyotime, Shanghai, China). The total proteins or nuclear and cytoplasmic proteins extract were separated by SDS-PAGE and blotted onto nitrocellulose (NC) membrane (Millipore, Billerica, USA). 5% BSA solution was used to block and then the membrane was incubated with primary antibody at 4 °C overnight. The primary antibodies were listed as follows: PKD2, GAPDH, FTH1, SQSTM1/P62, Beclin1, Bcl-2 (Santa Cruz, USA, 1:1000); LC3B, ULK1, TFEB, SLC40A1, GPX4, FTL (Proteintech, USA, 1:1000); PARP1 (Abgent, USA, 1:1000); P-ULK1 (S317), VPS34 (CST, USA, 1:2000). After washing with TBST solution, the membrane was incubated with corresponding HRP-labeled secondary antibody (Santa Cruz, CA, USA: 1:10000) for 1h. The ECL kit and FluorChem E system were used for detection (Proteinsimple, CA, USA).
2.7. IHC staining
The tissue sections fixed with formalin and embedded in paraffin blocks were used for IHC staining. The slides were deparaffinized in xylene, rehydrated through graded alcohol solutions, blocked in 3% H2O2 solution. Citrate-EDTA antigen retrieval solution (Beyotime, Shanghai, China) was used for antigen repairing at 96–98 °C for 15 min. Anti-PKD2 antibody (dilution 1:200, Santa Cruz, CA, USA), anti-Ki67 antibody (dilution 1:200, Santa Cruz, CA, USA) and anti-FTH1 antibody (dilution 1:200, Santa Cruz, CA, USA) were used as the primary antibodies at 4 °C overnight, respectively. Then, they were then probed with horseradish peroxidase-conjugated anti-rabbit secondary antibody for 30 min at 37 °C followed by incubation with horseradish peroxidase (HRP)-labeled streptavidin for 30 min. The slides were stained by (diaminobenzidine) DAB and counterstained by hematoxylin. Finally, the results were assessed by a light microscope. 将用福尔马林固定并包埋在石蜡块中的组织切片用于IHC染色。将载玻片在二甲苯中脱蜡，通过梯度醇溶液再水化，在3％H2 O 2溶液中封闭。使用柠檬酸盐-EDTA抗原修复液（Beyotime，上海，中国）在96-98℃下进行抗原修复15分钟。抗 PKD2 抗体（稀释 1:200，Santa Cruz，CA，USA）、抗 Ki67 抗体（稀释 1：200，Santa Cruz，CA，USA）和抗 FTH1 抗体（稀释 1：200，Santa Cruz，CA） ，美国）分别用作一抗，4℃过夜。然后，用辣根过氧化物酶缀合的抗兔二抗在 37°C 下探测 30 分钟，然后与辣根过氧化物酶 (HRP) 标记的链霉亲和素一起孵育 30 分钟。载玻片用（二氨基联苯胺）DAB 染色并用苏木精复染。最后，通过光学显微镜评估结果。
Two pathologists were invited to score these specimens according to the intensity of dyed color and the percentage of positive cells stained independently. The intensity of staining was graded as: 0, no color; 1, light yellow; 2, light brown; 3, deep brown, and the percentage of positive cells stained was graded as: 0 (<5%), 1 (5%–25%), 2 (25%–50%), 3 (50%∼75%), 4 (>75%). The IHC score calculated by proportion of positive cells multiplied by the corresponding intensity was used as the final staining score (0–12). The final staining score ≤4 was regarded as low expression and >4 was high expression. 邀请两名病理学家根据染色颜色的强度和染色的阳性细胞的百分比独立对这些标本进行评分。染色强度分级为：0，无颜色； 1、淡黄色； 2、浅棕色； 3，深棕色，阳性细胞染色百分比分级为：0（<5%）、1（5%~25%）、2（25%~50%）、3（50%~75%）、 4（>75%）。通过阳性细胞比例乘以相应强度计算出的 IHC 评分用作最终染色评分（0-12）。最终染色评分≤4为低表达，>4为高表达。
2.8. Proliferation assays 2.8.增殖分析
Cells were resuspended and seeded in 96-well plates with a density of 3000 cells per well. After adherence, the first plate was fixed with 10% cold trichloroacetic acid for at least 24 h. Then the other samples were collected in this way at the indicated time. After washing and drying, the plates were stained by Sulforhodamine B sodium salt (SRB) (Sigma, USA) for 30 min and washed by 1% (vol/vol) acetic acid. After drying, 150 μl 10 mmol/L Tris was added and the absorbance was measured at 562 nm in a microplate reader (Thermo Fisher, USA). 将细胞重悬并接种于96孔板中，密度为每孔3000个细胞。粘附后，将第一块板用 10% 冷三氯乙酸固定至少 24 小时。然后在指定时间以这种方式收集其他样本。洗涤和干燥后，用磺胺罗丹明 B钠盐 (SRB) (Sigma, USA) 对板染色 30 分钟，并用 1% (vol/vol) 乙酸洗涤。干燥后，加入150 μl 10 mmol/LTris ，在酶标仪（Thermo Fisher，美国）中于562 nm处测量吸光度。
The assay was performed using EdU Cell Proliferation Kit with Alexa Fluor488 (Beyotime, Shanghai, China) and EdU Cell-Light EdU Apollo567 in Vitro Flow Cytometry Kit (Ribobio, Guangzhou, China) according to the manufacturer's protocol. Cells were incubated with 5 μM EdU for 2 h (Carboplatin treatment: EdU for 3h) and then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton X-100 for 20 min. The incorporated EdU was visualized by means of a click reaction and the nuclear DNA was stained with Hoechst for 10 min. The results were observed by using a fluorescence microscope (Olympus, Milan, Italy) and the percentage of EdU positive cells was calculated by Image J. 根据制造商的方案，使用 EdU Cell Proliferation Kit with Alexa Fluor488（Beyotime，上海，中国）和 EdU Cell-Light EdU Apollo567 in Vitro Flow Cytometry Kit（Ribobio，广州，中国）进行测定。将细胞与 5 μM EdU 一起孵育 2 小时（卡铂处理：EdU 3 小时），然后用 4% 多聚甲醛固定 10 分钟，并用 0.3% Triton X-100 透化 20 分钟。掺入的 EdU 通过点击反应可视化，核 DNA 用 Hoechst 染色 10 分钟。使用荧光显微镜（Olympus，米兰，意大利）观察结果，并通过Image J计算EdU阳性细胞的百分比。
2.10. Colony formation assay 2.10.集落形成测定
Cells were seeded in 6-well plates at a density of 1000 cells per well and cultured at 37 °C for 14 days. Then, the plates were washed with phosphate-buffered saline (PBS) and stained with crystal violet for 30 min. The number of the colonies was counted. 将细胞以每孔1000个细胞的密度接种在6孔板中，并在37°C培养14天。然后，用磷酸盐缓冲盐水（PBS）洗涤板并用结晶紫染色30分钟。计算集落的数量。
2.11. Wound healing assays 2.11.伤口愈合测定
After transfection, cells were seeded in 12-well plates for adherent culture. Then a sterile 200 μl pipette tip was used to make the scratches. The culture medium with 2% fetal bovine serum was added. The scratch area was captured every 12 h and the migration rate were measured by Image J. 转染后，将细胞接种于12孔板中进行贴壁培养。然后用无菌200μl移液器吸头进行划痕。加入含2%胎牛血清的培养基。每 12 小时捕获一次划痕区域，并通过 Image J 测量迁移率。
2.12. Cell migration and invasion 2.12.细胞迁移和侵袭
Cell migration and invasion assays were analyzed using the Transwell chambers assay (Costar, Corning Inc., Corning, NY, USA), with or without coated Matrigel (Corning). 40,000 cells were plated in the upper chamber in in the serum-free medium. The lower chambers were filled with 600 μl of the medium with 10% FBS. At the indicated time point, the non-migrated and non-invaded cells in the upper chambers were removed and the migrated or invaded cells were stained with crystal violet for 30 min. The images were observed by microscope and the numbers of migrated and invaded cells were calculated by Image J. 使用Transwell小室测定法（Costar，Corning Inc.，康宁，纽约，美国）分析细胞迁移和侵袭测定，有或没有包被的基质胶（康宁）。将 40,000 个细胞置于上室的无血清培养基中。下室填充 600 μl 含 10% FBS 的培养基。在指定时间点，除去上室中未迁移和未侵袭的细胞，并将迁移或侵袭的细胞用结晶紫染色30分钟。通过显微镜观察图像，通过Image J计算迁移和侵袭细胞的数量。
2.13. Autophagic flux detection 2.13.自噬通量检测
StubRFP-sensGFP-LC3 adenovirus (Gene, Shanghai, China) was used to monitor autophagic flux. Cells were infected by adenovirus according to the manufacturer's instructions. After 48h, fluorescence images were captured and autophagosomes (yellow dots in fusion images) and autolysosomes (red dots in fusion images) were counted by image J. StubRFP-sensGFP-LC3腺病毒（Gene，上海，中国）用于监测自噬流。根据制造商的说明用腺病毒感染细胞。 48小时后，捕获荧光图像，并通过图像J对自噬体（融合图像中的黄点）和自溶酶体（融合图像中的红点）进行计数。
2.14. Immunofluorescence 2.14.免疫荧光
Cells were plated on glass coverslips in 24-well plates. Cells were fixed with ice-cold 4% Paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. Then cells were washed with PBS, blocked in 10% goat serum albumin for 30 min. Anti-FTL (Proteintech, USA: 1:200), anti-NCOA4 (Santa Cruz, USA, 1:200), anti-PKD2 (Santa Cruz, USA, 1:200), anti-LAMP2 (Santa Cruz, USA, 1:200), anti-LC3B (Proteintech, USA, 1:200) and anti-TFEB (Proteintech, USA, 1:200) were primary antibodies. Cells were incubated by the primary antibodies at 4 °C overnight. Next, cells were rinsed with PBS and incubated with Alexa Fluor 594 and Alexa Fluor 488 for 1 h at room temperature in the dark and counterstained with Hoechst (Beyotime, Shanghai, China), mounted and examined using a fluorescence microscope (Olympus, Tokyo, Japan). The colocalization was analyzed by image J. 将细胞铺在 24 孔板的玻璃盖玻片上。将细胞用冰冷的 4% 多聚甲醛固定 20 分钟，并用 0.2% Triton X-100 透化 10 分钟。然后用PBS洗涤细胞，用10%山羊血清白蛋白封闭30分钟。抗FTL（Proteintech，美国：1:200）、抗NCOA4（Santa Cruz，美国，1:200）、抗PKD2（Santa Cruz，美国，1:200）、抗LAMP2（Santa Cruz，美国， 1:200)、抗 LC3B (Proteintech, USA, 1:200) 和抗 TFEB (Proteintech, USA, 1:200) 为一抗。细胞在 4°C 下用一抗孵育过夜。接下来，用 PBS 冲洗细胞，并与 Alexa Fluor 594 和 Alexa Fluor 488 在室温下避光孵育 1 小时，并用 Hoechst（Beyotime，上海，中国）复染，安装并使用荧光显微镜（Olympus，Tokyo，日本）。通过图像 J 分析共定位。
