A TAZ–ANGPTL4–NOX2 Axis Regulates Ferroptotic Cell Death

Published OnlineFirst October 22, 2019; DOI: 10.1158/1541-7786.MCR-19-0691

MOLECULAR CANCER RESEARCH | CELL FATE DECISIONS

A TAZ–ANGPTL4–NOX2 Axis Regulates Ferroptotic Cell Death and Chemoresistance in Epithelial Ovarian Cancer Wen-Hsuan Yang1,2,3, Zhiqing Huang4,5, Jianli Wu1,2, Chien-Kuang C. Ding1,2, Susan K. Murphy4,5,and Jen-Tsan Chi1,2

ABSTRACT ◥ Ovarian cancer is the deadliest gynecologic cancer. Despite recent ovarian cancer. TAZ removal confers ferroptosis resistance, while advances, clinical outcomes remain poor, necessitating novel ther- TAZS89A overexpression sensitizes cells to ferroptosis. In addition, apeutic approaches. To investigate metabolic susceptibility, we we found that lower TAZ level in chemo-resistant recurrent ovarian performed nutrigenetic screens on a panel of clear cell and serous cancer is responsible for reduced ferroptosis susceptibility. The ovarian cancer cells and identified cystine addiction and vulnera- integrative genomic analysis identified ANGPTL4 as a direct TAZ- bility to ferroptosis, a novel form of regulated cell death. Our results regulated target gene that sensitizes ferroptosis by activating NOX2. may have therapeutic potential, but little is known about the Collectively, cell density–regulated ferroptosis in ovarian cancer is determinants of ferroptosis susceptibility in ovarian cancer. We mediated by TAZ through the regulation of the ANGPTL4–NOX2 found that vulnerability to ferroptosis in ovarian cancer cells is axis, suggesting therapeutic potentials for ovarian cancers and other enhanced by lower cell confluency. Because the Hippo pathway TAZ-activated tumors. effectors Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) are recognized as sensors of cell Implications: This study reveals that TAZ promotes ferroptosis in density, and TAZ is the predominant effector in the tested ovarian ovarian cancers by regulating ANGPTL4 and NOX, offering a novel cancer cell lines, we investigated the role of TAZ in ferroptosis of therapeutic potential for ovarian tumors with TAZ activation.

Introduction Ferroptosis as a novel cell death involving lipid peroxidation The impact of ovarian cancer and the critical need for novel One possible therapeutic approach is the induction of ferroptosis, a therapeutics novel and distinct form of iron-dependent programmed cell death (5, 6). Ferroptosis sensitivity is found to be affected by various Epithelial ovarian cancer is the deadliest gynecologic cancer and biological processes, such as loss of p53 (7), DNA damage pathway (8), claims the lives of approximately 150,000 women every year world- metabolisms (9–11), or epithelial–mesenchymal transition (EMT; wide (1). The symptoms of ovarian cancer are vague and often refs. 12, 13), which are often dysregulated in ovarian cancer. Ferrop- attributed to other more common ailments. As a result, the correct tosis can be induced by the small molecule, erastin (14), that reduces diagnosis usually only occurs after cancer has spread beyond the cystine import and result in a redox imbalance by reducing intracel- ovaries. Standard therapy involves surgical debulking followed by lular glutathione levels. Glutathione is a cofactor for glutathione chemotherapy with a platinum-taxane doublet (2). Although many peroxidase (GPX4), an enzyme that resolves the accumulation of patients initially respond favorably to this combined treatment, most lipid-based reactive oxygen species (ROS). Therefore, ferroptosis and patients relapse with a recurrent disease that is often resistant to lipid peroxidation can also be induced by chemical or genetic inhi- platinum and taxane drugs. Other chemotherapeutic options are used bition of GPX4 (6). A previous study has indicated that the levels of mainly in an effort to prolong survival. Recently, platinum-PARP GPX4, regulated by the EMT-activator ZEB1, may dictate ferroptosis inhibitor combinations have been proven to be beneﬁcial for ovarian sensitivity of drug-resistant cancer cells, implicating GPX4 as a major cancer regardless of the BRCA1/2 mutation status (3, 4). However, the determinant of ferroptosis (12, 13). On the other hand, accumulation outcomes for most women with ovarian cancer are still unsatisfactory, of lipid-based ROS and ferroptosis can also be induced by the therefore novel therapeutic options are still urgently needed. generation of superoxide and hydrogen peroxide upon upregulation of NADPH oxidases (NOX; ref. 5). In this study, we perform a nutrigenetic screen and show that most 1Department of Molecular Genetics and Microbiology, Duke University School of ovarian cancer cell lines are addicted to cystine and sensitive to Medicine, Durham, North Carolina. 2Center for Genomic and Computational ferroptosis. Furthermore, we found that ferroptosis susceptibility of Biology, Duke University School of Medicine, Durham, North Carolina. 3Depart- ovarian cancer cells is affected by cell density. Low density, but not ment of Biochemistry, Duke University School of Medicine, Durham, North high-density ovarian cancer cells, were highly susceptible to erastin- 4 Carolina. Department of Obstetrics and Gynecology, Division of Reproductive induced ferroptosis. The density-dependent phenotypes of cancer cells Sciences, Duke University School of Medicine, Durham, North Carolina. 5Depart- are sensed and regulated by the evolutionarily conserved Hippo ment of Obstetrics and Gynecology, Division of Gynecologic Oncology, Duke University School of Medicine, Durham, North Carolina. pathway (15) converging into two transcriptional coactivators, Yes- associated protein 1 (YAP) and transcriptional coactivator with PDZ- Corresponding Author: Jen-Tsan Chi, Duke Medical School, 101 Science Drive, 2141 CIEMAS, DUMC Box# 3382, Durham, NC 27705. Phone: 919-668-4759; binding motif (TAZ). YAP/TAZ activities are regulated by their Fax: 919-668-4777; E-mail: [email protected] phosphorylation and intracellular localization. When grown at high cell density, YAP/TAZ are phosphorylated, retained in the cytosol, Mol Cancer Res 2019;XX:XX–XX and subjected to proteasomal degradation. Upon shifting to low cell doi: 10.1158/1541-7786.MCR-19-0691 density, YAP/TAZ become dephosphorylated and translocate into 2019 American Association for Cancer Research. the nucleus to associate with transcriptional factors to drive

