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YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis

索拉非尼 癌症研究 肝细胞癌 ATF4 医学 内科学 化疗 切碎
作者
Ruize Gao,Ravi Kiran Reddy Kalathur,Mairene Coto‐Llerena,Caner Ercan,David Buechel,Shuang Song,Salvatore Piscuoglio,Michael T. Dill,Fernando D. Camargo,Gerhard Christofori,Fengyuan Tang
出处
期刊:Embo Molecular Medicine [EMBO]
卷期号:13 (12) 被引量:327
标识
DOI:10.15252/emmm.202114351
摘要

Article19 October 2021Open Access Source DataTransparent process YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis Ruize Gao Ruize Gao Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Ravi K R Kalathur Ravi K R Kalathur Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Mairene Coto-Llerena Mairene Coto-Llerena Institute of Pathology, University Hospital Basel, Basel, Switzerland Search for more papers by this author Caner Ercan Caner Ercan orcid.org/0000-0002-5611-2699 Institute of Pathology, University Hospital Basel, Basel, Switzerland Search for more papers by this author David Buechel David Buechel Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Song Shuang Song Shuang Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Salvatore Piscuoglio Salvatore Piscuoglio orcid.org/0000-0003-2686-2939 Institute of Pathology, University Hospital Basel, Basel, Switzerland Search for more papers by this author Michael T Dill Michael T Dill Stem Cell Program, Boston Children's Hospital, Boston, MA, USA Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Fernando D Camargo Fernando D Camargo orcid.org/0000-0002-5630-5909 Stem Cell Program, Boston Children's Hospital, Boston, MA, USA Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Gerhard Christofori Corresponding Author Gerhard Christofori [email protected] orcid.org/0000-0002-8696-9896 Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Fengyuan Tang Corresponding Author Fengyuan Tang [email protected] orcid.org/0000-0001-6921-8479 Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Ruize Gao Ruize Gao Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Ravi K R Kalathur Ravi K R Kalathur Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Mairene Coto-Llerena Mairene Coto-Llerena Institute of Pathology, University Hospital Basel, Basel, Switzerland Search for more papers by this author Caner Ercan Caner Ercan orcid.org/0000-0002-5611-2699 Institute of Pathology, University Hospital Basel, Basel, Switzerland Search for more papers by this author David Buechel David Buechel Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Song Shuang Song Shuang Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Salvatore Piscuoglio Salvatore Piscuoglio orcid.org/0000-0003-2686-2939 Institute of Pathology, University Hospital Basel, Basel, Switzerland Search for more papers by this author Michael T Dill Michael T Dill Stem Cell Program, Boston Children's Hospital, Boston, MA, USA Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Fernando D Camargo Fernando D Camargo orcid.org/0000-0002-5630-5909 Stem Cell Program, Boston Children's Hospital, Boston, MA, USA Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Gerhard Christofori Corresponding Author Gerhard Christofori [email protected] orcid.org/0000-0002-8696-9896 Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Fengyuan Tang Corresponding Author Fengyuan Tang [email protected] orcid.org/0000-0001-6921-8479 Department of Biomedicine, University of Basel, Basel, Switzerland Search for more papers by this author Author Information Ruize Gao1, Ravi K R Kalathur1, Mairene Coto-Llerena2, Caner Ercan2, David Buechel1, Song Shuang3, Salvatore Piscuoglio2, Michael T Dill4,5, Fernando D Camargo4,5, Gerhard Christofori *,1 and Fengyuan Tang *,1 1Department of Biomedicine, University of Basel, Basel, Switzerland 2Institute of Pathology, University Hospital Basel, Basel, Switzerland 3Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 4Stem Cell Program, Boston Children's Hospital, Boston, MA, USA 5Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA *Corresponding