2.15. MDA, ROS and iron detection 2.15。 MDA、ROS 和铁检测
MDA content was detected according to the manufacturer's protocol (Beyotime, Shanghai, China). Protein concentration was measured by BCA protein assay kit (Beyotime, Shanghai, China). Finally, the ratio of MDA to protein concentration was calculated. ROS content was detected according to the manufacturer's protocol (Beyotime, Shanghai, China). Then, the ROS content was observed by a fluorescence microscope or flow cytometry. An iron assay kit (MAK025 Sigma‐Aldrich) was subsequently used to quantify total iron in the cell lysates according to manufacturer's instructions. Briefly, cells can be lysed in 4 volume of iron assay buffer, centrifuge at 16,000×g for 10 min to remove insoluble materials. Five μl of iron reducer were added into 50 μl samples for total iron (Fe3+ plus Fe2+) assay. Next, 100 μl iron probe solution was added and incubated at 25 °C for 60 min protected from light. Finally, the absorbance was detected at 593 nm wavelength. MDA含量根据制造商的方案进行检测（Beyotime，上海，中国）。采用BCA蛋白测定试剂盒（Beyotime，上海，中国）测定蛋白浓度。最后，计算MDA与蛋白质浓度的比率。根据制造商的方案（Beyotime，上海，中国）检测ROS含量。然后通过荧光显微镜或流式细胞术观察ROS含量。随后根据制造商的说明，使用铁测定试剂盒（MAK025 Sigma-Aldrich）对细胞裂解物中的总铁进行定量。简而言之，可以将细胞溶解在 4 倍体积的铁测定缓冲液中，以 16,000× g离心 10 分钟以除去不溶物质。将 5 μl 铁还原剂添加到 50 μl 样品中进行总铁（Fe 3+加 Fe 2+ ）测定。接下来，添加100μl铁探针溶液并在25℃避光孵育60分钟。最后在593 nm波长处检测吸光度。
2.16. Cell apoptosis 2.16。细胞凋亡
Apoptotic cells were detected by flow cytometry analysis, using the Annexin V-FITC/Propidium (PI) apoptosis detection kit (YEASEN, Shanghai, China) according to the manufacturer's protocol. Annexin V-FITC and PI were used as fluorescent identification dyes for apoptotic cells. The results were processed by FlowJo software. 根据制造商的方案，使用Annexin V-FITC/Propidium (PI)凋亡检测试剂盒(YEASEN，上海，中国)通过流式细胞术分析检测凋亡细胞。使用Annexin V-FITC和PI作为凋亡细胞的荧光识别染料。结果由FlowJo软件处理。
2.17. Calcein-AM and PI staining 2.17。钙黄绿素-AM 和 PI 染色
A549 and PC9 cells were seeded in 6-well plates and treated with corresponding chemicals. Cell viability and death were assessed using calcein-AM and PI double staining kit according to the manufacturer's protocol (Elabscience Biotechnology Co., Wuhan, China). The positive cells were observed by a fluorescence microscope and counted by Image J. A549 和 PC9 细胞接种在 6 孔板中并用相应的化学物质处理。根据制造商的方案（Elabscience Biotechnology Co.，Wuhan，China），使用钙黄绿素-AM和PI双染色试剂盒评估细胞活力和死亡。荧光显微镜观察阳性细胞，Image J计数。
2.18. Xenograft model
All mice were maintained under SPF housing and with a maximum of five mice per cage. The animal house was maintained at temperature of 22 ± 2 °C with relative humidity of 50 ± 15% and 12 h dark/light cycle. All experimental mice drank and ate freely. The sterilized lab rat and mouse diet was purchased from Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., China. We conducted all animal care and experiments in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines (http://www.aaalac.org) and with approval from the Committee for Ethics of Animal Experiments of Shandong Provincial Hospital on March 1st, 2023 (approval number: NO.NSFC 2023-405). BALB/c nude mice were obtained from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). A549 cells transfected with shNT or shPKD2 at a concentration of 2 × 106 mixed with 100 μl 50% Matrigel were implanted subcutaneously in the thighs of 4-week-old male nude mice. The tumor volumes were monitored every 6 days by Vernier caliper and tumor volume was indicated by a × b2/2 (a: long diameter; b: short diameter). The tumors were harvested on the 42nd day and analyzed.
2.19. Statistical analysis
The statistical analyses in this study were conducted by R (version 4.1.3), SPSS (version 26.0), Graphpad Prism 8. When data were quantitative, statistical significance for normally distributed variables was estimated by Student's t-tests, and nonnormally distributed variables were analyzed by the Wilcoxon rank sum test. When comparing more than two groups, Kruskal-Wallis tests and one-way analysis of variance were used as nonparametric and parametric methods, respectively. The correlation was tested by Spearman or Pearson Correlation test. Two-sided P < 0.05 was considered statistically significant unless otherwise stipulated. 本研究中的统计分析是通过R（版本4.1.3）、SPSS（版本26.0）、Graphpad Prism 8进行的。当数据是定量的时，正态分布变量的统计显着性通过Student's t检验估计，非正态分布变量通过 Wilcoxon 秩和检验进行分析。当比较两组以上时，分别使用 Kruskal-Wallis 检验和单因素方差分析作为非参数和参数方法。通过 Spearman 或 Pearson 相关性检验来检验相关性。除非另有规定，两侧P < 0.05 被认为具有统计学意义。
3. Results 3. 结果
3.1. PKD2 was highly expressed in LUAD and related to ferroptosis 3.1. PKD2 在 LUAD 中高表达并与铁死亡相关
To identify the expression of PKD2 across cancers, we first analyzed PKD2 mRNA expression in 32 cancers by TIMER 2.0 tool. PKD2 had significantly upregulated expression in most malignant tumors than corresponding normal tissues, including bladder cancer, breast cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma, LUAD etc. (P < 0.05). Moreover, metastatic skin cutaneous melanoma possessed higher PKD2 expression comparing to the primary one (Fig. 1A). Eight pairs of LUAD and paracarcinoma tissues were collected from Shandong Provincial Hospital to analyze PKD2 expression by qRT-PCR. Among them, 6 pairs tissues (75%) showed that PKD2 was upregulated in LUAD (P < 0.05) (Fig. 1B). Tumor microenvironment is complex and composed of tumor cells and non-cancerous host cells including fibroblasts, endothelial cells, multiple immune cells [21]. Considering PKD2 mRNA level in LUAD mass by qRT-PCR only reflected the mean level of various cells in tumor microenvironment and even a few normal lung tissues, the immunohistochemical staining was further conducted to detect PKD2 expression and distribution in tumor microenvironment. The surgical specimens of LUAD patients from Shandong provincial hospital were collected for the immunohistochemical staining. The results suggested that LUAD cells had the highest PKD2 expression followed by alveolus, stromal cells, which implied PKD2 expression was dependent on cell types (Fig. 1C). Moreover, PKD2 was mainly distributed in cytoplasm whether in LUAD cells or alveolus. A IHC microarray including 14 normal tissues and 61 LUAD tissues was obtained and the demographics was showed in Table 1. There was no statistically significant difference of PKD2 expression between age, sex, tumor stage subgroups, grade and histological subtype. However, the results of IHC microarray of LUAD exhibited that LUAD and paracarcinoma tissues both possessed higher PKD2 expression than normal lung tissues (P < 0.05) (Fig. 1D) and tumors in high PKD2 IHC scores group were characterized by bigger volume (P < 0.05) (Fig. 1E). Our previous study has suggested that high PKD2 expression promoted epithelial-mesenchymal transition and indicated worse prognoses of LUAD patients [18]. To further explore the underlying mechanism, GSEA enrichment analysis was conducted between high and low PKD2 expression groups. The results showed many malignant tumors initiation including endometrial cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer and multiple oncogenic pathways such as MAPK and Wnt signaling pathway were obviously enriched in high PKD2 expression group (Fig. 1F). Intriguingly, high PKD2 expression was also related to negative regulation of ferroptosis and low PKD2 expression group was predicted to be more sensitive to sorafenib, a ferroptosis inducer (P = 0.018) (Fig. 1G). Taken together, we speculated regulating ferroptosis may be another mechanism of PKD2 affecting LUAD progression and prognosis. 为了确定 PKD2 在不同癌症中的表达，我们首先使用 TIMER 2.0 工具分析了 32 种癌症中 PKD2 mRNA 的表达。 PKD2在大多数恶性肿瘤中的表达均较相应的正常组织显着上调，包括膀胱癌、乳腺癌、宫颈鳞癌、宫颈内膜腺癌、LUAD等(P<<0.05)。此外，与原发性皮肤黑色素瘤相比，转移性皮肤黑色素瘤具有更高的 PKD2 表达（图 1 A）。收集山东省立医院的8对LUAD和癌旁组织，通过qRT-PCR分析PKD2的表达。其中，6对组织（75%）显示PKD2在LUAD中表达上调（ P <0.05）（图1B）。肿瘤微环境复杂，由肿瘤细胞和非癌宿主细胞（包括成纤维细胞、内皮细胞、多种免疫细胞）组成[21 ]。考虑到qRT-PCR检测的LUAD肿块中PKD2 mRNA水平仅反映了肿瘤微环境中各种细胞甚至少数正常肺组织的平均水平，因此进一步进行免疫组化染色来检测肿瘤微环境中PKD2的表达和分布。采集山东省立医院LUAD患者的手术标本进行免疫组化染色。 结果表明，LUAD细胞的PKD2表达最高，其次是肺泡细胞、基质细胞，这意味着PKD2表达依赖于细胞类型（图1C）。此外，无论是在LUAD细胞还是在肺泡中，PKD2主要分布在细胞质中。获得了包括14个正常组织和61个LUAD组织的IHC微阵列，其人口统计数据显示在表1中。 PKD2表达在年龄、性别、肿瘤分期亚组、分级和组织学亚型之间无统计学差异。然而，LUAD的IHC微阵列结果显示，LUAD和癌旁组织均比正常肺组织具有更高的PKD2表达（ P <0.05）（图1D ），并且高PKD2IHC评分组的肿瘤体积较大（ P < 0.05) (图 1 E)。我们之前的研究表明，PKD2 高表达会促进上皮间质转化，并表明 LUAD 患者的预后较差[ 18]。为了进一步探讨其潜在机制，我们在 PKD2 高表达组和低表达组之间进行了 GSEA 富集分析。 结果显示，子宫内膜癌、非小细胞肺癌、胰腺癌、前列腺癌等多种恶性肿瘤的起始，以及MAPK、Wnt信号通路等多种致癌通路在PKD2高表达组中明显富集（图1F）。有趣的是，高PKD2表达也与铁死亡的负调节有关，并且低PKD2表达组预计对铁死亡诱导剂索拉非尼更敏感（P =0.018）（图1G ）。综上所述，我们推测调节铁死亡可能是 PKD2 影响 LUAD 进展和预后的另一种机制。