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gene expression regulating cell proliferation, differentiation, and CAT GTT GAT CCA GCC CAT); siNOX2 #1 (target sequence: GAA migration (16). Recent studies have also identified the novel role of GAC AAC TGG ACA GGA A); and siNOX2 #2 (target sequence: GAA YAP and TAZ in regulating ferroptosis (17, 18). However, the rele- ACT ACC TAA GAT AGC G). Knockdown efficacy was validated by vance of these findings for ovarian cancer remains unknown. Here, we qRT-PCR and/or Western blot analysis. have established the role of cell density and TAZ in regulating ferroptosis of ovarian cancer. In addition, we found that TAZ regulates RNA isolation and qRT-PCR erastin-induced ferroptosis through the induction of ANGPTL4, which Total RNAs from cultured cells were extracted using the RNeasy in turn activates NOX2, resulting in ferroptosis. Thus, these data Mini Kit (Qiagen, #74104) with DNase I treatment (Qiagen, #79254). support the role of TAZ in regulating ferroptosis through ANGPTL4– cDNAs were synthesized from 1 mg of total RNA using SuperScript II NOX2 and that inducing ferroptosis may be a novel therapeutic Reverse Transcriptase (Thermo Fisher Scientific, #18064) with ran- strategy for ovarian cancer and other TAZ-activated tumors. dom hexamers following protocols from the manufacturer. The levels of gene expression were measured by qPCR with Power SYBR Green PCR Mix (Applied Biosystems; Thermo Fisher Scientific, #4367659). Materials and Methods Primers used included (listed 50–30): b-actin-F': GGG GTG TTG AAG Materials and reagents GTC TCA AA; b-actin-R': GGC ATC CTC ACC CTG AAG TA; TAZ- Erastin was obtained from the Duke University Small Molecule F': TGC TAC AGT GTC CCC ACA AC; TAZ-R': GAA ACG GGT Synthesis Facility. The following antibodies, their catalog numbers, CTG TTG GGG AT; ANGPTL4-F': GGC TCA GTG GAC TTC AAC sources, and dilutions were indicated below: YAP/TAZ (#8418; Cell CG; ANGPTL4-R': CCG TGA TGC TAT GCA CCT TCT; NOX2-F': Signaling Technology, 1:1,000), b-tubulin (#86298; Cell Signaling TGG AGT TGT CAT CAC GCT GTG; NOX2-R': CTG CCC ACG Technology, 1:2,000), vinculin (sc-73614; Santa Cruz Biotechnology, TAC AAT TCG TTC; and 18S-F': CTG GAT ACC GCA GCT AGG 1:2,000), V5 tag (#13202; Cell Signaling Technology, 1:2,000), H3 AA; 18S-R': CCC TCT TAA TCA TGG CCT CA. (#4499; Cell Signaling Technology, 1:2,000), GAPDH (sc-25778; Santa Cruz Biotechnology, 1:2,000), ANGPTL4 (#40-9800; Thermo Western blot analysis Fisher Scientific, 1:1,000), NOX2 (sc-130543; Santa Cruz Biotechnol- For immunoblotting, please refer to previous publication for ogy, 1:1,000), anti-rabbit IgG, horseradish peroxidase (HRP)-linked details (17). In short, cells were collected and quantified by BCA antibody (#7074; Cell Signaling Technology, 1:2,000–1:4,000), and protein assay. After separating by SDS-PAGE, the proteins were anti-mouse IgG, HRP-linked Antibody (#7072; Cell Signaling Tech- transferred to polyvinylidene difluoride membranes that were further nology, 1:2,000–1:4,000). Plasmids were obtained from Addgene blocked with 5% nonfat milk or BSA and probed with indicated (TAZS89A #52084; ANGPTL4-V5 #102446). The NOX2 inhibitor, antibodies following by HRP-conjugated secondary antibodies. The gp91 ds-tat, was purchased from Eurogentec (catalog no. AS-63818) signals were developed and detected by Amersham ECL prime West- and recombinant human ANGPTL4 protein was purchased from ern blotting detection reagent and the Bio-Rad ChemiDoc Imaging Novus Biologicals (4487-AN). VAS2870 (Calbiochem-492000), System. GKT136901 (Calbiochem-534032), Z-VAD-FMK (SML0583), ferrostatin-1 (SML0583), and liproxstatin-1 (SML1414) were purchased Cell viability and cytotoxicity assays from Sigma. After seeded and transfected with indicated siRNAs for 2 days, cells (60–70% confluence) were treated with erastin or cystine deprivation Cell culture and transfection for another 24 to 48 hours. Cell viability was evaluated using crystal Cell lines were maintained as part of the cell line repository within violet staining or the CellTiter Glo Luminescent Cell Viability Assay the Division of Reproductive Sciences at Duke University. For the Kit (Promega G7571) according to the manufacturer's instructions. details, please refer to ref. 17. Short tandem repeat profiling was The CellTiter Glo Luminescent Cell Viability Assay is based on performed at the Duke University DNA Analysis Facility each time quantitation of the cellular ATP levels as an indicator of metabolically when frozen stocks are prepared; they were last genotyped on June 5, active cells and cellular viability. The cytotoxicity and cell death of 2018. Mycoplasma testing was conducted at the Cell Culture Facility, treated cells were determined by CytoTox-Fluor Cytotoxicity Assay Duke University on June 5, 2018. For nutrigenetic screens, all ovarian (Promega G9260), which measured the released DNA as indicator of cancer cells were cultured in a humidified incubator at 37C and 5% cell death according to the manufacturer's instructions. CO2 using custom-made DMEM (11995-DMEM; Thermo Fisher Scientific) with 10% heat-inactivated and dialyzed FBS (HyCloneTM ELISA FBS; GE Healthcare Life Sciences, #SH30070.03HI) with the indicated Quantification of secreted ANGPTL4 in the culture media from amino acid removed as described previously (19, 20). Transfections cells following a 2-day incubation was performed by Human were performed according to the manufacturer's instructions with ANGPTL4 ELISA Kit (RAB0017; Sigma-Aldrich) according to the TransIT-LT1 transfection reagent (Mirus Bio) or RNAiMax transfec- manufacturer's instruction. tion reagent (Thermo Fisher Scientific). Chromatin immunoprecipitation analysis siRNA-mediated gene knockdown Chromatin immunoprecipitation (ChIP)-qPCR experiment was All human siRNAs were purchased from Dharmacon or Qiagen: carried out according to the Myers Lab ChIP-seq protocol (21). Please nontargeting control, siNT (Qiagen, SI03650318); siTAZ (Dharma- refer to the previous publication for the experimental details (17). con, M-016083); siANGPTL4 (Dharmacon, M-007807); siNOX2 Primers targeting chromosome 10 was used as a negative control for (Dharmacon, M-011021); siTAZ#1 (target sequence: AGA CAT GAG potential nonspecific binding (22). Primer sequences are as follows ATC CAT CAC TAA); siTAZ#2 (target sequence: ACA GTA GTA (listed 50–30): CTGF-F': GCC AAT GAG CTG AAT GGA GT; CTGF- CCA AAT GCT TTA); siANGPTL4 #1 (target sequence: CTG CGA R': CAA TCC GGT GTG AGT TGA TG; ANGPTL4-F': GTC TCC ATT CAG CAT CTG CAA); siANGPTL4 #2 (target sequence: CAC CAC GGT TCG TAG AG; ANGPTL4-R': TAT AAG TTG GGT GCG