author. Tel: +41 61 207 35 62; Fax: +41 61 207 35 66; E-mail: [email protected] *Corresponding author. Tel: +41 61 207 35 62; Fax: +41 61 207 35 66; E-mail: [email protected] EMBO Mol Med (2021)13:e14351https://doi.org/10.15252/emmm.202114351 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In this study, a combination of shRNA-mediated synthetic lethality screening and transcriptomic analysis revealed the transcription factors YAP/TAZ as key drivers of Sorafenib resistance in hepatocellular carcinoma (HCC) by repressing Sorafenib-induced ferroptosis. Mechanistically, in a TEAD-dependent manner, YAP/TAZ induce the expression of SLC7A11, a key transporter maintaining intracellular glutathione homeostasis, thus enabling HCC cells to overcome Sorafenib-induced ferroptosis. At the same time, YAP/TAZ sustain the protein stability, nuclear localization, and transcriptional activity of ATF4 which in turn cooperates to induce SLC7A11 expression. Our study uncovers a critical role of YAP/TAZ in the repression of ferroptosis and thus in the establishment of Sorafenib resistance in HCC, highlighting YAP/TAZ-based rewiring strategies as potential approaches to overcome HCC therapy resistance. SYNOPSIS Resistance to therapy occurs in most liver cancer patients treated with Sorafenib, and patients succumb to the disease. A synthetic lethal screen identified a regulatory circuit, which prevents ferroptosis and promotes cancer cell survival, thus promoting resistance to Sorafenib. The transcription factors YAP and TAZ stabilize ATF4 by promoting its nuclear import to cooperatively induce expression of SLC7A11, a cystine importer critical for glutathione synthesis. Glutathione synthesis and homeostasis are required to repress ferroptosis and to maintain Sorafenib resistance in liver cancer cells. Inhibition of Glutathione synthesis re-sensitizes Sorafenib-resistant cancer cells to Sorafenib therapy, which then induces ferroptosis and represses tumor growth in murine liver cancer models. Pharmacological repression of the anti-oxidant pathways regulated by YAP/TAZ and ATF4 could re-sensitize therapy-resistant liver cancers to Sorafenib treatment. The paper explained Problem While treatment of liver cancer patients with Sorafenib, the current treatment of choice for advanced hepatocellular carcinoma, induces in most cases initial beneficial effects, resistance to Sorafenib therapy eventually occurs, tumors relapse, and patients succumb to the disease. Results We investigated the molecular mechanism underlying the development and maintenance of resistance to Sorafenib therapy in liver cancer cells. We found that the transcriptional regulators YAP/TAZ and ATF4 cooperatively induce the expression of genes required for antioxidant pathways, which are critical to prevent cancer cell death by ferroptosis. These pathways are also upregulated in tumors of Sorafenib-resistant liver cancer patients. Proof-of-concept experiments with cultured liver cancer cells and in liver cancer mouse models revealed that inhibition of these pathways prevents the development of resistance to Sorafenib therapy. Impact These results suggest the possibility to re-sensitize therapy-resistant liver cancers to Sorafenib treatment by pharmacologically repressing the antioxidant pathways regulated by YAP/TAZ and ATF4. Introduction Liver cancer is the second leading cause of cancer-related mortality in patients. Hepatocellular carcinoma (HCC) represents about 90% of all cases of primary liver cancer (Llovet et al, 2008, 2016). Although cancer therapies have substantially improved clinical outcome (Kudo et al, 2018; Lee et al, 2020), patients invariably experience cancer relapse. Thus, delineating the mechanisms underlying evasive resistance of HCC is an unmet medical need and may add to a general understanding of therapy resistance in other cancer types as well. Ferroptosis is an emerging type of cell death induced by metal iron and reactive oxygen species (ROS) and driven by lipid peroxidation (Dixon et al, 2012; Jiang et