Fig. 1. PKD2 was highly expressed in LUAD and associated with ferroptosis. A PKD2 mRNA expression in TCGA tumors vs normal tissues (if available) by TIMER 2.0. B PKD2 mRNA expression in 8 pairs of LUAD and paracarcinoma tissues by qRT-PCR. C Representative immunohistochemical images of PKD2 in alveolus, mesenchyme, LUAD tissues. Left: 100X, scale bar, 200 μm; Right: 400X, scale bar, 50 μm. D IHC score of PKD2 in normal, paracarcinoma and LUAD tissues. E Comparison of tumor volume between PKD2 low- and high-score groups. F GSEA enrichment analyses. G Comparison of sensitivity of sorafenib between PKD2 high and low expression groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 图1 . PKD2 在 LUAD 中高表达并与铁死亡相关。通过 TIMER 2.0 比较TCGA肿瘤与正常组织（如果有）中的 PKD2 mRNA 表达。B通过 qRT-PCR 检测 8 对 LUAD 和癌旁组织中 PKD2 mRNA 的表达。 C肺泡、间充质、LUAD 组织中 PKD2 的代表性免疫组织化学图像。左：100X，比例尺，200 μm；右：400X，比例尺，50 μm。D正常组织、癌旁组织和 LUAD 组织中 PKD2 的 IHC 评分。 E PKD2低分组和高分组之间肿瘤体积的比较。 F GSEA 富集分析。 GPKD2高、低表达组索拉非尼敏感性比较。 *P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.0001。
Table 1. Demographics and clinicopathologic characteristics of LUAD patients from IHC microarray. 表 1 . IHC 微阵列中 LUAD 患者的人口统计学和临床病理特征。
To validate our hypothesis, we first established PKD2 overexpression and knockdown systems by using siRNA, shRNA and plasmid in A549 and PC9 cell lines, respectively. As shown in Supplementary Figs. 1A–1E, the systems were successfully established with high silencing and overexpression efficiency. The increasing of intracellular ROS levels is one of the most important hallmarks of ferroptosis. We detected the ROS levels by immunofluorescence assay. The results showed that erastin could significantly increase the ROS levels in A549 and PC9 cells. PKD2-deficient A549 cells had higher ROS levels than the control with erastin treatment and PC9 cells with PKD2 overexpression had lower ROS levels than the control with erastin treatment (Fig. 2A and B). ROS detected by flow cytometry also showed the consistent results that silencing PKD2 would accelerate ROS accumulation in A549 and PC9 cells treated by erastin (Fig. 2C and D). CRT0066101 is a specific PKD inhibitor and commonly used in the study on PKD function. When A549 and PC9 cells were treated by 10 μM CRT0066101 for 24h, there was an obvious elevation of ROS level (Fig. 2E and F). Increased MDA level was another marker of occurrence of ferroptosis. We found silencing PKD2 would significantly increase the MDA level in A549 and PC9 cells with erastin stimulation (Fig. 2G and H). In order to observe the cell death more intuitively, we detected the ratio of dead cells to the total cells by PI staining and flow cytometry. It was observed that silencing PKD2 could predominantly increase the ratio of dead cells to the total cells in A549 and PC9 cells (Fig. 2I and J). Excessive iron promotes ROS generation by the Fenton reaction and accelerates lipid peroxidation [22]. Intracellular free iron was measured by iron detection assay and the results showed that silencing PKD2 could significantly increase iron content in A549 and PC9 cells with erastin treatment (Fig. 2K, L). PKD2 overexpression showed the opposite results in A549 and PC9 cells (Fig. 2M, N). To sum up, the above results suggested that PKD2 could alleviate erastin-induced ferroptosis of LUAD cells. 为了验证我们的假设，我们首先分别在 A549 和 PC9 细胞系中使用siRNA 、shRNA 和质粒建立 PKD2 过表达和敲低系统。如补充图中所示。如图1A-1E所示，系统成功建立，具有高沉默和过表达效率。细胞内ROS水平的增加是铁死亡最重要的标志之一。我们通过免疫荧光法检测ROS水平。结果表明erastin可以显着提高A549和PC9细胞中ROS的水平。 PKD2缺陷的A549细胞的ROS水平高于erastin处理的对照细胞，而PKD2过表达的PC9细胞的ROS水平低于erastin处理的对照细胞（图2A和B）。流式细胞仪检测的ROS也显示出一致的结果，即沉默PKD2会加速erastin处理的A549和PC9细胞中ROS的积累（图2C和D）。 CRT0066101是一种特异性PKD抑制剂，常用于PKD功能研究。当A549和PC9细胞用10μM CRT0066101处理24小时时，ROS水平明显升高（图2E和F）。 MDA水平升高是铁死亡发生的另一个标志。我们发现，在erastin刺激下，沉默PKD2会显着增加A549和PC9细胞中的MDA水平（图2G和H）。 为了更直观地观察细胞死亡情况，我们通过PI染色和流式细胞仪检测死亡细胞占细胞总数的比例。据观察，沉默 PKD2 可显着增加 A549 和 PC9 细胞中死亡细胞与总细胞的比例（图 2I 和 J）。过量的铁会促进芬顿反应产生ROS并加速脂质过氧化[22 ]。通过铁检测测定细胞内游离铁，结果表明沉默PKD2可以显着增加erastin处理的A549和PC9细胞中的铁含量（图2K，L）。 PKD2过表达在A549和PC9细胞中显示出相反的结果（图2M ，N）。综上所述，上述结果表明PKD2可以减轻erastin诱导的LUAD细胞铁死亡。
Fig. 2. Abrogation of PKD2 promoted erastin-induced ferroptosis of LUAD cells. A The ROS content in A549 cells with silencing PKD2 with or without erastin treatment (20 μM, 24h). B The ROS content in PC9 cells with PKD2 overexpression with or without erastin treatment (20 μM, 24h). C, D Erastin-induced ROS content detected by flow cytometry in A549 and PC9 cells with or without PKD2 knockdown treated by erastin treatment (20 μM, 24h). E, F Erastin-induced ROS content detected by flow cytometry in A549 and PC9 cells with or without PKD2 inhibitor CRT0066101. G, H Relative MDA content in A549 and PC9 cells with erastin (20 μM, 24h) treatment when PKD2 was knocked down. I, J Percentage of PI staining positive cells in A549 and PC9 cells with or without PKD2 knockdown treated by erastin (50 μM, 24h). K, L Relative total iron content in A549 and PC9 cells with PKD2 knockdown stimulated by erastin (20 μM, 24h). M, N Relative total iron content in A549 and PC9 cells with PKD2 overexpression stimulated by erastin (20 μM, 24h). All statistical data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 图2 . PKD2 的废除促进了erastin 诱导的LUAD 细胞铁死亡。 A沉默 PKD2 的 A549 细胞中 ROS 含量，无论是否经过erastin 处理（20 μM，24 小时）。 B PKD2 过表达的 PC9 细胞中 ROS 含量，无论是否经过erastin 处理（20 μM，24 小时）。 C、D通过流式细胞术检测经过erastin处理（20μM，24小时）的PKD2敲低或未敲除的A549和PC9细胞中Erastin诱导的ROS含量。 E、F通过流式细胞术检测有或没有 PKD2 抑制剂 CRT0066101 的 A549 和 PC9 细胞中 Erastin 诱导的 ROS 含量。 G、H当 PKD2 被敲低时，用erastin（20 μM，24 小时）处理的 A549 和 PC9 细胞中的相对 MDA 含量。 I、J经过erastin（50 μM，24 小时）处理或未敲除PKD2 的A549 和PC9 细胞中PI 染色阳性细胞的百分比。 K、L经erastin（20 μM，24 小时）刺激PKD2 敲低的A549 和PC9 细胞中的相对总铁含量。 M、N经erastin（20 μM，24 小时）刺激PKD2 过表达的A549 和PC9 细胞中的相对总铁含量。所有统计数据均以平均值±标准差表示； n = 3。* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.0001。
3.3. Abrogation of PKD2 could promote autophagic degradation of ferritin 3.3.废除 PKD2 可促进铁蛋白的自噬降解
To find how PKD2 regulated ferroptosis, we analyzed a series of key ferroptosis-related genes expression by western blot. Glutathione peroxidase 4 (GPX4) is a key enzyme responsible for glutathione (GSH) synthesis and prevents ferroptosis [23]. Solute carrier family 40 member 1 (SLC40A1, also known as FPN1) is the only discovered iron export protein in mammals, and inhibiting SLC40A1 induces ferroptosis [24]. Silencing or overexpressed PKD2 couldn't alter the protein levels of GPX4 and SLC40A1 in PC9 cells treated by erastin (Fig. 3A). Interestingly, silencing PKD2 could downregulate FTL expression and ectopically expressing PKD2 could upregulate FTL expression in PC9 cells with erastin treatment, which was also verified in A549 cells (Fig. 3B and C). To explore whether PKD2 regulate transcription of ferritin, we detect the mRNA levels of FTL, FTH1 by qRT-PCR. Next, we found silencing or overexpressing PKD2 couldn't obviously change FTL, FTH1 mRNA levels in A549 cells, which suggested that PKD2 regulated ferritin expression in post-transcriptional stage (Fig. 3D and E). The consistent results were observed in PC9 cells with PKD2 knockdown or overexpression (Supplementary Figs. 2A and 2B). It has been demonstrated that the autophagic degradation of ferritin (also known as ferritinophagy) can promote ferroptosis, and NCOA4 is a selective cargo receptor that mediates ferritin degradation in lysosomes [25]. We investigated whether PKD2 regulated degradation of ferritin by ferritinophagy in LUAD. As a ferritinophagy marker, NCOA4 mRNA and protein expression was detected by qRT-PCR and western blot, respectively. In A549 and PC9 cells, silencing or overexpressed PKD2 couldn't obviously alter NCOA4 mRNA expression (Fig. 3D and E; Supplementary Figs. 2A and 2B). However, we further found that silencing PKD2 could downregulate FTL, FTH1, NCOA4 protein expression (Fig. 3F and G) and PKD2 overexpression showed the opposite results in A549 and PC9 cells with erastin treatment (Fig. 3H and I). Furthermore, the inhibitor, bafilomycin A1, blocking the fusion of autophagosome and lysosome, could rescue the erastin-induced decrease of FTH1, NCOA4 protein expression in A549 and PC9 cells with PKD2 knockdown (Fig. 3F and G) and impaired the difference of FTH1, NCOA4 protein expression between PKD2 overexpression and the control groups in A549 and PC9 (Fig. 3H and I). The phenomenon was also validated in A549 and PC9 cells with PKD2 knockdown treated by another autophagic inhibitor, CQ (Supplementary Figs. 2C and 2D). In addition, we detected the ferritin levels in LUAD tissue. We found LUAD cells had the highest FTH1 expression and the stromal cells had the least expression. The protein level of FTH1 in alveolus was somewhere in between, which was consistent with PKD2 expression in LUAD tissues (Supplementary Fig. 2E). Moreover, the immunohistochemical landscape of LUAD also displayed that there existed a positive relation between PKD2 and FTH1 expression (Fig. 3J). To observe the effects of PKD2 expression on ferritinophagy intuitively, we tracked ferritin and lysosomes with FTL and LAMP2 respectively by immunofluorescence. The results showed that silencing PKD2 would increase the colocalization of ferritin and lysosomes in A549 and PC9 cells exposed to erastin, indicating enhancing ferritinophagy (Fig. 3K, Supplementary Fig. 2F). Thus, above results suggested that PKD2 could prevent ferritinophagy of LUAD cells. 为了了解 PKD2 如何调节铁死亡，我们通过蛋白质印迹分析了一系列关键的铁死亡相关基因的表达。谷胱甘肽过氧化物酶 4 (GPX4) 是负责谷胱甘肽(GSH) 合成并防止铁死亡的关键酶 [23]。溶质载体家族40成员1（SLC40A1，也称为FPN1）是哺乳动物中唯一发现的铁输出蛋白，抑制SLC40A1会诱导铁死亡[24 ]。沉默或过表达PKD2不能改变erastin处理的PC9细胞中GPX4和SLC40A1的蛋白水平（图3A ）。有趣的是，在erastin处理的PC9细胞中，沉默PKD2可以下调FTL表达，异位表达PKD2可以上调FTL表达，这也在A549细胞中得到了验证（图3B和C）。为了探讨PKD2是否调节铁蛋白的转录，我们通过qRT-PCR检测FTL、FTH1的mRNA水平。接下来，我们发现沉默或过表达PKD2不能明显改变A549细胞中FTL、FTH1 mRNA水平，这表明PKD2在转录后阶段调节铁蛋白表达（图3D和E）。在 PKD2 敲低或过表达的 PC9 细胞中观察到一致的结果（补充图 2A 和 2B）。 已经证明铁蛋白的自噬降解（也称为铁蛋白自噬）可以促进铁死亡，NCOA4是介导溶酶体中铁蛋白降解的选择性货物受体[ 25]。我们研究了 LUAD 中 PKD2 是否通过铁蛋白自噬调节铁蛋白的降解。作为铁蛋白自噬标记物，分别通过 qRT-PCR 和蛋白质印迹检测NCOA4 mRNA 和蛋白表达。在A549和PC9细胞中，沉默或过表达PKD2不能明显改变NCOA4 mRNA表达（图3D和E；补充图2A和2B）。然而，我们进一步发现沉默PKD2可以下调FTL、FTH1、NCOA4蛋白表达（图3F和G），而PKD2过表达在erastin处理的A549和PC9细胞中显示出相反的结果（图3H和I）。此外，抑制剂巴弗洛霉素A1阻断自噬体和溶酶体的融合，可以在PKD2敲低的A549和PC9细胞中挽救erastin诱导的FTH1、NCOA4蛋白表达的降低（图3F和G），并损害A549和PC9中PKD2过表达组与对照组之间的FTH1、NCOA4蛋白表达（图3H和I）。 这种现象也在另一种自噬抑制剂 CQ 处理的 PKD2 敲低的 A549 和 PC9 细胞中得到了验证（补充图 2C 和 2D）。此外，我们检测了 LUAD 组织中的铁蛋白水平。我们发现 LUAD 细胞的 FTH1 表达最高，基质细胞的表达最低。肺泡中FTH1的蛋白水平介于两者之间，这与LUAD组织中PKD2的表达一致（补充图2E ）。此外，LUAD的免疫组织化学图谱也显示PKD2和FTH1表达之间存在正相关关系（图3J）。为了直观地观察PKD2表达对铁蛋白自噬的影响，我们分别用FTL和LAMP2通过免疫荧光追踪铁蛋白和溶酶体。结果表明，沉默PKD2会增加暴露于erastin的A549和PC9细胞中铁蛋白和溶酶体的共定位，表明铁蛋白自噬增强（图3K ，补充图2F ）。因此，上述结果表明PKD2可以阻止LUAD细胞的铁蛋白自噬。
Fig. 3. PKD2 inhibited autophagic degradation of ferritin. AImmunoblots of ferroptosis regulator in PC9 cells with PKD2 knockdown or overexpression with erastin (20 μM, 24h) treatment. B, C Immunoblots of FTL protein in A549 and PC9 cells with PKD2 knockdown or overexpression treated by erastin (20 μM, 24h). D, E mRNA expression of NCOA4, FTH1, FTL in A549 cells with PKD2 knockdown or overexpression. F–I Immunoblots of FTH1 and NCOA4 in A549 and PC9 cells with PKD2 knockdown or overexpression treated by erastin (20 μM, 24h) with or without bafilomycin A1 (100 nM, 4h). J Representative immunohistochemical images of PKD2, FTH1 in LUAD tissue from two cases. 100X, scale bar, 200 μm; 400X, scale bar, 50 μm. H Representative confocal images show the colocalization of FTL and LAMP2 in experimental cells. The coefficient of colocalization was calculated by Image J. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 图3 . PKD2 抑制铁蛋白的自噬降解。 APKD2 敲低或用erastin（20 μM，24 小时）处理过表达的 PC9 细胞中铁死亡调节因子的免疫印迹。B、C经过erastin（20 μM，24 小时）处理的PKD2 敲低或过表达的A549 和PC9 细胞中FTL 蛋白的免疫印迹。 D、E PKD2 敲低或过表达的 A549 细胞中 NCOA4、FTH1、FTL 的 mRNA 表达。 PKD2 敲低或过度表达的 A549 和 PC9 细胞中 FTH1 和 NCOA4 的F–I免疫印迹，经erastin（20 μM，24 小时）、加或不加巴弗洛霉素 A1（100 nM，4 小时）处理。 J两例 LUAD 组织中 PKD2、FTH1 的代表性免疫组织化学图像。 100X，比例尺，200μm； 400X，比例尺，50 μm。 H代表性共焦图像显示实验细胞中 FTL 和 LAMP2 的共定位。通过Image J计算共定位系数。数据以平均值±SD表示； n = 3。* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.0001。
3.4. PKD2 inhibited autophagic flux in LUAD via preventing autophagosome-lysosome fusion 3.4. PKD2 通过阻止自噬体-溶酶体融合抑制 LUAD 中的自噬流
To further explore the mechanism of blockade of ferritinophagy by PKD2, GO enrichment analyses were performed. The results demonstrated that multiple activities related to DNA replication were activated including centriole assembly, chromatin assembly. Response to IFN-γ and Th17 cell differentiation were also relative to PKD2 expression (Fig. 4A). Interestingly, we also found that negative regulation of autophagy and vesicle activities were regulated by PKD2. Moreover, GSVA analysis showed PDK2 negatively regulated autophagy (R = 0.21, P = 6.9e-07) (Fig. 4B; Supplementary Fig. 3A). The above results implied that PKD2 may participate in negative regulation of autophagy. Next, we further investigated expression of key molecules in autophagy signaling. We found that there were not obvious changes of ATG7, Beclin1, ULK1, VPS34, ATG5 mRNA levels between PKD2 knockdown group and the control in both A549 and PC9 cells (Fig. 4C and D). The similar results were obtained when PKD2 was overexpressed in A549 and PC9 cells (Fig. 4E and F). The markers of autophagic flux, P62 and LC3B mRNA expression were also not regulated by PKD2 in A549 and PC9 cells (Supplementary Figs. 3B–3G). The transformation of LC3B–I to LC3B-II and P62 protein level can reflect the change of autophagic flux [26]. However, we observed that P62 and LC3B-II protein expression were decreased in A549 cells with PKD2 knockdown stimulated by autophagy inducers, rapamycin and EBSS (Fig. 4G and H). Similarly, the change of P62 protein and LC3B-II expression was also observed in PC9 cells with PKD2 knockdown under the condition of serum-free-medium (SFM) starvation or rapamycin stimulation (Fig. 4I, Supplementary 3H). Ectopic PKD2 expression could result in the accumulation of P62 and LC3B-II protein in A549 and PC9 cells when autophagy was activated by rapamycin stimulation or SFM starvation. (Fig. 4J and K; Supplementary Fig. 3I). Moreover, bafilomycin A1 could impair the decrease of P62 and LC3B-II protein in A549 and PC9 cells with PKD2 knockdown when autophagy was activated (Fig. 4L, M; Supplementary Fig. 3H). The differences of P62 and LC3B-II protein between PKD2 overexpression group and control group were also reduced by bafilomycin A1 in A549 and PC9 cells (Fig. 4J; Supplementary Fig. 3I). Additionally, in A549 and PC9 cells, silencing PKD2 didn't change the protein levels of ULK1, p-ULK1, VPS34, Beclin1 and Bcl-2 under starvation conditions (Fig. 4N, O). To more intuitively observe the autophagic flux, RFP-GFP-LC3B assay was conducted. In RFP-GFP-LC3B assay, GFP and RFP are both fluorescence-emitting markers in autophagosomes, while in autolysosomes, the GFP signal is lost due to the low pH environment, allowing only RFP to fluoresce. The ratio of (RFP-GFP) to RFP reflects the rate of autophagosome-lysosome fusion. In A549 cells, silencing PKD2 increased (RFP-GFP)/RFP values under starvation conditions (P < 0.05), which indicated silencing PKD2 promoted the transition of autophagosome to autolysosome (Fig. 4P). Furthermore, PKD2 overexpression in A549 cells decreased (RFP-GFP)/RFP values under starvation conditions, which suggested that PKD2 overexpression prevented the transition of autophagosome to autolysosome (P < 0.01) (Supplementary Fig. 3J). In addition, we also monitored the colocalization of autophagosomes and lysosomes by immunofluorescence. We labeled LC3B and LAMP2 to track the autophagosome and lysosome, respectively. The yellow puncta represented the colocalization of autophagosomes and lysosomes. The results showed that PKD2 knockout would increase the colocalization of autophagosomes and lysosomes in A549 cells under starvation conditions (Fig. 4Q). The opposite results were observed in A549 cells with PKD2 overexpression under starvation conditions (Supplementary Fig. 3K). Consequently, these indicated PKD2 blocked ferritinophagy in LUAD cells via inhibiting autophagosome-lysosome fusion. 