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GAG TGG; and Ch10-F': ACC AAC ACT CTT CCC TCA GC; Ch10- The statistical data were represented as mean SEM. The number of R': TTA TTT TGG TTC AGG TGG TTG A. biological replicates is listed in each ﬁgure.

Lipid ROS assay using flow cytometry Data availability Ten mmol/L of C11-BODIPY dye (D3861; Thermo Fisher Scientific) RNA-sequencing (RNA-seq) for transcriptional profiles of CAOV2 was used for lipid ROS staining according to the manufacturer's primary and CAOV2 recurrent cells (GSE133663) and CAOV2 ovar- instructions. Please refer to the previous publication for the experi- ian cancer cells with TAZ silencing (GSE133664) have been deposited mental details (17). In short, cells were collected after siRNAs treat- in the NCBI Genome Expression Omnibus under SuperSeries GSE ments for 2 days, 10 mmol/L erastin treatment for 18 hours, and 10 133673. mmol/L C11-BODIPY-containing medium for 1 hour and the ROS levels were determined by flow cytometry analysis (FACSCanto II; BD Biosciences). Results Ovarian cancer cells are sensitive to cystine deprivation Statistical analyses Many tumor cells have dysregulated metabolisms that render them The results were evaluated with two-tailed Student t test or ANOVA addicted to certain nutrients, such as amino acids (23). To reveal such (one- or two-way) using GraphPad Prism version 8.0.1 (GraphPad nutrient addiction in ovarian cancers, we established a nutrigenetic Software). Statistically significant differences were set to P < 0.05 screen by removing glucose or each of the 15 individual amino acids between experimental groups (, P < 0.05; , P < 0.01; , P < 0.001). usually included in DMEM (19, 24). The nutrigenetic screen was

Figure 1. Ovarian cancer cells are sensitive to cystine deprivation. A, Normalized cell viability of the indicated ovarian cancer cells after deprivation of individual amino acids or glucose. Error bars represent mean SEM (n ¼ 3, biological replicates). Two ferroptosis inhibitors rescued cystine deprivation–induced cell death. TOV-21G cells were seeded in 10% (20 mmol/L of cystine) or 20% (40 mmol/L of cystine) of full DMEM media supplied with or without either 200 nmol/L liproxstatin-1 (Lip; B)or2mmol/L ferrostatin-1 (Fer-1; C). After 2 days, the cell viability was determined by CellTiter Glo and normalized to the signal of full DMEM media (200 mmol/L of cystine) of each group (n ¼ 3 per group; mean SEM; two-way ANOVA). D, Cell viability of CAOV2 was determined by Cell- titerGlo after cells were treated with 16 mmol/L erastin, 20 mmol/L Z-VAD-FMK (Z-VAD), or 1 mmol/L Fer-1. , P < 0.001.