al, 2021). Among the core regulatory components, the cystine-glutamate antiporter known as system Xc- or xCT (SLC7A11, encoded by the gene SLC7A11) imports cystine for the de novo synthesis of the important antioxidant peptide glutathione (GSH). GSH, among many functions, is also used as a substrate of phospholipid-hydroxyperoxide-glutathione-peroxidase (GPX4) to catalyze the detoxification of phospholipid hydroperoxides (Lachaier et al, 2014). Hence, ferroptosis can be potently induced by cysteine deprivation and GPX4 inhibition. Small pharmacological inhibitors, including the GPX4 inhibitor RSL3, and Erastin and Sorafenib as direct inhibitors of xCT-mediated import function, are widely used for the induction of ferroptosis (Dixon et al, 2012; Lachaier et al, 2014). YAP/TAZ are well-characterized transcriptional effectors of Hippo signaling involved in a variety of physio-pathological processes, including tumorigenesis and tissue regeneration (Pan, 2010; Harvey et al, 2013). Previous studies have suggested the Hippo-YAP/TAZ pathway is a key driver of ferroptosis in epithelial tumors (Wu et al, 2019; Yang et al, 2019). Here, we aimed at dissecting the molecular drivers of Sorafenib resistance in HCC and identified YAP/TAZ as negative regulators of ferroptosis. In a TEAD- and ATF4-dependent manner, YAP/TAZ induce the expression of SLC7A11, thus assisting cells in overcoming Sorafenib-induced ferroptosis. Moreover, the data suggest YAP/TAZ as key chaperones in stabilizing ATF4 protein and sustaining its nuclear transcriptional activity. Our study highlights YAP/TAZ as novel repressors of ferroptosis and, thus, as attractive therapeutic targets to overcome therapy resistance. Results Identification of YAP/TAZ as key drivers of Sorafenib resistance To identify the molecular mechanisms underlying therapy resistance in HCC, we had previously established Sorafenib-resistant HCC cell lines, called Huh7-IR and Huh7-CR (IC50 of 10.7 and 10.8 µM, respectively) and Hep3B-IR and Hep3B-CR (IC50 of 7.2 and 8.3 µM, respectively), as compared to their Sorafenib-sensitive parental cell lines Huh7 and Hep3B (IC50 of 1.7 and 3.0 µM, respectively) (Appendix Fig S1A–C) (Tang et al, 2019; Gao et al, 2021). Global transcriptomic analysis between the Sorafenib-sensitive parental cells and their Sorafenib-resistant derivatives revealed that, in addition to changes in various signaling pathways, cellular metabolism pathways had dynamically shifted in HCC cells upon the establishment of Sorafenib resistance, including amino acid metabolism (Appendix Fig S1D; Dataset EV1). To functionally identify intrinsic drivers of Sorafenib resistance in HCC cells, we performed a genome-wide shRNA-mediated synthetic lethality screen in Sorafenib-resistant HCC cells (Fig 1A). Notably, we turned our focus on the identification of factors and pathways involved in the establishment of adaptive resistance to Sorafenib, as opposed to the factors and pathways activated during an acute response to Sorafenib treatment. Huh7-IR and Huh7-CR cells were transfected with a shRNA library selected to target all genes known to play a role in cell signaling and covering each gene with at least 8 independent shRNA sequences. Cells were then cultured for 4 weeks under selection conditions in the presence of Sorafenib. Then, genomic DNA was extracted, and shRNA barcodes were amplified for next-generation sequencing to identify shRNAs and their target genes which have been lost during long-term culture, indicating that these genes may be critical for the maintenance of Sorafenib resistance. shRNAs which had dropped out in parental Huh7 cells upon acute treatment with Sorafenib were subtracted from the list, to only enrich for the genes critical for the maintenance of Sorafenib resistance, and a total of 1,072 genes were identified (Appendix Fig S1E; Dataset EV2). We also reasoned that critical drivers of Sorafenib resistance were deregulated at a transcriptional level during the adaptive reprogramming. Thus, we overlaid the hits from the