为了进一步探讨 PKD2 阻断铁蛋白自噬的机制，进行了GO富集分析。结果表明，与DNA复制相关的多种活动被激活，包括中心粒组装、染色质组装。对 IFN-γ 和 Th17细胞分化的反应也与 PKD2 表达相关（图 4A）。有趣的是，我们还发现自噬和囊泡活性的负调节受到 PKD2 的调节。此外，GSVA分析显示PDK2负向调节自噬（R =0.21， P =6.9e-07）（图4B ；补充图3A）。上述结果提示PKD2可能参与自噬的负调控。接下来，我们进一步研究了自噬信号传导中关键分子的表达。我们发现A549和PC9细胞中PKD2敲低组与对照组之间ATG7、 Beclin1 、ULK1、VPS34、ATG5 mRNA水平没有明显变化（图4C和D）。当PKD2在A549和PC9细胞中过表达时，获得了类似的结果（图4E和F）。 在 A549 和 PC9 细胞中，自噬流标记物、P62 和 LC3B mRNA 表达也不受 PKD2 的调节（补充图 3B-3G）。 LC3B-I向LC3B-II的转变以及P62蛋白水平可以反映自噬通量的变化[26]。然而，我们观察到，在自噬诱导剂、雷帕霉素和EBSS刺激下，PKD2敲低的A549细胞中P62和LC3B-II蛋白表达降低（图4G和H）。同样，在无血清培养基（SFM）饥饿或雷帕霉素刺激的条件下，在PKD2敲低的PC9细胞中也观察到P62蛋白和LC3B-II表达的变化（图4I，补充3H）。当雷帕霉素刺激或 SFM 饥饿激活自噬时，异位 PKD2 表达可能导致 A549 和 PC9 细胞中 P62 和 LC3B-II 蛋白的积累。 （图4 J和K；补充图3I ）。此外，当自噬被激活时，巴弗洛霉素 A1 可以削弱 PKD2 敲低的 A549 和 PC9 细胞中 P62 和 LC3B-II 蛋白的减少（图 4 L，M；补充图 3H ）。 在A549和PC9细胞中，巴弗洛霉素A1也减少了PKD2过表达组和对照组之间P62和LC3B-II蛋白的差异（图4J ；补充图3I）。此外，在A549和PC9细胞中，饥饿条件下沉默PKD2不会改变ULK1、p-ULK1、VPS34、 Beclin1和Bcl-2的蛋白水平（图4N、O）。为了更直观地观察自噬流，进行了RFP-GFP-LC3B测定。在RFP-GFP-LC3B检测中，GFP和RFP都是自噬体中的荧光发射标记，而在自溶酶体中，由于低pH环境，GFP信号丢失，仅允许RFP发出荧光。 (RFP-GFP)与RFP的比值反映了自噬体-溶酶体的融合率。在A549细胞中，沉默PKD2在饥饿条件下增加了（RFP-GFP）/RFP值（P < 0.05），这表明沉默PKD2促进了自噬体向自溶酶体的转变（图4 P）。此外，A549细胞中的PKD2过表达在饥饿条件下降低了(RFP-GFP)/RFP值，这表明PKD2过表达阻止了自噬体向自溶酶体的转变( P <0.01)(补充图3J)。此外，我们还通过免疫荧光监测了自噬体和溶酶体的共定位。我们标记 LC3B 和LAMP2分别跟踪自噬体和溶酶体。 黄色点代表自噬体和溶酶体的共定位。结果表明，PKD2敲除会增加饥饿条件下A549细胞中自噬体和溶酶体的共定位（图4 Q）。在饥饿条件下 PKD2 过度表达的 A549 细胞中观察到相反的结果（补充图 3K ）。因此，这些表明 PKD2 通过抑制自噬体-溶酶体融合来阻断 LUAD 细胞中的铁蛋白自噬。
Fig. 4. Augmenting PKD2 blocked the fusion of autophagosome and lysosome in LUAD. A GO enrichment analyses, including BP (biological process), CC (cellular component), MF (molecular function). B The correlation analysis of PKD2 expression and ssGSEA score of negative regulation of autophagy. C, D The alteration of autophagy-related genes in mRNA level in A549 and PC9 cells when PKD2 was knocked down. E, F The alteration of other autophagy-related genes in mRNA level in A549 and PC9 cells when PKD2 was overexpressed. G-MImmunoblots of LC3 and P62 proteins in A549 or PC9 cells with PKD2 knockdown or overexpression treated by rapamycin (5 μM, 4h), EBSS (4h), serum-free medium (SFM) starvation (6h) with or without bafilomycin A1 (100 nM, 4h). N, O Immunoblots of autophagy-related genes in A549 and PC9 cells with PKD2 knockdown treated by SFM starvation (6h). P A549 cells with PKD2 knockdown were infected by StubRFP-sensGFP-LC3 adenovirus and starved for 6 h. Representative confocal images show GFP puncta, RFP puncta and merged puncta in experimental cells. The data were presented as the mean ± SD; n = 3. Q Representative confocal images show the colocalization of LC3B and LAMP2 in experimental cells. The coefficient of colocalization was calculated by Image J. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 图4 .增强 PKD2 可阻断 LUAD 中自噬体和溶酶体的融合。 GO富集分析，包括BP（生物过程）、CC（细胞成分）、MF（分子功能）。 BPKD2表达与自噬负调控ssGSEA评分的相关性分析。C、D当 PKD2 被敲低时，A549 和 PC9 细胞中自噬相关基因 mRNA 水平的变化。 E、F PKD2过表达时，A549和PC9细胞中其他自噬相关基因mRNA水平的变化。 PKD2 敲低或过表达的 A549 或 PC9 细胞中 LC3 和 P62 蛋白的GM免疫印迹，经雷帕霉素（5 μM，4 小时）、EBSS（4 小时）、无血清培养基 (SFM) 饥饿（6 小时）、加或不加巴弗洛霉素 A1（100）处理纳米，4 小时）。通过 SFM 饥饿（6 小时）处理 PKD2 敲低的 A549 和 PC9 细胞中自噬相关基因的N、O免疫印迹。 PKD2敲低的PA549细胞被StubRFP-sensGFP-LC3腺病毒感染并饥饿6小时。代表性共焦图像显示实验细胞中的 GFP 点、RFP 点和合并点。数据以平均值±标准差表示； n = 3。Q代表性共焦图像显示实验细胞中 LC3B 和 LAMP2 的共定位。通过Image J计算共定位系数。数据以平均值±SD表示； n = 3。* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.0001。
3.5. PKD2-mediated blocking of autophagosome-lysosome fusion was independent of TFEB 3.5. PKD2 介导的自噬体-溶酶体融合阻断不依赖于 TFEB
In order to explore how PKD2 regulated autophagic flux, we focused on regulators of autophagosome-lysosome fusion. TFEB, a transcription factor of the MiT-TFE family, is a master regulator of the transcription of genes involved in autophagosome-lysosome fusion. Its activity is extensively regulated by phosphorylation status, controlled by various kinases and phosphatases. The phosphorylation status will inhibit its cytoplasm-to-nucleus trafficking [27]. To identify whether PKD2 blocked autophagosome-lysosome fusion via inhibiting TFEB's function, we detected the protein expression of TFEB when PKD2 was overexpressed. As showed in Fig. 5A, PKD2 overexpression couldn't affect the protein level of TFEB in A549 cells. Next, we wondered whether PKD2 regulated the shuttling between cytoplasm and nucleus. Subcellular fractionation studies in HEK cells showed that PKD2 overexpression couldn't alter subcellular localization of TFEB (Fig. 5B). To exhibit the effects of PKD2 on subcellular location of TFEB more vividly, immunofluorescence assay was conducted. The results showed that rapamycin could promote the expression of TFEB but alteration of PKD2 expression couldn't change fluorescence intensity and location of TFEB in A549 cells (Fig. 5C and D). Together, these results suggested that the role of PKD2 on autophagy was independent of TFEB in LUAD. 为了探索 PKD2 如何调节自噬流，我们重点关注自噬体-溶酶体融合的调节因子。 TFEB 是 MiT-TFE 家族的转录因子，是参与自噬体-溶酶体融合的基因转录的主要调节因子。其活性受到磷酸化状态的广泛调节，并由各种激酶和磷酸酶控制。磷酸化状态将抑制其细胞质到细胞核的运输[27 ]。为了确定PKD2是否通过抑制TFEB的功能来阻断自噬体-溶酶体融合，我们检测了PKD2过表达时TFEB的蛋白表达。如图5A所示，PKD2过表达不影响A549细胞中TFEB的蛋白水平。接下来，我们想知道 PKD2 是否调节细胞质和细胞核之间的穿梭。 HEK 细胞中的亚细胞分级分离研究表明 PKD2 过表达不能改变TFEB 的亚细胞定位（图 5 B）。为了更生动地展示PKD2对TFEB亚细胞定位的影响，进行了免疫荧光测定。结果显示，雷帕霉素可以促进A549细胞中TFEB的表达，但改变PKD2的表达并不能改变TFEB在A549细胞中的荧光强度和位置（图5C和D）。总之，这些结果表明 LUAD 中 PKD2 对自噬的作用独立于 TFEB。
Fig. 5. PKD2 blocked autophagosome-lysosome fusion in a TFEB-independent way. A Immunoblots of TFEB in A549 cells with PKD2 overexpression. B Extraction of cytoplasmic and nuclear protein was to detect subcellular location of TFEB in HEK cells with rapamycin (5 μM, 4h) treatment when PKD2 was overexpressed. C, D Representative immunofluorescence images show PKD2 (green), TFEB (red), nucleus (blue) and merged images in A549 cells with or without rapamycin (5 μM, 4h) treatment when PKD2 was overexpressed or knocked down. 图5 。 PKD2 以不依赖 TFEB 的方式阻断自噬体-溶酶体融合。 A PKD2 过表达的 A549 细胞中 TFEB 的免疫印迹。 B提取细胞质和核蛋白，检测 PKD2 过表达时用雷帕霉素（5 μM，4h）处理的 HEK 细胞中 TFEB 的亚细胞定位。C、D代表性免疫荧光图像显示当 PKD2 过度表达或敲低时，用或不用雷帕霉素（5 μM，4 小时）处理的 A549 细胞中的 PKD2（绿色）、TFEB（红色）、细胞核（蓝色）和合并图像。
3.6. PKD2 knockdown-mediated ferroptosis could be impaired by inhibiting autophagosome-lysosome fusion or regulating FTH1
To verify inhibition of ferroptosis by PKD2 was dependent on autophagy blockade, we utilized autophagy inhibitor bafilomycin A1 to stimulate A549 and PC9 cells. ROS accumulation mediated by silencing PKD2 was impaired in the group with bafilomycin A1 treatment (Fig. 6A-C). Moreover, the difference of erastin-induced MDA between PKD2-deficiency group and control group could be reduced by bafilomycin A1 in A549 and PC9 cells (Fig. 6D and E). Additionally, FTH1 knockdown could restore ROS accumulation triggered by PKD2 overexpression in PC9 cells (Fig. 6F). Silencing FTH1 could also restore death of A549 cells caused by PKD2 overexpression (Fig. 6G). To sum up, ferroptosis caused by PKD2 knockdown could be impaired by inhibiting autophagosome-lysosome fusion or regulating FTH1.