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applied to a panel of eight ovarian cancer cell lines including four cell down by siRNAs reduces sensitivity to erastin (Fig. 2F; Supple- lines of clear-cell subtype (TOV-21G, ES-2, RMG-2, and RMG-V) and mentary Fig. S1E). Collectively, these data strongly indicate that four cell lines of serous subtype (OVCA432, OVCA429, OVCA420, TAZ regulates the sensitivity of ovarian cancer cells to erastin- and 41M). The screen revealed that the removal of cystine, the dimeric induced ferroptosis. form of cysteine, dramatically reduced the viability of most clear-cell and serous subtypes of ovarian cancer cells (Fig. 1A). Glutamine or Resistance to ferroptosis following treatment with carboplatin glucose deprivation also decreases cell viability, especially in the clear- in vivo cell subtype, reminiscent of the glutamine addiction of basal-type We performed our experiments in CAOV2 because we have breast cancer cells (25). Knowing that cystine deprivation is reported to established paired CAOV2 xenograft cells from a mouse model of trigger ferroptosis (5), we determined whether ferroptosis inhibitors ovarian cancer that mimics the course of disease (26). In short, the can rescue the cell death triggered by cystine deprivation. Both CAOV2 cells were stably transduced with a construct containing GFP liproxstatin-1 and ferrostatin-1, two ferroptosis inhibitors, significant- and luciferase (pGreenFire plasmid constructs, SBI) to generate ly rescued cystine deprivation-induced cell death of TOV-21G cells CAOV2-GFP/LUC cells, allowing us to monitor tumor growth using (Fig. 1B and C), indicating that cystine deprivation indeed induces in vivo bioluminescence imaging. CAOV2-GFP/LUC cells (3.5 105 ferroptosis. We further confirm that the erastin-induced cell death can per mouse) were injected intraperitoneally into female nude mice with be rescued by ferrostatin-1, but not apoptosis inhibitor, Z-VAD-FMK tumor formation monitored weekly using the IVIS in vivo imaging (Fig. 1D). Therefore, we used erastin, a canonical inducer of ferrop- system. Once tumor formation was evident, we initiated treatment tosis, to investigate the ferroptosis mechanisms of ovarian cancer in with carboplatin (60 mg/kg i.p. twice a week for 2 weeks) and our subsequent studies. monitored tumor volume based on the luciferase flux. In this model, tumor signal was reduced following carboplatin treatment, but then Cell density affects the sensitivity of ovarian cancers to erastin- recurred approximately 2 to 4 weeks after carboplatin treatment had induced ferroptosis stopped. Therefore, we refer to the residual tumor cells that While investigating ferroptosis in ovarian cancer cells, we eventually emerged after carboplatin treatment as CAOV2R (recur- observed that cell density affects the ferroptosis sensitivity of rent). We then compared cystine dependency (Fig. 3A and B)and CAOV2, a serous ovarian cancer cell line. When CAOV2 cells erastin sensitivity (Fig. 3C and D) between the CAOV2 and were plated at low density, they were highly sensitive to erastin CAOV2R cells. These experiments revealed that CAOV2R was based on CellTiter Glo assay (Fig. 2A) and crystal violet staining more resistant to ferroptosis based on crystal violet staining (Fig. 2B; Supplementary Fig. S1A). On the other hand, when plated (Fig. 3A and C), cytotoxicity assay (Fig. 3B), and CellTiter Glo at higher densities, CAOV2 cells were much less sensitive to erastin- viabilityassay(Fig. 3D). Because TAZ regulates ferroptosis sensi- induced death using CellTiter Glo and crystal violet assays (Fig. 2A tivity (Fig 2), we compared TAZ protein abundance in CAOV2 and and B; Supplementary Fig. S1A). To exclude the possibility that CAOV2R cells and found that CAOV2R contained a lower level of these differences are caused by different levels of available erastin TAZ protein (Fig. 3E). We, therefore, overexpressed the constitu- per cell, the same number of TOV-21G cells were seeded in larger or tively active TAZS89A (27) in the CAOV2R cells (Fig. 3F)and smaller areas to recreate lower or higher cell densities. We found found that increased TAZ significantly sensitized the CAOV2R cells that cells plated at lower cell density were consistently more to erastin (Fig. 3G). These findings suggest that comparisons of sensitive to ferroptosis (Supplementary Fig. S1B). Therefore, cell CAOV2 versus CAOV2R can be used to elucidate the molecular density impacts ferroptosis sensitivity. mechanisms of TAZ-regulated ferroptosis. Because the two closely related Hippo pathway paralogues, YAP/ TAZ, sense and mediate cell density–dependent responses (15), we ANGPTL4 is a direct target gene of TAZ that regulates sensitivity investigated the potential role of YAP/TAZ in regulating ferroptosis in to ferroptosis ovarian cancers. Among the two paralogues (YAP and TAZ), Western TAZ is a transcriptional coactivator that affects cellular pheno- blot analysis reveals that TAZ is the predominantly expressed protein types through regulating the expression of target genes upon associ- in both TOV-21G (clear cell subtype) and CAOV2 cells (serous ation with transcriptional factors such as transcriptional enhanced subtype; Fig. 2C). In contrast, YAP is the predominant Hippo effector associate domain (TEAD). Assuming that knockdown of TAZ may protein in the breast cancer cell, MDA-MB-231, which serves as a repress the relevant target genes essential for ferroptosis, we deter- control for YAP expression and detection (Fig. 2C). We further mined the transcriptional response to knockdown of TAZ in performed cytosolic and nuclear fractionations of CAOV2 cells under CAOV2 by RNA-seq (Supplementary Fig. S2A; GSE133673). Next, low or high cell densities. Consistently, TAZ is the main protein whose we integrated these TAZ-affected genes with the genes that were nuclear levels were elevated when grown in low cell density (Fig. 2D). downregulated in CAOV2R when compared with CAOV2 (Fig. 4A) Therefore, we focused on the potential role of TAZ as the major to identify 1,179 candidate genes.ToidentifytheTAZ-regulated effector of the Hippo pathway in regulating density-dependent fer- genes that are essential for ferroptosis across different cells, we roptosis in ovarian cancers. further narrowed the list of candidate genes by comparing with the gene lists identified from our renal cell studies through both RNAi TAZ regulates sensitivity to erastin-induced ferroptosis screen and TAZ-affected genes (Supplementary Fig. S2B; ref. 17). To test the possibility that TAZ regulates susceptibility of ovarian Among the 3 candidate genes from these analyses, ATP6V1B2 was cancer cells to erastin, we first reduced TAZ expression (>85% not pursued because it was only one subunit of a multicomponent knockdown) by siRNAs in CAOV2 cells (Supplementary Fig. S1C protein complex. The knockdown of GPSM3 did not confer erastin and S1D). Knockdown of TAZ by pool siRNA as well as two more resistance (Supplementary Fig. S2C and S2D). Therefore, Angio- independent siRNAs all reduce erastin-induced cell death based on poietin-Like 4 (ANGPTL4) emerged as the most promising candi- CellTiter Glo assays (Fig. 2E). To determine whether TAZ also date gene based on the following evidence. First, ANGPTL4 mRNA regulates ferroptosis in TOV-21G cells, we found that TAZ knock- was downregulated upon TAZ knockdown, which was confirmed by

