Role of the yap/taz-tead transcriptional complex in the metabolic control of trail sensitivity by the mevalonate pathway in cancer cells

Citation: El Yousfi, Y.; Mora-Molina, R.; López-Rivas, A.; Yerbes, R. Role of the YAP/TAZ-TEAD Transcriptional Complex in the Metabolic Control of TRAIL Sensitivity by the Mevalonate Pathway in Cancer Cells. Cells 2023, 12, 2370. https://doi.org/ 10.3390/cells12192370 Academic Editor: Jung-Hye Choi Received: 31 August 2023 Revised: 19 September 2023 Accepted: 25 September 2023 Published: 27 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Role of the YAP/TAZ-TEAD Transcriptional Complex in the Metabolic Control of TRAIL Sensitivity by the Mevalonate Pathway in Cancer Cells Younes El Yousfi 1, Rocío Mora-Molina 1, Abelardo López-Rivas 1and Rosario Yerbes 1,2,* 1Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, CSIC-Universidad de Sevilla-Universidad Pablo de Olavide, 41092 Seville, Spain; [email protected] (Y.E.Y.); [email protected] (R.M.-M.); [email protected] (A.L.-R.) 2Medical Physiology and Biophysics Department, Universidad de Sevilla and Instituto de Biomedicina de Sevilla (IBiS) (Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla), 41013 Seville, Spain *Correspondence: [email protected] Abstract: Different studies have reported that inhibiting the mevalonate pathway with statins may increase the sensitivity of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although the signaling mechanism leading to this sensitization remains largely unknown. We investigated the role of the YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif)-TEAD (TEA/ATTS domain) transcriptional complex in the metabolic control of TRAIL sensitivity by the mevalonate pathway. We show that depleting nuclear YAP/TAZ in tumor cells, either via treatment with statins or by silencing YAP/TAZ expression with siRNAs, facilitates the activation of apoptosis by TRAIL. Furthermore, the blockage of TEAD transcriptional activity either pharmacologically or through the ectopic expression of a disruptor of the YAP/TAZ interaction with TEAD transcription factors, overcomes the resistance of tumor cells to the induction of apoptosis by TRAIL. Our results show that the mevalonate pathway controls cellular the FLICE-inhibitory protein (cFLIP) expression in tumor cells. Importantly, inhibiting the YAP/TAZ-TEAD signaling pathway induces cFLIP down-regulation, leading to a marked sensitization of tumor cells to apoptosis induction by TRAIL. Our data suggest that a combined strategy of targeting TEAD activity and selectively activating apoptosis signaling by agonists of apoptotic TRAIL receptors could be explored as a potential therapeutic approach in cancer treatment. Keywords: statins; mevalonate pathway; TNF-related apoptosis-inducing ligand; YAP/TAZ-TEAD; cellular FLICE-like inhibitory protein 1. Introduction TRAIL is a member of the tumor necrosis factor (TNF) family that activates an apoptotic program in a broad variety of tumor cells upon binding to its pro-apoptotic receptors, sparing most normal cells [ 1 , 2 ]. The binding of TRAIL to its pro-apoptotic receptors induces the formation of the death-inducing signaling complex (DISC), recruiting the adapter protein Fas-associated death domain (FADD) and procaspase-8 [ 3 , 4 ]. Procaspase-8 is processed and activated to initiate an apoptotic cascade that results in cell death [ 3 , 4 ]. At the DISC, apoptosis signaling may be hindered by the caspase-8 inhibitor cFLIP [ 5 ]. The down-regulation of cFLIP levels is a frequent observation upon different treatments that sensitize tumor cells to TRAIL-induced apoptosis [6–10]. The mevalonate pathway is an essential metabolic pathway involved in the synthesis of different isoprenoids, including cholesterol, lipoproteins and vitamin D, among others [ 11 ]. Statins are inhibitors of the 3-OH-3-methyl-glutaryl CoA (HMG-CoA) reductase, a rate-limiting step in mevalonate synthesis [ 12 , 13 ]. Statins are the