Fig. 6. PKD2 knockdown-mediated ferroptosis could be impaired by bafilomycin A1 stimulation or regulating FTH1. A The ROS content in PKD2-depleted PC9 cells with or without erastin, bafilomycin A1 treatment. B The data of the ROS content in PC9 cells were presented as the mean ± SD; n = 3. C The ROS content in PKD2-depleted A549 cells with or without erastin, bafilomycin A1 treatment was detected by flow cytometry. D, E Relative MDA content in A549 and PC9 cells with erastin (20 μM, 24h) and bafilomycin A1 (100 nM, 4h) treatment when PKD2 was knocked down. The data were presented as the mean ± SD; n = 3. F The ROS content in PKD2 overexpressed PC9 cells treated by erastin with or without FTH1 knockdown was detected by flow cytometry. G Percentage of PI staining positive cells in PKD2 overexpressed A549 cells with or without FTH1 knockdown. All statistical data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
3.7. PKD2 promoted proliferation, migration and invasion of LUAD cells in vitro and vivo
To further investigate the pathologic roles of PKD2 in LUAD, we verified the effects of PKD2 overexpression and knockdown on malignant phenotypes of LUAD cells. The results of cell proliferation determined by SRB staining demonstrated that overexpression of PKD2 increased LUAD cells (A549, PC9) proliferation and knockdown of PKD2 markedly inhibited cells proliferation (P < 0.05) (Fig. 7A-D). Ki67, as a proliferation marker, was positively related to PKD2 expression in the LUAD landscape of immunohistochemical staining (Supplementary Fig. 4A). Moreover, the results of EdU staining also supported that ectopic PKD2 expression in A549 and PC9 cells could promote their proliferation and knockdown of PKD2 showed the opposite results (P < 0.05) (Fig. 7E and F; Supplementary Figs. 4B and 4C). Subsequently, colony formation assay was performed to identify the proliferative ability of LUAD cells when PKD2 was knocked down or up-regulated. The number of colonies in PKD2 knockdown group was obviously decreased than that in control (P < 0.05) (Fig. 7G). On the contrary, the number of colonies in PKD2 overexpression group was increased (P < 0.05) (Fig. 7H). Wound healing assay was used to determine migration of LUAD cells. When PKD2 was knocked down, the ability of migration in A549 and PC9 cells was significantly impaired (Fig. 7I and J) and PKD2 overexpression could promote migration of A549 and PC9 cells (P < 0.05) (Supplementary Figs. 4D and 4E). In addition, Transwell assay also suggested that down-regulated PKD2 expression would impair migration and invasion of A549 and PC9 cells and PKD2 overexpression exerted the opposite effect (Fig. 7K, L; Supplementary Figs. 4F and 4G). 为了进一步研究 PKD2 在 LUAD 中的病理作用，我们验证了 PKD2 过表达和敲低对 LUAD 细胞恶性表型的影响。 SRB染色测定的细胞增殖结果表明，PKD2的过表达增加了LUAD细胞（A549，PC9）的增殖，而PKD2的敲低显着抑制了细胞增殖（P <0.05）（图7AD ）。 Ki67作为增殖标志物，在免疫组织化学染色的LUAD图谱中与PKD2表达呈正相关（补充图4A）。此外， EdU染色的结果也支持A549和PC9细胞中异位PKD2表达可以促进其增殖，而PKD2的敲低显示相反的结果（P <0.05）（图7E和F；补充图4B和4C ） 。随后，进行集落形成实验来鉴定当PKD2被敲低或上调时LUAD细胞的增殖能力。 PKD2敲低组的集落数明显低于对照组（ P <0.05）（图7G ）。相反，PKD2过表达组的集落数增加( P <0.05)(图7H)。伤口愈合测定用于确定 LUAD 细胞的迁移。 当PKD2被敲低时，A549和PC9细胞的迁移能力显着受损（图7 I和J），PKD2过表达可以促进A549和PC9细胞的迁移（ P <0.05）（补充图4D和4E ） 。此外，Transwell实验还表明，下调的PKD2表达会损害A549和PC9细胞的迁移和侵袭，而PKD2的过表达则发挥相反的作用（图7K ，L；补充图4F和4G ）。
Fig. 7. PKD2 promoted proliferation, migration, invasion of LUAD cells in vitro. A, B Effects of silencing PKD2 on the proliferative ability of A549 and PC9 cells by SRB staining assay. C, D Effects of PKD2 overexpression on the proliferative ability of A549 and PC9 cells by SRB staining assay. E, F The EdU assay detected the effects of silencing PKD2 on the proliferative ability of A549 and PC9 cells. G Effects of PKD2 knockdown on the colony-formation ability of A549 and PC9 cells. H Effects of PKD2 overexpression on the colony-formation ability of A549 and PC9 cells. I, J Wound healing assay validated that the role of silencing PKD2 on migrative ability of A549 and PC9 cells. K, L Effects of silencing or overexpressed PKD2 on migrative and invasive ability of PC9 cells by Transwell assay. All statistical data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 图7 . PKD2在体外促进LUAD细胞的增殖、迁移、侵袭。 A、B通过SRB染色测定沉默PKD2对A549和PC9细胞增殖能力的影响。 C、D通过SRB染色测定PKD2过表达对A549和PC9细胞增殖能力的影响。 E、F EdU 测定检测了沉默 PKD2 对 A549 和 PC9 细胞增殖能力的影响。 G PKD2 敲低对 A549 和 PC9 细胞集落形成能力的影响。 H PKD2 过表达对 A549 和 PC9 细胞集落形成能力的影响。 I、J伤口愈合实验验证了沉默 PKD2 对 A549 和 PC9 细胞迁移能力的作用。 K、L通过 Transwell 实验观察沉默或过表达 PKD2 对 PC9 细胞迁移和侵袭能力的影响。所有统计数据均以平均值±标准差表示； n = 3。* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.0001。
To further verify the role of PKD2 in regulating cell growth, we next examined the effect of PKD2 on cell proliferation in vivo. A549 cells infected with lentivirus containing shPKD2 or shNT as a control were subcutaneously injected into nude mice. Compared to the control, PKD2 knockdown slowed down the tumor growth and reduced tumor weight in BALB/c-nu mice (Fig. 8A-D). Therefore, PKD2 knockdown significantly inhibited growth of A549 xenografts. Together, the above results showed that PKD2 promoted proliferation, migration and invasion of LUAD cells in vitro and vivo. 为了进一步验证PKD2在调节细胞生长中的作用，我们接下来检查了PKD2对体内细胞增殖的影响。将含有shPKD2或shNT的慢病毒感染的A549细胞作为对照，皮下注射到裸鼠体内。与对照相比，PKD2敲低减慢了BALB/c-nu小鼠中的肿瘤生长并减轻了肿瘤重量（图8AD）。因此，PKD2敲低显着抑制A549异种移植物的生长。综上所述，上述结果表明PKD2在体外和体内促进LUAD细胞的增殖、迁移和侵袭。
Fig. 8. PKD2 knockdown suppressed LUAD progression in vivo. A A549 cells, infected with lentivirus containing shPKD2 or shNT, at a concentration of 2 × 106 mixed with 50% Matrigel were implanted subcutaneously in 4-week-old male nude mice. Mice were sacrificed at 42 days after injection with no tumor volume reaching 2000 mm3. B Tumor volumes were recorded every 6 days. C Tumor samples separated surgically from mice. D Tumor weight of samples separated surgically from mice was compared between shPKD2 and shNT groups. 图8 . PKD2 敲低可抑制体内 LUAD 进展。将含有shPKD2或shNT的慢病毒感染的A549细胞以2×10 6的浓度与50％ Matrigel混合，植入4周龄雄性裸鼠皮下。注射后42天处死小鼠，肿瘤体积未达到2000mm3。B每6天记录一次肿瘤体积。 C通过手术从小鼠中分离出肿瘤样本。 D比较 shPKD2 组和 shNT 组之间通过手术从小鼠分离的样品的肿瘤重量。
3.8. Targeting PKD2 could enhance the sensitivity of LUAD cells to carboplatin via inducing ferroptosis and apoptosis 3.8.靶向PKD2可通过诱导铁死亡和细胞凋亡增强LUAD细胞对卡铂的敏感性
Carboplatin-based chemotherapies are the primary treatments for advanced LUAD, but resistance to carboplatin has become increasingly challenging [28]. Some previous studies reported that ferroptosis could be induced by carboplatin [29]. Considering oncogenic role and inhibition of ferroptosis, we wondered whether targeting PKD2 could enhance the efficacy of carboplatin in LUAD. As we expected, silencing PKD2 could further decrease A549 and PC9 cells viability and enhance their death induced by carboplatin (Fig. 9A-D). IC50 assay showed that PKD2 knockdown increased the sensitivity of A549 and PC9 cells to carboplatin (Fig. 9E and F). Moreover, we observed an increase in ROS production by carboplatin, and combination of PKD inhibitor CRT0066101 and carboplatin could further enhance ROS production in A549 and PC9 cells (Fig. 9G and H). The similar phenomenon was also observed in MDA production. Carboplatin could induce MDA production and combination of carboplatin and PKD2 inhibitor further enhanced MDA production in LUAD cells (Fig. 9I and J). In addition, EdU assay suggested that silencing PKD2 would further inhibit proliferation of LUAD cells treated by carboplatin (Supplementary Figs. 5A and 5B). Previous studies reported that PKD2 could inhibit cell apoptosis to promote progression of tumor. So, we investigated whether apoptosis involved in PKD2-mediated the sensitivity of LUAD cells to carboplatin. Cell apoptosis assay was performed and demonstrated that silencing PKD2 or pharmacological inhibition of PKD2 by CRT0066101 would promote carboplatin-induced cell apoptosis of A549 cells (Supplementary Figs. 5C–5E). Moreover, combination of PKD2 inhibitor and carboplatin killed tumor cells in an inhibitor-concentration dependent manner. The same phenomenon could be observed in PC9 cells (Supplementary Figs. 5F–5H). The above results manifested that targeting PKD2 could enhance the sensitivity of LUAD cells to carboplatin via inducing ferroptosis and apoptosis. 基于卡铂的化疗是晚期 LUAD 的主要治疗方法，但对卡铂的耐药性变得越来越具有挑战性[ 28 ]。之前的一些研究报道卡铂可以诱导铁死亡[ 29]。考虑到致癌作用和铁死亡的抑制作用，我们想知道靶向 PKD2 是否可以增强卡铂在 LUAD 中的疗效。正如我们所预期的，沉默 PKD2 可以进一步降低 A549 和 PC9细胞的活力并增强卡铂诱导的死亡（图 9 AD）。 IC50测定显示PKD2敲低增加了A549和PC9细胞对卡铂的敏感性（图9E和F）。此外，我们观察到卡铂增加了ROS的产生，并且PKD抑制剂CRT0066101和卡铂的组合可以进一步增强A549和PC9细胞中ROS的产生（图9G和H）。在MDA生产中也观察到类似的现象。卡铂可以诱导MDA产生，并且卡铂和PKD2抑制剂的组合进一步增强了LUAD细胞中MDA的产生（图9I和J）。此外， EdU测定表明沉默PKD2将进一步抑制卡铂处理的LUAD细胞的增殖（补充图5A和5B）。前期研究报道PKD2可以抑制细胞凋亡，促进肿瘤进展。 因此，我们研究了细胞凋亡是否参与PKD2介导的LUAD细胞对卡铂的敏感性。进行细胞凋亡测定并证明，CRT0066101沉默PKD2或药理抑制PKD2会促进卡铂诱导的A549细胞凋亡（补充图5C-5E ）。此外，PKD2抑制剂和卡铂的组合以抑制剂浓度依赖性方式杀死肿瘤细胞。在 PC9 细胞中也可以观察到相同的现象（补充图 5F-5H ）。上述结果表明，靶向PKD2可以通过诱导铁死亡和细胞凋亡来增强LUAD细胞对卡铂的敏感性。
Fig. 9. Targeting PKD2 could enhance sensitivity of LUAD cells to carboplatin. A, B The cell viability of A549 and PC9 cells with PKD2 knockdown with or without carboplatin treatment. The data were presented as the mean ± SD; n = 3. C, D The cell death of A549 and PC9 cells with PKD2 knockdown with or without carboplatin treatment. The data were presented as the mean ± SD; n = 3. E, F IC50 experiment was conducted to detect cell viability of A549 and PC9 cells with carboplatin treatment when PKD2 was knocked down or not. G, H The ROS content in A549 and PC9 cells in the indicated treatment was detected by flow cytometry. The data were presented as the mean ± SD; n = 3. I, J Relative MDA content in A549 and PC9 cells in the indicated treatment was detected. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 图9 .靶向 PKD2 可以增强 LUAD 细胞对卡铂的敏感性。 A、B PKD2 敲低的 A549 和 PC9 细胞在有或没有卡铂处理的情况下的细胞活力。数据以平均值±标准差表示； n = 3。C 、D PKD2 敲低的 A549 和 PC9 细胞在有或没有卡铂治疗的情况下的细胞死亡。数据以平均值±标准差表示； n = 3。E 、F IC50实验检测卡铂处理的A549和PC9细胞在PKD2敲低与否时的细胞活力。 G、H通过流式细胞术检测指定处理中的 A549 和 PC9 细胞中的 ROS 含量。数据以平均值±标准差表示； n = 3. I、J检测指定处理中 A549 和 PC9 细胞中的相对 MDA 含量。数据以平均值±标准差表示； n = 3。* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.0001。