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Figure 2. TAZ regulates the sensitivity of CAOV2 to erastin-induced ferroptosis. A, The relative cell viability of CAOV2 cells grown at low/medium/high (4,000/8,000/16,000 cells per 96-well) cell densities after treated with indicated concentrations of erastin. Data are represented as mean SEM with three biological replicates per group; two-way ANOVA (, P < 0.001). B, Crystal violet staining of CAOV2 cultured at low/medium/high (1.6 105/3.2 105/6.4 105 cells per 6-well) cell densities and treated with 16 mmol/L erastin. C, Western blot analysis of YAP and TAZ proteins in the ovarian cancer cells (TOV-21G, CAOV2, and CAOV2R) and one breast cancer cell line (MDA-MB-231). Representative data of at least three independent experiments. D, Western blot analysis measuring YAP and TAZ protein levels in the cytosolic (Cyto)/nucleic (Nuc) fractions of CAOV2 extracts when grown at low (L) or high (H) cell density. Histone H3 and GAPDH serve as nuclear and cytosolic marker, respectively. E, The relative cell viability of CAOV2 cells after silencing of control (siNT), TAZ (siTAZ), or two individual TAZ-targeting siRNAs (siTAZ #1 and siTAZ #2) for 1 day before they were treated with indicated dosages of erastin for 2 days (n ¼ 2; mean SEM; two-way ANOVA; , P < 0.001). F, The relative cell viability of TOV-21G cells after silencing of control (siNT) or TAZ (siTAZ) for 2 days before they were treated by indicated dosages of erastin treatment for 1 day (n ¼ 3; mean SEM; two-way ANOVA; , P < 0.001). See also Supplementary Fig. S1. qRT-PCR (Fig. 4B). Second, there is a signiﬁcant correlation tively, these data indicate that ANGPTL4 is a prominent TAZ- between the expression of ANGPTL4 and TAZ (encoded by regulated determinant of ferroptosis in ovarian cancer. WWTR1) in the TCGA ovarian tumor data set. The expression of Next, we analyzed previous ChIP-seq studies and found the WWTR1 is also consistently correlated with CTGF (28) and regulatory regions of ANGPTL4 were physically associated with CYR61 (29), 2 canonical YAP/TAZ target genes (Supplementary YAP/TAZ/TEADcomplexes(22,30).Tofurthervalidatethat Fig. S2E–S2G).Third,theknockdownofANGPTL4 in both CAOV2 ANGPTL4 is a direct target gene of TAZ, we performed ChIP- and TOV-21G protects cells from erastin-induced death (Fig. 4C qPCR using an antibody speciﬁcforendogenousTAZprotein.As and D; Supplementary Fig. S2H and S2I). This protective effect was shown in Fig. 4G, we found that the promoter region of ANGPTL4, further validated by using two more independent ANGPTL4-target- similar to the CTGF (positive control), was enriched in the TAZ ing siRNAs in CAOV2 cells (Fig. 4E; Supplementary Fig. S2J). pull-down, indicating that the ANGPTL4 promoter is associated Consistently, overexpression of V5-tag ANGPTL4 sensitized TOV- with TAZ. Thus, ANGPTL4 is a direct downstream target gene of 21G cells to ferroptosis (Fig. 4F; Supplementary Fig. S2K). Collec- TAZ, which may contribute to TAZ-regulated ferroptosis.