synthetic lethality screen with the list of genes differentially regulated during the establishment of therapy resistance. This analytical combination revealed a total of 38 significant hits, among which eight genes were retrieved by more than three independent shRNA sequences and their gene regulation was highly significant (Fig 1B; Dataset EV3). Interestingly, among these eight top candidate genes was the Hippo pathway transducer WWTR1, also known as TAZ. Figure 1. YAP/TAZ are key drivers of Sorafenib resistance by inhibiting ferroptosis Scheme of the shRNA-mediated synthetic lethal screen. Huh7-IR and CR cells were infected with lentiviral vectors (MOI = 0.5) expressing the shRNA library (human genome-wide pooled lentiviral shRNA library module 1, vector: pRSI16cb, Cellecta) and cultured with 2 μg/ml puromycin for the selection of shRNA-expressing cells plus 7 μM Sorafenib (Srf). After 4 weeks of culture, genomic DNA was extracted, and shRNA barcodes were amplified for next-generation sequencing to uncover the critical genes for Sorafenib resistance. Combinatorial analysis of genes differentially expressed between Sorafenib-sensitive and resistant cells and genes with depleted barcodes in the synthetic lethal screen in Huh7-IR and CR cells. Thirty-eight common genes were identified, among which was WWTR1 coding for TAZ. Huh7-parental, IR and CR, and Hep3B-parental, and IR and CR cells were treated with different concentrations of Sorafenib (0, 3, 6, 9 μM) for Huh7-P/IR/CR and Sorafenib (0, 2, 4, 6 μM) for Hep3B-P/IR/CR for 18 h before harvest. Protein levels of YAP and TAZ were determined by immunoblotting illustrating higher protein levels of YAP/TAZ in Sorafenib-resistant cells. GAPDH served as loading control. Results represent three independent experiments. Colony formation assay showing that shRNA-mediated depletion of YAP/TAZ leads to cell numbers in response to Sorafenib treatment. Huh7 IR and CR cells either expressing a control shRNA (shLuc, non-targeting shRNA) or shRNA against both YAP and TAZ (shY/T) were treated with different concentrations of Sorafenib (0, 4, 8 μM) for 2 weeks and colonies were visualized by crystal violet staining. Results represent three independent experiments. Gene Set Enrichment Analysis (GSEA) of the genes differentially expressed between YAP/TAZ-deficient (siY/T) and control siRNA (siCtrl) transfected HLE cells showed an enrichment for genes involved in the regulation of lipid peroxidation. Basal reactive oxygen (ROS) levels increased upon loss of YAP/TAZ. HLE-shLuc and HLE-shY/T cell lines were stained with CellROX™ Green Flow Cytometry Assay Kit, and ROS levels were measured by flow cytometry using a 488 nm laser. Results represent three independent experiments. Basal lipid peroxidation levels increased with the loss of function of YAP/TAZ. HLE-shLuc and HLE-shY/T cells were stained with C11-BODIPY 581/591. Reduced-Bodipy was measured by flow cytometry using a 488 nm laser, and oxidized-Bodipy was measured with a 561 nm laser. A significant shift of oxidized-Bodipy occurred upon depletion of YAP/TAZ. Results represent three independent experiments. Colony formation assay demonstrating that the ferroptosis inhibitor Ferrostatin-1 (Fer) reversed Sorafenib-induced cell death in YAP/TAZ-deficient HCC cells. HLE-shLuc and shY/T cells were treated with different concentrations of Sorafenib (0, 2, 4 μM) and either DMSO or Ferrostatin-1 (Fer; 5 μM) for 2 weeks. Results represent three independent experiments. Source data are available online for this figure. Source Data for Figure 1 [emmm202114351-sup-0007-SDataFig1.jpg] Download figure Download PowerPoint The mammalian Hippo pathway has been previously implicated in tumorigenesis and therapy response in liver cancers (Harvey et al, 2013), and YAP and TAZ are well-established Hippo transducers sharing redundant functional read-outs (Totaro et al, 2018). Thus, we next focused our analysis on examining the expression of YAP and TAZ in Sorafenib-sensitive and resistant HCC cell lines. Indeed, we