To sum up, this study presented here demonstrated that PKD2 suppressed ferroptosis by blocking autophagosome-lysosome fusion in a TFEB-independent way to suppress ferritinophagy and targeting PKD2 could enhance efficacy of carboplatin on LUAD (Fig. 10). 综上所述，本文提出的这项研究表明，PKD2通过以不依赖于TFEB的方式阻断自噬体-溶酶体融合来抑制铁蛋白自噬，从而抑制铁死亡，并且靶向PKD2可以增强卡铂对LUAD的功效（图10 ）。
Fig. 10. Proposed model of PKD2-mediated ferroptosis suppression by blocking autophagosome-lysosome fusion to suppress ferritinophagy and targeting PKD2 could enhance efficacy of carboplatin on LUAD. 图10 。提出的通过阻断自噬体-溶酶体融合来抑制铁蛋白自噬和靶向 PKD2 的 PKD2 介导的铁死亡抑制模型可以增强卡铂对 LUAD 的疗效。
4. Discussion 4. 讨论
Ferroptosis is an iron-dependent regulated cell death [30]. Emerging evidence has demonstrated that ferroptosis can be harnessed for cancer therapy, particularly for those refractory malignancies that are resistant to the traditional therapies [31]. Inducing ferroptosis not only results in destruction of malignant cells themselves, but also increases the vulnerability of tumor cells to other therapies. A dual-targeting PI3K and HDAC inhibitor BEBT-908 has been reported to promote ferroptosis of cancer cells and shape a proinflammatory tumor microenvironment, thus potentiating cancer immune checkpoint therapy [32]. Immunotherapy-activated CD8+T cells can release interferon gamma (IFN-γ) to downregulate the expression of SLC3A2 and SLC7A11, two subunits of glutamate-cystine antiporter system Xc−, impairs the uptake of cystine by tumor cells and as a result, leads to tumor cell lipid peroxidation and ferroptosis [33]. RAS mutations are common in about half of all metastatic colorectal cancer and limit the efficacy of anti-EGFR antibodies. β-Elemene, a new ferroptosis inducer, can sensitize KRAS mutant metastatic colorectal cancer cells to cetuximab (anti-EGFR antibodies) [34]. Consequently, combination of inducing ferroptosis and other therapies shows broad prospect in the future cancer therapy. It is vital to explore more high-efficiency targets to trigger ferroptosis in tumor cells at present. 铁死亡是一种铁依赖性调节细胞死亡[ 30]。新的证据表明，铁死亡可用于癌症治疗，特别是那些对传统疗法有抵抗力的难治性恶性肿瘤[31]。诱导铁死亡不仅会导致恶性细胞本身的破坏，还会增加肿瘤细胞对其他疗法的脆弱性。据报道，双靶点 PI3K 和HDAC 抑制剂BEBT-908 可促进癌细胞铁死亡并形成促炎性肿瘤微环境，从而增强癌症免疫检查点治疗[32]。免疫治疗激活的CD8+T 细胞可以释放干扰素 γ(IFN-γ) 来下调谷氨酸-胱氨酸逆向转运蛋白系统Xc−的两个亚基 SLC3A2 和 SLC7A11 的表达，从而损害肿瘤细胞对胱氨酸的摄取，从而导致肿瘤细胞脂质过氧化和铁死亡[33]。 RAS 突变在大约一半的转移性结直肠癌中很常见，并且限制了抗 EGFR 抗体的功效。 β-榄香烯是一种新的铁死亡诱导剂，可以使KRAS突变的转移性结直肠癌细胞对西妥昔单抗（抗 EGFR 抗体）敏感[ 34 ]。因此，诱导铁死亡与其他疗法的结合在未来的癌症治疗中显示出广阔的前景。目前探索更多高效靶点来引发肿瘤细胞铁死亡至关重要。
In this study, we have revealed that PKD2 could alleviate erastin-induced ferroptosis. Silencing PKD2 could increase accumulation of ROS, MDA and iron and death of LUAD cells exposed to erastin. It has been reported that PKD2 inhibition could induced apoptosis of human colorectal cancer cells [16]. PKD knockdown or pharmacological inhibition could ameliorate necrosis and severity of pancreatitis [35]. These studies implied that PKD2 may be involved in regulation of cell death. Our study demonstrated PKD2 also regulated ferroptosis of LUAD for the first time. Mechanistically, PKD2 could alleviate ferritinophagy-mediated ferroptosis of LUAD cells by blocking fusion of autophagosome and lysosome. Autophagy is evolutionarily-conserved degradative system via lysosome and maintains intracellular homeostasis [36]. It has been involved in regulation of many physiological and pathological activities and orchestrated by a complex network at the same time. Many kinases have been reported to participate in autophagy regulation. DAP-kinase (DAPK) is a Ca2+/calmodulin regulated Ser/Thr kinase and phosphorylates multiple kinases including PKD [37]. DAPK-mediated phosphorylation of beclin 1 can promote its dissociation from Bcl-XL, thus inducing autophagy [38]. We found PKD2 was initially related to vesicle activity and regulation of autophagy by bioinformatic analysis in this article. By detecting LC3B and P62 expression, we found ectopic PKD2 expression could promote the accumulation of LC3B-II and P62. In addition, RFP-GFP-LC3B assay and the colocalization of LC3B and LAMP2 vividly showed that silencing PKD2 could accelerate the transition of autophagosome to autolysosome in LUAD cells. The results suggested PKD2 could block autophagosome-lysosome fusion in LUAD. Previous study has suggested that PKD-depletion can increase pro-survival Bcl-2 family protein expression and promote autophagy in mouse model of pancreatitis [39]. PKD was also reported to be able phosphorylate vps34 and promote ROS-induced autophagy. The roles of PKD on autophagy seems to be paradoxical. The contradictory results may be explained as follows. On the one hand, PKD may take part in multiple nodes of autophagy, and autophagy promotion or inhibition may be context-dependent. On the other hand, three members of PKD family, PKD1, PKD2, PKD3 have high homology in structure but heterogeneity in function. They have different dominant expression in different tissues and organs. 在这项研究中，我们发现 PKD2 可以减轻erastin诱导的铁死亡。沉默 PKD2 可以增加 ROS、MDA 和铁的积累以及暴露于erastin 的 LUAD 细胞的死亡。据报道，抑制PKD2可诱导人结直肠癌细胞凋亡[ 16]。 PKD 敲除或药物抑制可以改善胰腺炎的坏死和严重程度[35]。这些研究表明 PKD2 可能参与细胞死亡的调节。我们的研究首次证明 PKD2 还调节 LUAD 的铁死亡。从机制上讲，PKD2 可以通过阻断自噬体和溶酶体的融合来减轻铁蛋白自噬介导的 LUAD 细胞铁死亡。自噬是进化上保守的降解系统，通过溶酶体维持细胞内稳态[36 ]。它参与了许多生理和病理活动的调节，同时由一个复杂的网络精心策划。据报道，许多激酶参与自噬调节。 DAP 激酶 (DAPK) 是一种 Ca 2+ /钙调蛋白调节的 Ser/Thr 激酶，可磷酸化包括 PKD 在内的多种激酶 [ 37 ]。 DAPK介导的beclin 1磷酸化可以促进其与Bcl-XL解离，从而诱导自噬[ 38 ]。本文通过生物信息学分析发现PKD2最初与囊泡活性和自噬调节有关。 通过检测LC3B和P62的表达，我们发现异位PKD2表达可以促进LC3B-II和P62的积累。此外，RFP-GFP-LC3B测定以及LC3B和LAMP2的共定位生动地表明，沉默PKD2可以加速LUAD细胞中自噬体向自溶酶体的转变。结果表明 PKD2 可以阻断 LUAD 中的自噬体-溶酶体融合。先前的研究表明，PKD 缺失可以增加胰腺炎小鼠模型中促生存 Bcl-2 家族蛋白的表达并促进自噬 [ 39 ]。据报道，PKD 能够磷酸化 vps34 并促进 ROS 诱导的自噬。 PKD 对自噬的作用似乎是矛盾的。矛盾的结果可以解释如下。一方面，PKD可能参与自噬的多个节点，并且自噬的促进或抑制可能具有上下文依赖性。另一方面，PKD家族的三个成员PKD1、PKD2、PKD3在结构上具有高度同源性，但在功能上具有异质性。它们在不同的组织和器官中具有不同的显性表达。
Ferroptosis has been reported to be regulated by autophagy in many ways, especially selective autophagy, such as ferritinophagy, lipophagy, clockophagy, and chaperone-mediated autophagy [40]. Intracellular iron level is a switch of ferroptosis, which is regulated by ferritin, ferroportin-1 and transferrin receptor 1 (TfR1). ROS-induced autophagy leads to iron-dependent ferroptosis by degradation of ferritin and induction of transferrin receptor 1 (TfR1) expression [41]. Ferroportin-1 is responsible for intracellular iron exporter and resists ferroptosis. It can be degraded by P62 through autophagy-lysosome pathway to trigger ferroptosis [42]. NCOA4 is the receptor of ferritin in autophagic degradation. Coatomer protein complex subunit zeta 1 (COPZ1) knockdown can increase NCOA4 expression to promote autophagic degradation of ferritin, finally leading to ferroptosis [43]. Herein, we found silencing PKD2 would decrease ferritin protein level but not mRNA level. The effect could be impaired by bafilomycin A1. Consequently, we speculate PKD2 can inhibit degradation of ferritin by preventing autophagosome-lysosome fusion. Moreover, bafilomycin A1 could impaired the role of PKD2 knockdown on ferroptosis and silencing FTH1 can also restore ferroptosis of LUAD cells with ectopic PKD2 expression. Our previous studies have demonstrated that PKD2 can accelerate LUAD metastasis via promoting EMT [18]. It is also reported that PKD2 can stabilize Aurora A kinase to modulate cell cycle, thus promoting cancer cell proliferation [44]. Inhibition of PKD2 in B cells suppresses tumor growth and promotes effector T cell function [45]. We also demonstrated that PKD2 could promote proliferation, migration and invasion by in vitro and vivo experiments. Given the oncogenic role of PKD2, PKD2 is promising to be a target for cancer therapy. Now we revealed that PKD2 also suppressed ferroptosis of LUAD, which enriched its oncogenic roles and underscored its potential in cancer therapy again. 据报道，铁死亡在许多方面受到自噬的调节，特别是选择性自噬，例如铁蛋白自噬、脂肪自噬、时钟自噬和分子伴侣介导的自噬[40]。细胞内铁水平是铁死亡的开关，由铁蛋白、铁转运蛋白-1 和转铁蛋白受体 1(TfR1) 调节。 ROS 诱导的自噬通过铁蛋白降解和诱导转铁蛋白受体 1 (TfR1) 表达导致铁依赖性铁死亡[41 ]。 Ferroportin-1 负责细胞内铁输出并抵抗铁死亡。它可以通过自噬-溶酶体途径被 P62 降解，引发铁死亡[ 42]。 NCOA4是自噬降解中铁蛋白的受体。涂层蛋白复合物亚基zeta 1（COPZ1）敲低可以增加NCOA4的表达，促进铁蛋白的自噬降解，最终导致铁死亡[43 ]。在此，我们发现沉默 PKD2 会降低铁蛋白水平，但不会降低 mRNA 水平。巴弗洛霉素 A1 可能会削弱该作用。因此，我们推测 PKD2 可以通过阻止自噬体-溶酶体融合来抑制铁蛋白的降解。此外，巴弗洛霉素 A1 可以削弱 PKD2 敲低对铁死亡的作用，沉默 FTH1 也可以恢复具有异位 PKD2 表达的 LUAD 细胞的铁死亡。 我们前期的研究表明PKD2可以通过促进EMT来加速LUAD转移[ 18]。另据报道，PKD2可以稳定Aurora A激酶来调节细胞周期，从而促进癌细胞增殖[44]。抑制 B 细胞中的 PKD2 可抑制肿瘤生长并促进效应T 细胞功能 [45 ]。我们还通过体外和体内实验证明PKD2可以促进增殖、迁移和侵袭。鉴于 PKD2 的致癌作用，PKD2 有希望成为癌症治疗的靶点。现在我们发现 PKD2 还可以抑制 LUAD 的铁死亡，这丰富了它的致癌作用，并再次强调了它在癌症治疗中的潜力。
Carboplatin is still the basic of chemotherapy regimen for LUAD patients. However, acquired drug resistance has greatly limited its application in clinical practice. Recent evidence has demonstrated that activation of ferroptosis is a part of antitumor function of multiple antitumor regimens and inducing ferroptosis can reverse their resistance including chemotherapy, targeted therapy and immunotherapy. Anti-PD-1/PD-L1 therapy is able to trigger ferroptosis of tumor cells but high TYRO3 expression will inhibit this effect and lead to resistant to anti-PD-1/PD-L1 therapy [46]. Cisplatin combined with paclitaxel promotes miR-522 secretion from CAFs to suppress ALOX15 and decrease lipid-ROS accumulation in gastric cancer, and ultimately lead to decreased chemo-sensitivity. It reveals that ferroptosis suppression is new mechanism of acquired chemo-resistance in gastric cancer [47]. Considering the role of PKD2 on ferroptosis, we validated that targeting PKD2 could enhance sensitivity of LUAD cells to carboplatin. PKD2 knockdown would further inhibit proliferation of A549 and PC9 cells and silencing PKD2 or pharmacological inhibition of PKD2 would increase carboplatin-induced apoptosis of LUAD cells. In addition, we validated that inhibition of apoptosis was also a basic of carboplatin resistance triggered by PKD2. Targeting PKD2 could enhance the sensitivity of LUAD cells to carboplatin via inducing ferroptosis and apoptosis in the meantime. Consequently, our study provided a rationale to target PKD2 to enhance sensitivity of carboplatin and improve clinical outcomes of LUAD patients. 卡铂仍然是LUAD患者化疗方案的基础。然而获得性耐药极大地限制了其在临床实践中的应用。最近的证据表明，铁死亡的激活是多种抗肿瘤方案抗肿瘤功能的一部分，诱导铁死亡可以逆转其耐药性，包括化疗、靶向治疗和免疫治疗。抗PD-1/PD-L1治疗能够引发肿瘤细胞的铁死亡，但TYRO3的高表达会抑制这种作用并导致抗PD-1/PD-L1治疗耐药[ 46]。顺铂联合紫杉醇促进CAF 分泌 miR-522，抑制 ALOX15 并减少胃癌中脂质-ROS 的积累，最终导致化疗敏感性降低。它揭示了铁死亡抑制是胃癌获得性化疗耐药的新机制[47 ]。考虑到 PKD2 对铁死亡的作用，我们验证了靶向 PKD2 可以增强 LUAD 细胞对卡铂的敏感性。敲低 PKD2 将进一步抑制 A549 和 PC9 细胞的增殖，沉默 PKD2 或药理抑制 PKD2 将增加卡铂诱导的 LUAD 细胞凋亡。此外，我们验证了细胞凋亡的抑制也是PKD2引发卡铂耐药的基础。靶向PKD2可以同时诱导铁死亡和细胞凋亡，从而增强LUAD细胞对卡铂的敏感性。因此，我们的研究为靶向 PKD2 以增强卡铂敏感性并改善 LUAD 患者的临床结果提供了理论基础。