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Figure 3. Regrowth of chemoresidual tumor cells are more resistant to ferroptosis. A, Crystal violet staining of CAOV2 and CAOV2R cells when culture with full or cystine- deprived (Cys) media. Representative data of at least three independent experiments. B, Cytotoxicity was determined by CytoTox-Fluor assay after treatment of 10% (20 mmol/L of cystine) of full media (n ¼ 3; two-way ANOVA; , P < 0.001; ns, not signiﬁcant). C, Crystal violet staining of CAOV2 and CAOV2R cells when cultured with DMSO or 15 mmol/L erastin. Representative data of at least three independent experiments. D, The relative cell viability of CAOV2 and CAOV2R cells when they were treated with the indicated dosage of erastin (n ¼ 3; mean SEM; two-way ANOVA; , P < 0.001). E, Western blot analysis of TAZ and Vinculin (control) protein levels in CAOV2 and CAOV2R cells. Representative data of at least three independent experiments. F, Validation of TAZS89A overexpression by Western blot analysis in CAOV2R cells. Representative data of at least three independent experiments. G, The relative cell viability of CAOV2R cells after the transfection of control vector or constitutively active TAZ, TAZS89A, following by treating with indicated dosages of erastin. Data are represented as mean SEM, n ¼ 3 biological replicates; two-way ANOVA (, P < 0.001).

Differential ANGPTL4 expression contributes to the distinct test the possibility that the lower expression of ANGPTL4 in ferroptosis sensitivities among CAOV2 and CAOV2R cells CAOV2R may contribute to its relative ferroptosis resistance, we We next determined whether the differential expression of determined whether the addition of recombinant ANGPTL4 proteins ANGPTL4 could explain the different ferroptosis sensitivity between may enhance the ferroptosis sensitivity of CAOV2R. As shown CAOV2 and CAOV2R cells. From the results of qRT-PCR and in Fig. 5D, soluble ANGPTL4 sensitized CAOV2R, but not CAOV2, Western blot analysis, we found CAOV2R has a lower ANGPTL4 cells to ferroptosis. expression at both mRNA level and protein level (Fig. 5A and B). Because ANGPTL4 encodes a secreted protein, we also examined ANGPTL4 regulates ferroptosis through NOX2 the level of extracellular ANGPTL4 in the culture media by ELISA. With regard to the mechanistic link between ANGPTL4 and Consistently, extracellular ANGPTL4 proteins are less abundant in ferroptosis, it is interesting to note that ANGPTL4 has been reported the CAOV2R cells as compared with the CAOV2 cells (Fig. 5C). To to activate the NADPH oxidase 1, NOX1, in the keratinocyte

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Figure 4. ANGPTL4 is a direct target gene of TAZ that regulates ferroptosis sensitivity. A, Venn diagram showing genes that are both downregulated upon TAZ knockdown in CAOV2 cells (3,370 þ 1,179 genes) and downregulated genes (2,540 þ 1,179 genes) in CAOV2R cells compared with CAOV2 cells by RNA-seq. B, Validation of downregulated ANGPTL4 mRNA level by qRT-PCR upon TAZ knockdown (n ¼ 3; mean SEM; Student t test; , P < 0.05). The relative cell viability of CAOV2 (C)and TOV-21G (D) cells after knockdown of ANGPTL4 following by treating with indicated dosages of erastin. Data are represented as mean SEM, n ¼ 3 biological replicates (, P < 0.001). E, The relative cell viability of CAOV2 cells after silencing of ANGPTL4 by two individual siRNAs for 2 days before treated with indicated dosages of erastin for 1 day. Data are represented as meanSEM, n ¼ 3 biological replicates (, P < 0.001). F, The relative cell viability of TOV-21G cells after overexpression of V5-tagged ANGPTL4, followed by treating indicated dosages of erastin. Data are represented as meanSEM, n ¼ 3 biological replicates (, P < 0.001). G, The relative levels of TAZ-associated genomic DNA in the indicated promoters associated with endogenous TAZ protein using ChIP- qPCR with TAZ antibody in CAOV2 protein lysate. CTGF promoter serves as a positive control for a TAZ target gene, whereas Ch10 serves as a negative control. See also Supplementary Fig. S2.