found high expression of YAP and TAZ in Sorafenib-resistant cells as compared to their sensitive counterparts (Fig 1C). Importantly, shRNA-mediated ablation of YAP and TAZ expression revealed that they were required to maintain the acquired resistance to Sorafenib, as determined by colony formation assays (Fig 1D). Together, these data indicate that YAP and TAZ represent critical drivers of acquired resistance to Sorafenib in HCC cells. YAP/TAZ promote resistance by antagonizing Sorafenib-induced ferroptosis To investigate the molecular mechanisms underlying YAP/TAZ-driven Sorafenib resistance, we analyzed the global transcriptomic changes upon loss of YAP and TAZ in intrinsically Sorafenib-resistant HLE cells (IC50 = 3.9 µM; Appendix Fig S1A) by RNA sequencing. This cell line was not highly resistant to Sorafenib, yet expressed substantial levels of YAP and TAZ proteins as compared to Sorafenib-sensitive cell lines (Appendix Fig S1F) and exhibited a much higher transfection rate as compared to the more resistant SNU cell lines. Interestingly, among the deregulated pathways, the genes involved in the regulation of lipid peroxidation, a hallmark of ferroptosis, were found to be significantly enriched (Fig 1E). Notably, Sorafenib is known to potently promote ferroptosis by blocking SLC7A11-mediated cellular cystine import (Dixon et al, 2014). We thus sought to validate the role of YAP and TAZ in the regulation of Sorafenib-induced lipid ROS. Indeed, siRNA-mediated loss of YAP and TAZ in HLE cells resulted in upregulated basal levels of ROS in HLE cells (Fig 1F). Moreover, loss of function of YAP and TAZ resulted in increased lipid peroxidation even in the absence of any treatment (Fig 1G), and even more so under treatment with Sorafenib or H2O2 (Appendix Fig S1G). Notably, Ferrostatin-1, a specific inhibitor of ferroptosis (Dixon et al, 2012), prevented YAP/TAZ deficiency-induced lipid peroxidation (Appendix Fig S1G). Consistent with these notions, intracellular GSH levels were decreased upon downregulation of YAP and TAZ (Appendix Fig S1H). Based on these observations, we hypothesized that YAP and TAZ promoted Sorafenib resistance by detoxifying Sorafenib-induced ferroptosis. We thus assessed whether Ferrostatin-1 can prevent the cell death induced by Sorafenib upon loss of YAP and TAZ. Indeed, Ferrostatin-1 fully restored the viability of YAP/TAZ-deficient cells in the presence of Sorafenib, as determined by colony formation assay (Fig 1H). We further asked whether YAP/TAZ were able to antagonize ferroptosis in a general manner, not only in Sorafenib-induced ferroptosis. To this end, we treated HLE cells with Erastin, a well-known inducer of ferroptosis (Dixon et al, 2012) and observed that Erastin induced ferroptosis in shLuc-transfected cells, which was further enhanced upon shRNA-mediated ablation of YAP and TAZ. In both conditions, cell death could be blocked by treatment with Ferrostatin-1, indicating that cell death was due to ferroptosis (Appendix Fig S1I). To further elaborate on the role of YAP and TAZ in supporting cell viability upon Sorafenib treatment of Sorafenib-resistant HCC cells, we further employed the Promega Celltiter GloTM assay which confirmed the conclusion that YAP/TAZ promotes Sorafenib resistance via blocking ferroptosis in YAP/TAZhigh cells (Appendix Fig S1J and K). Treatment of Huh7 parental, IR, and CR cells with other inducers of ferroptosis, including Erastin, RSL3, FIN58, and FINO2 all resulted into reduced cell viability of Sorafenib-sensitive Huh7-parental cells as compared to the Sorafenib-resistant Huh7-IR and Huh7-CR cells (Appendix Fig S2A), further indicating that Sorafenib-resistant cells are also more resistant to ferroptosis induction. To further demonstrate the specificity of a role of YAP/TAZ in ferroptotic cell death, yet to exclude apoptosis or necroptosis, we treated siCtrl and siYAP/TAZ-transfected HLE cells with Sorafenib and with the ferroptosis inhibitor Ferrostatin-1, with the pan-caspase inhibitor Z-VAD-FMK to repress