Although our study validated PKD2 could alleviate ferritinophagy-mediated ferroptosis via preventing the fusion of autophagosome and lysosome from multiple angles, there were still some limitations in our study. Firstly, the effects of PKD2 on chemotherapy resistance remains to be validate in vivo. Secondly, the elaborate mechanism how PKD2 prevented the fusion of autophagosome and lysosome was not illuminated, which would be our main task in our future work. 尽管我们的研究从多个角度验证了PKD2可以通过阻止自噬体和溶酶体的融合来缓解铁蛋白自噬介导的铁死亡，但我们的研究仍然存在一些局限性。首先，PKD2对化疗耐药的影响仍有待体内验证。其次，PKD2如何阻止自噬体和溶酶体融合的详细机制尚未阐明，这将是我们未来工作的主要任务。
5. Conclusion 5. 结论
Our study validated that augmenting PKD2 can alleviate ferritinophagy-mediated ferroptosis of LUAD cells via inhibiting the fusion of autophagosome and lysosome in a TFEB-independent way. Targeting PKD2 is promising to develop new regimens to enhance sensitivity of carboplatin in LUAD treatment. 我们的研究证实，增强 PKD2 可以通过以不依赖 TFEB 的方式抑制自噬体和溶酶体的融合，从而减轻铁蛋白自噬介导的 LUAD 细胞铁死亡。以 PKD2 为靶点有望开发新的治疗方案，以增强 LUAD 治疗中卡铂的敏感性。
This work is supported by National Natural Science Foundation of China (Grant No. 82303653) and Natural Science Foundation of Shandong Province (Grant No. ZR2021MH192).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank all patients, investigators, and institutions involved in these studies, especially the TCGA database and Shandong Province Hospital.
Supplementary Fig. 1. PKD2 overexpression and knockdown systems were established by using siRNA, shRNA and plasmid in A549 and PC9 cell lines. A, B Immunoblots of A549 and PC9 cells with PKD2 knockdown by PKD siRNAs. C Immunoblots of A549 cells with PKD2 knockdown by PKD shRNA. D, E Immunoblots of A549 cells with PKD2 overexpression by PKD plasmid.
Supplementary Fig. 2. PKD2 blocked autophagic degradation of ferritin in LUAD. A, B The mRNA expression of FTL, FTH1, NCOA4 in PC9 cells with PKD2 knockdown or overexpression by qRT-PCR. C, D Immunoblots of NCOA4, FTL in A549 and PC9 cells with PKD2 knockdown with erastin (20 μM, 24h) and chloroquine (40 μM, 6h) treatment. E Representative immunohistochemical images of FTH1 in alveolus, mesenchyme, LUAD tissues. Left: 100X, scale bar, 200 μm; Right: 400X, scale bar, 50 μm. F Representative confocal images show the colocalization of FTL and LAMP2 in experimental cells. The coefficient of colocalization was calculated by Image J. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplementary Fig. 3. Regulation of PKD2 on autophagy of LUAD. A GSEA enrichment analysis. B, C P62 mRNA expression of A549 and PC9 cells with silencing PKD2 by qRT-PCR. D, E P62 mRNA expression of A549 and PC9 cells with PKD2 overexpression by qRT-PCR. F, G LC3 mRNA expression of A549 cells with PKD2 knockdown and PC9 cells with PKD2 overexpression. H, I Immunoblots of LC3 and P62 proteins in PC9 cells with PKD2 knockdown or overexpression treated by rapamycin (5 μM, 4h) with or without bafilomycin A1 (100 nM, 4h). J A549 cells with PKD2 overexpression were infected by StubRFP-sensGFP-LC3 adenovirus and starved for 6 h. Representative confocal images show GFP puncta, RFP puncta and merged puncta in experimental cells. The data were presented as the mean ± SD; n = 3. K Representative confocal images show the colocalization of LC3B and LAMP2 in experimental cells. The coefficient of colocalization was calculated by Image J. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplementary Fig. 4. PKD2 promote proliferation, migration, invasion of LUAD cells in vitro. A Representative immunohistochemical images of PKD2, Ki67 in LUAD tissue from two cases. 100X, scale bar, 200 μm; 400X, scale bar, 50 μm. B, C The EdU assay was used to detect proliferative ability of A549 and PC9 cells with PKD2 overexpression. D, E Wound healing assay was used to detect migrative ability of A549 and PC9 cells with PKD2 overexpression. F, G The role of PKD2 on A549 cell migration and invasion was validated by Transwell assay. All statistical data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplementary Fig. 5. Targeting PKD2 could enhance sensitivity of LUAD cells to carboplatin. A, B The EdU assay detected proliferation of A549 and PC9 cells with PKD2 knockdown when cells were treated by carboplatin (20 μM, 24h). C The effects of silencing PKD2 or pharmacological inhibition of PKD2 on carboplatin (20 μM, 24h)-induced apoptosis of A549 cell. D, E The data were presented as the mean ± SD; n = 3. F The effects of silencing PKD2 or pharmacological inhibition of PKD2 on carboplatin (20 μM, 24h)-induced apoptosis of PC9 cells. G, H The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
PKD2过表达的J A549细胞被StubRFP-sensGFP-LC3腺病毒感染并饥饿6小时。代表性共焦图像显示实验细胞中的 GFP 点、RFP 点和合并点。数据以平均值±标准差表示； n = 3. K代表性共焦图像显示实验细胞中 LC3B 和 LAMP2 的共定位。通过Image J计算共定位系数。数据以平均值±SD表示； n = 3。* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.0001。
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Inhibition of ferroptosis alleviates atherosclerosis through attenuating lipid peroxidation and endothelial dysfunction in mouse aortic endothelial cell
Combinative treatment of β-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation
With the increasing prevalence of microplastics (MPs) in the environment, human health has become a growing concern. After entering the human body, MPs accumulate in the kidneys, indicating that the kidneys are their major target organs. This study investigated nephrotoxicity associated with MPs, with a specific focus on polystyrene (PS) MPs and amino-functionalized polystyrene (PS-NH2) MPs. Although previous studies have documented the nephrotoxic effects associated with short-term exposure to MPs, the mechanisms of kidney toxicity caused by chronic long-term exposure to MPs remain largely unclear. In animal models, mice were exposed to MPs (10 mg/L) at concentrations that are accessible to humans, administered via drinking water over a period of six months. These findings indicate that MPs can induce renal fibrosis by facilitating the onset of inflammation and accumulation of a substantial number of inflammatory cells. Our in vitro study showed that long-term exposure to MPs (60 μg/mL) induced ferroptosis in renal tubular epithelial cells via ferritinophagy and secreted TGF-β1, leading to renal fibroblast activation. Conversely, the application of Fer-1, a ferroptosis inhibitor, prevents ferroptosis in renal epithelial cells and reverses the activation of renal fibroblasts. Our study identified a novel toxicity mechanism for renal fibrosis induced by MPs exposure, offering new insights into the detrimental effects of environmental MPs on human health.
2024, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
For example, GCH1 knockdown induced ferroptosis and activated ferritinophagy in colorectal cancer [17], while d-Borneol enhanced cisplatin sensitivity in NSCLC by promoting ferritinophagy [18]. Recent studies have reported that PKD2 deletion in LUAD exacerbates ferritinophagy and weakens resistance to chemotherapeutic drugs [51]. In our study, NCOA4 was downregulated in LUAD clinical samples.
Ferroptosis, a type of autophagy-dependent cell death, has been implicated in the pathogenesis of lung adenocarcinoma (LUAD). This study aimed to investigate the involvement of coatomer protein complex I subunit zeta 1 (COPZ1) in ferroptosis and ferritinophagy in LUAD.
Publicly available human LUAD sample data were obtained from the TCGA database to analyze the association of COPZ1 expression with LUAD grade and patient survival. Clinical samples of LUAD and para-carcinoma tissues were collected. COPZ1-deficient LUAD cell model and xenograft model were established. These models were analyzed to evaluate tumor growth, lipid peroxidation levels, mitochondrial structure, autophagy activation, and iron metabolism.
High expression of COPZ1 was indicative of malignancy and poor overall survival. Clinical LUAD tissues showed increased COPZ1 expression and decreased nuclear receptor coactivator 4 (NCOA4) expression. COPZ1 knockdown inhibited xenograft tumor growth and induced apoptosis. COPZ1 knockdown elevated the levels of ROS, Fe2+ and lipid peroxidation. COPZ1 knockdown also caused mitochondrial shrinkage. Liproxstatin-1, deferoxamine, and z-VAD-FMK reversed the effects of COPZ1 knockdown on LUAD cell proliferation and ferroptosis. Furthermore, COPZ1 was directly bound to NCOA4. COPZ1 knockdown restricted FTH1 expression and promoted NCOA4 and LC3 expression. NCOA4 knockdown reversed the regulation of iron metabolism, lipid peroxidation, and mitochondrial structure induced by COPZ1 knockdown. COPZ1 knockdown induced the translocation of ferritin to lysosomes for degradation, whereas NCOA4 knockdown disrupted this process.
This study provides novel evidence that COPZ1 regulates NCOA4-mediated ferritinophagy and ferroptosis. These findings provide new insights into the pathogenesis and potential treatment of LUAD.