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Figure 5. Differential ANGPTL4 expression contributes to the different ferroptosis sensitivities among CAOV2 and CAOV2R cells. A, The relative levels of ANGPTL4 mRNA were measured by qRT-PCR in CAOV2 and CAOV2R cells (n ¼ 3; mean SEM; Student t test; , P < 0.05). B, Western blot analysis of ANGPTL4 and b-tubulin proteins in CAOV2 and CAOV2R cells. Representative data of at least three independent experiments. C, ELISA of extracellular ANGPTL4 proteins in the culture media of CAOV2 and CAOV2R cells incubated for 2 days (n ¼ 4, mean SEM; Student t test; , P < 0.05). D, Relative cell viability of CAOV2 and CAOV2R cells when treated with recombinant ANGPTL4 protein in media followed by 8 or 16 mmol/L erastin and then normalized to DMSO control group (n ¼ 3, mean SEM; two-way ANOVA; , P < 0.05; , P < 0.01; , P < 0.001).

carcinoma cells (31). The NADPH oxidases are recognized to generate CAOV2 and CAOV2R, we found that gp91dstat protected CAOV2, ROS (32), promoting lipid peroxidation and ferroptosis (5). Therefore, but not CAOV2R, from ferroptosis (Fig. 6F). Furthermore, NOX we investigated the expression levels of all seven members of the NOX inhibitor GKT136901 also abolishes the ferroptosis-sensitizing effect protein family (NOX1-5 and DUOX1-2) in ovarian cancer. Among when TAZS89A was overexpressed in CAOV2R cells (Fig. 6G). different NOXs, we found that NOX2 is the most abundantly expressed Therefore, NOX2 activity is essential for ferroptosis regulation by member from the analysis of RNA-seq data from the TCGA data set TAZ. Finally, we measured the lipid-based reactive oxygen species (Supplementary Fig. S3A). We also noticed NOX2 protein was lower in (lipid ROS), the hallmark of ferroptosis, by C11-BODIPY staining and CAOV2R when compared with CAOV2 cells (Supplementary validated that knockdown of TAZ, ANGPTL4, or NOX2 decreases the Fig. S3B). Therefore, we focused on the role of NOX2 protein. First, erastin-induced lipid peroxidation (Fig. 7A). Taken together, we we found a pan NOX inhibitor, VAS2870, protected CAOV2 cells from propose a signaling mechanism (Fig. 7B) by which TAZ is a cell ferroptosis (Fig. 6A). Another NOX inhibitor, GKT136901, also density–dependent determinant of ferroptosis sensitivity in ovarian protected cells from ferroptosis (Fig. 6B). We further used a cancer through regulating levels of ANGPTL4, which in turn regulates NOX2-speciﬁc inhibitor, gp91dstat peptide, in the CAOV2 cells and NOX2 activity and ferroptotic death. found it protected ferroptosis as well (Fig. 6C). In addition, silencing of NOX2 by siRNA also conferred ferroptosis resistance in both CAOV2 cells (Fig. 6D; Supplementary Fig. S3C) and TOV-21G cells (Fig. 6E; Discussion Supplementary Fig. S3D). To examine the relevance of TAZ– Here, we employed nutrigenetic screens and identiﬁed cystine ANGPTL4–NOX2 for the varying ferroptosis sensitivity between addiction and ferroptosis susceptibility of ovarian cancer cells,

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Figure 6. ANGPTL4 regulates ferroptosis through NOX2. A–E, The relative cell viability of erastin-treated CAOV2 cells (A–D) or TOV-21G cells (E) when NOXs was inhibited by 20 mmol/L pan-NOX inhibitor VAS2870 (A); 20 mmol/L NOX inhibitor, GKT136901 (B); speciﬁc NOX2 inhibitor, gp91dstat (33 mg/mL; C); or pooled siRNAs (siNOX2) and/or two independent NOX2-targeting siRNAs (siNOX2 #1 and #2; D–E, n ¼ 3, mean SEM; two-way ANOVA; , P < 0.001). F, The relative cell viability of CAOV2 or CAOV2R cells was determined by CelltiterGlo after 24 hours of 8 mmol/L erastin with or without NOX2 inhibitor, gp91dstat (33 mg/mL). Data are represented as meanSEM, n ¼ 3 after normalized to the DMSO controls (two-way ANOVA; , P < 0.05; , P < 0.01; , P < 0.001; ns, not signiﬁcant). G, The relative cell viability of CAOV2R expressing control vector or TAZS89A was determined by CelltiterGlo after 24 hours of 10 mmol/L erastin with or without 20 mmol/L NOX inhibitor, GKT136901. Data are represented as mean SEM, n ¼ 6 after normalized to the DMSO controls (two-way ANOVA; , P < 0.001). See also Supplementary Fig. S3.

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Figure 7. The proposed molecular mechanism on how TAZ-regulated ferroptosis sensitivity through ANGPTL4–NOX2 axis in ovarian cancer cells. A, Inhibition of TAZ, ANGPTL4, or NOX2 abolishes the elevated lipid ROS induced by erastin treatment. Lipid ROS in CAOV2 cells was assessed by ﬂow cytometry using C11-BODIPY after transfected with siTAZ, siANGPTL4, or siNOX2. Repre- sentative data from one of three independent experiments are shown. B, Schematic representing the model of TAZ-regulated ferroptosis of ovarian cancer through ANGPTL4–NOX2.

implying that ferroptosis-inducing agents may hold therapeutic poten- This study enhances our understanding of the role of TAZ and how tial for ovarian cancer. However, little is known about ferroptosis in it regulates ferroptosis in ovarian cancer. ovarian cancer other than being brieﬂy mentioned in two recent YAP/TAZ and other components of the Hippo signaling pathway articles (33, 34) without mechanistic investigation. By studying how are important in oncogenesis and migration of ovarian cancer cell density regulates ferroptosis sensitivity, as described for other cells (35). In our experiments, TAZ, rather than YAP, is the dominant cancer types, we have elucidated the role of TAZ in regulating effector in the tested ovarian cancer cells. Although the role of YAP was ferroptosis through the ANGPTL4–NOX2 axis in ovarian cancer. the ﬁrst recognized, many studies have also supported the role of TAZ