apoptosis or with the RIPK3 inhibitor GSK-872 to suppress necroptosis. Interestingly, only Ferrostatin-1 rescued cell viability, thus confirming a specific role of YAP/TAZ in regulating Sorafenib-induced ferroptosis (Appendix Fig S2B). In contrast, cell death induced by acute Sorafenib treatment of Sorafenib-sensitive (parental) Huh7 and Hep3B cells could not be reversed by treatment with Ferrostatin-1 (Appendix Fig S3A and B). Given that YAP/TAZ expression was significantly lower in Sorafenib-sensitive Huh7 and Hep3B cells as compared to their resistant counterparts (Fig 1C), we also assessed whether the forced expression of YAP/TAZ could overcome cell death induced by Sorafenib treatment of Sorafenib-sensitive cells. Indeed, the forced expression of a constitutively active version of YAP (YAP-5SA) prevented Sorafenib-induced cell death (Appendix Fig S3C and D) and increased GSH levels (Appendix Fig S3E). This increased resistance to Sorafenib observed in YAP-5SA-expressing Huh7 and Hep3B cells as compared to empty vector control-transfected cells was maintained in the presence of Erastin, although the overall levels of cell viability were dramatically reduced by Erastin (Appendix Fig S3F). These results are consistent with a previous report by our laboratory demonstrating that the acute treatment of Sorafenib-sensitive HCC cells with Sorafenib induces autophagy and apoptosis which is prevented by YAP/TAZ activities (Tang et al, 2019). Yet, these results also indicates that YAP/TAZ not only counteract ferroptosis but also apoptosis in the context of Sorafenib treatment. Altogether, the results demonstrate that YAP and TAZ act as general inhibitors of ferroptosis and apoptosis, thereby promoting Sorafenib resistance. YAP/TAZ transcriptionally upregulate SLC7A11 To further investigate how the transcription factors YAP/TAZ restrict ferroptosis, we set out to identify their transcriptional target genes in the context of Sorafenib resistance of HCC cells. To this end, we overlaid the list of genes downregulated in their expression in YAP/TAZ-depleted HLE cells and the list of genes upregulated in Sorafenib-resistant cells, which led to the identification of 56 genes (Dataset EV4). Of note, SLC7A11, coding for the cystine-glutamate antiporter known to regulate ferroptosis, was among these genes (Fig 2A). Figure 2. YAP/TAZ transcriptionally upregulate SLC7A11 expression Combinatorial analysis of the genes upregulated in Sorafenib-resistant cells and the genes downregulated upon YAP/TAZ depletion uncovered 56 common genes, among which was SLC7A11. Quantitative RT-PCR analysis confirmed the dependency of SLC7A11 gene expression on YAP/TAZ. HLE cells were transfected with control siRNA (siCtrl) or siRNA against YAP/TAZ (siY/T) and cultured with DMSO or 6 μM Sorafenib for 18 h. RNA was extracted and analyzed by quantitative RT-PCR. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments. SLC7A11 protein levels were upregulated by the exposure to Sorafenib, yet downregulated by siRNA-mediated depletion of YAP/TAZ. HLE cells were transfected with siCtrl or siY/T and cultured with DMSO or 6 μM Sorafenib for 18 h before harvest, followed by immunoblotting for YAP/TAZ and SLC7A1. GAPDH served as loading control. Results represent three independent experiments. siRNA-mediated ablation of YAP/TAZ significantly reduced SLC7A11 promoter activity, as determined by SLC7A11-promoter-luciferase reporter assay. HLE cells were transfected with SLC7A11-promoter firefly luciferase reporter construct and a constitutive-active Renilla luciferase reporter construct (pRL-CMV) and with siCtrl or siY/T. Relative luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega E1980). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments. A potential TEAD binding motif was predicted at – 400 bp within the promoter region of SLC7A11. PCR primers SLC7A11-CH1 (P1) and SLC7A11-CH2 (P2) were designed to examine the potential binding of transcription factors to the TEAD binding motif by chromatin immunoprecipitation (ChIP). Binding of YAP and TAZ to a DNA fragment containing the TEAD binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for TEAD binding motif in the SLC7A11 promoter region shown in (E). The non-coding region NC10 served as negative control, and the bona fide TEAD target gene CYR61 served as positive control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments. Knockdown of YAP/TAZ impairs cystine uptake either with or without Sorafenib treatment, and cystine uptake decreased with the exposure to Sorafenib. HLE-shLuc and HLE-shY/T cells were cultured with DMSO or 6 μM Sorafenib for 18 h, cystine-FITC was added to cells, and after incubation at 37°C for 30 min, intracellular Cystine-FITC levels were measured by flow cytometry using a 488 nm laser. Results represent three independent experiments. Colony formation assay showing that stable overexpression of SLC7A11 reversed Sorafenib-induced cell death in YAP/TAZ-deficient HCC cells. HLE-shLuc and HLE-shY/T were or were not transfected to overexpress SLC7A11 (SLC7A11 OE) and treated with DMSO or 6 μM Sorafenib for 2 weeks as indicated. Results represent three independent experiments. Representative images of immunohistochemical staining of SLC7A11 and YAP proteins in HCC samples from patients. Scale bars, 50 μm. Quantification of the immunohistochemical stainings shown in (I) revealed a positive correlation of YAP and SLC7A11 expression (N = 10). Statistical significance was calculated using Pearson correlation analysis. Source data are available online for this figure. Source Data for Figure 2 [emmm202114351-sup-0008-SDataFig2.zip] Download figure Download PowerPoint As a key regulator of lipid peroxidation and ferroptosis, we confirmed the functional importance of SLC7A11 for Sorafenib resistance in HCC cells. Loss of SLC7A11 resulted in an increase of intracellular ROS levels (Appendix Fig S4A) as well as of lipid peroxidation (Appendix Fig S4B). Moreover, cystine uptake was reduced by siRNA-mediated depletion of SLC7A11 (Appendix Fig S4C) and, as a consequence, the levels of intracellular GSH were diminished (Appendix Fig S4D). Finally, the depletion of SLC7A11 resulted in increased cell death in a colony formation assay which was fully rescued by Ferrostatin-1 (Appendix Fig S4E and F). These results confirm SLC7A11 as a key regulator of ferroptosis in response to Sorafenib. We next explored the expression of SLC7A11 in HCC of patients. Of note, SLC7A11 mRNA was highly upregulated in primary HCC tumors. Further, immunohistochemical analysis of HCC tumor sections revealed that SLC7A11 was upregulated in HCC of patients as compared with adjacent liver parenchyma (Appendix Fig S4G). Statistical analysis using multi-tissue arrays with non-tumor and tumor tissues confirmed its high expression in HCC (Appendix Fig S4H). Interestingly, SLC7A11 protein significantly correlated with reduced cell differentiation of HCC samples, as classified by Edmondson grades III and IV (Appendix Fig S4I). Even more important, using the expression data retrieved from the TCGA liver dataset (Cancer Genome Atlas Research Network. Electronic address & Cancer Genome Atlas Research, 2017), we observed that SLC7A11 expression predicted patient survival, with high expression of SLC7A11 significantly correlating with poor clinical outcome (Appendix Fig S4J). We next sought to validate whether YAP/TAZ directly affected SLC7A11 gene expression. Indeed, knockdown of YAP and TAZ together resulted in a significant downregulation of SLC7A11 at both mRNA level and protein level (Appendix Fig S5A; Fig 2B and C). Moreover, overexpression of a constitutive-active form of YAP induced an upregulation of SLC7A11 (Appendix Fig S5B). The transcriptional activity of YAP/TAZ was best known to be regulated by cell–cell contact, and we thus further explored whether SLC7A11 gene expression was also sensitive to changes in cell density. Indeed, the expression of SLC7A11 was significantly downregulated with increasi
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