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in ovarian cancer. For example, increased expression of TAZ mRNA is Our results may have therapeutic implications. While inducing correlated with poor prognosis and TAZ manipulation affects migra- ferroptosis may have substantial antitumor potential, challenges tion, proliferation, treatment response, and EMT of ovarian can- remain. It is not clear which tumors would respond to ferroptosis- cer (36, 37). Therefore, our findings suggest that inducing ferroptosis inducing agents. Our results indicate that TAZ-activated tumors may will be an effective strategy for eradicating TAZ-activated tumors, be particularly sensitive to various ferroptosis-inducing therapies. which are particularly aggressive and resistant to current standard Thus, TAZ activation and induction of its canonical target genes may treatments. In the future, it will be interesting to test whether YAP or serve as predictive biomarkers. In addition, it is important to consider other regulators of the Hippo pathway also play a role in regulating the compounds or therapeutic agents that would be most effective for ferroptosis. inducing ferroptosis. Although erastin and various GPX4 inhibitors ANGPTL4 is a member of the angiopoietin family and the can induce ferroptosis, their toxicity and stability may limit in vivo members of which act as regulators of lipid and glucose metabo- application and translation potential. One promising agent is recom- lism (38, 39). ANGPTL4 also plays a role in tumor biology. binant human cyst(e)inase that can trigger ferroptosis by depleting ANGPTL4 is upregulated in several human cancers and this is plasma cystine (47). Because its antitumor efficacy and in vivo safety associated with metastasis and poor outcome (40, 41). In addition, have been demonstrated in murine models for multiple tumors, it may ANGPTL4 is induced by various oncogenic pathways to promote have the potentials to trigger in vivo ferroptosis of ovarian cancer and angiogenesis, invasion, and metastasis (42). Especially relevant is a improve clinical outcomes. study that ANGPTL4 stimulates oncogenic ROS and anoikis resis- tancethroughtheactivationofNADPHoxidase(31).Ourcurrent Disclosure of Potential Conflicts of Interest fl study reveals that ANGPTL4 is a direct transcriptional target of No potential con icts of interest were disclosed. TAZ, consistent with the repression of ANGPTL4 by the TAZ/YAP Authors’ Contributions inhibitor, verteporfin (43). On the basis of the previous studies, Conception and design: W.-H. Yang, C.-K.C. Ding, J.-T. Chi ANGPTL4 is expected to increase ROS and predispose cells to signal Development of methodology: W.-H. Yang, Z. Huang, S.K. Murphy, J.-T. Chi that induces ferroptosis. Therefore, our findings implicate high Acquisition of data (provided animals, acquired and managed patients, provided ANGPTL4 levels and anoikis resistance as novel and highly relevant facilities, etc.): W.-H. Yang, J. Wu, C.-K.C. Ding, S.K. Murphy oncogenic properties that can be targeted by inducing ferroptosis. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Because anoikis resistance is essential for tumor metastasis (44), our computational analysis): W.-H. Yang, J. Wu, J.-T. Chi results imply that ferroptosis may target tumor cells at different Writing, review, and/or revision of the manuscript: W.-H. Yang, Z. Huang, C.- K.C. Ding, S.K. Murphy, J.-T. Chi stages of tumor progression. Anoikis resistance is one of the Administrative, technical, or material support (i.e., reporting or organizing data, phenotypic changes that occur during EMT (45, 46). Our findings constructing databases): W.-H. Yang, C.-K.C. Ding, J.-T. Chi are also in agreement with other studies showing increased ferrop- Study supervision: J.-T. Chi tosis sensitivity during EMT that is in part due to the regulation of Other (one of the main people for experiment performance): Z. Huang GPX4 by ZEB1 (12). Acknowledgments We have found that the carboplatin-treated CAOV2R cells are less We thank David Corcoran for RNA-seq data analysis and Carole Grenier for sensitive to ferroptosis and have a lower level of TAZ. These results technical assistance with experiments. This work was supported by the Duke Cancer seemingly contradict previous reports on the ferroptosis sensitivity of Institute Pilot Project, Department of Defense grants (W81XWH-17-1-0143, persister cells (12) due to increased GPX4 expression. However, our W81XWH-15-1-0486, and W81XWH-19-1-0842) and NIH (1R01GM124062 and results are consistent with the data in Hangauer et al. (13), which 1R01NS111588) to J.-T. Chi. show cells regrowing at 2 months, similar to the timeframe of carboplatin-treated CAOV2R cells in our studies, are much less The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance responsive to GPX4 inhibitor RSL3-induced cell death. Of course, the with 18 U.S.C. Section 1734 solely to indicate this fact. discrepancy between results may also be due to the different cell lines used, different cancer drugs, or different ferroptosis inducers in Received July 4, 2019; revised September 17, 2019; accepted October 17, 2019; these studies. published first October 22, 2019.

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A TAZ−ANGPTL4−NOX2 Axis Regulates Ferroptotic Cell Death and Chemoresistance in Epithelial Ovarian Cancer

Wen-Hsuan Yang, Zhiqing Huang, Jianli Wu, et al.

Mol Cancer Res Published OnlineFirst October 22, 2019.

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