standard of care in the treatment of hypercholesterolemia and the prevention of cardiovascular pathologies [ 12 , 13 ]. Cells 2023,12, 2370. https://doi.org/10.3390/cells12192370 https://www.mdpi.com/journal/cells Cells 2023,12, 2370 2 of 19 In addition to reducing cholesterol levels, statins also affect the synthesis of other isoprenoids, essential constituents of signaling proteins implicated in cancer [ 14 , 15 ]. In this regard, different clinical studies have indicated the beneficial effect of statins in cancer patients both on their own and in combination with other anti-tumor treatments [ 16 , 17 ]. Interestingly, different works have shown that statins enhanced apoptosis activation by TRAIL, although the mechanism involved has not been revealed [18–20]. The Hippo pathway is an evolutionarily conserved protein kinase cascade that is involved in tumorigenesis and tumor progression [ 21 , 22 ]. YAP and TAZ transcriptional coactivators promote tissue proliferation and organ growth and are negatively regulated by the Hippo pathway [ 23 ]. Indeed, YAP has been shown to play a key role in inhibiting apoptosis in cancer cells [ 24 – 27 ], although a pro-apoptotic function of YAP has also been reported [ 28 – 30 ]. Importantly, several studies have demonstrated that YAP/TAZ nuclear localization and function can be controlled via the mevalonate pathway [ 31 – 33 ] through the geranylgeranylation and activation of RhoA GTPases [33]. We have examined the function of the YAP/TAZ-TEAD transcriptional system in the regulation of sensitivity to TRAIL via the mevalonate pathway in tumor cells. Herein, we report that inhibiting the mevalonate pathway using statin treatment markedly sensitized tumor cells to TRAIL. Sensitization to TRAIL through statins was abrogated in tumor cells overexpressing a constitutively active form of YAP. In this way, we show that both statins and YAP/TAZ knockdown reduced the cellular levels of the caspase-8 inhibitor cFLIP and sensitized cancer cells to a TRAIL-induced and mitochondria-regulated apoptotic process. 2. Materials and Methods 2.1. Cell Culture HCT116 cells were kindly provided by Dr. J.A. Pintor-Toro (CABIMER, Seville, Spain). HCT116 Bax/Bak K.O. cells were a donation from Dr. C. Muñoz-Pinedo (Idibell, Barcelona, Spain). Both cell lines were maintained in McCoy’s medium containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, penicillin 50 U/mL and streptomycin 50 µg/mL . A549 cells were a gift from Dr. FJ. Oliver, Institute of Parasitology and Biomedicine CSIC, Granada, Spain. A549, MDA-MB468 (a gift by Dr. J. Arribas, VallD’Hebron Institute of Oncology, Barcelona, Spain) and HeLa cell lines were maintained in complete DMEM medium containing 10% fetal bovine serum, 2 mM Lglutamine, penicillin (50 U/mL) and streptomycin (50 µ g/mL). Complete medium of A549 cells was also supplemented with glucose (4.5 g/L). All cell lines were regularly tested for mycoplasma contamination. 2.2. Reagents Reagents for cell culture, buffer preparation and molecular biology were purchased from Merck/Sigma-Aldrich, (St. Louis, MO, USA). Simvastatin, cerivastatin, propidium iodide, puromycin, hygromycin, doxycycline and DAPI were purchased from Merck/SigmaAldrich. Geneticin (G-418) was from Gibco (Fisher Scientific, Loughborough, UK). XAV939 was obtained from Selleckchem (Houston, TX, USA). Human His-tagged recombinant TRAIL was produced as described [34]. 2.3. Antibodies Anti-Yap (SC-101199) and anti-GAPDH (SC-47724) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-caspase 8 (1C12) (9746), was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-cFLIP monoclonal antibody 7F10 (ALX-804-961-0100) was from Enzo Life Sciences (Farmingdale, NY, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from DAKO (P0447, P0448, P0449) (Cambridge, UK). Alexa 488-conjugated secondary antibody was obtained from Jackson ImmunoResearch (Baltimore Pike, PA, USA). Cells 2023,12, 2370 3 of 19 2.4. Determination of Hypodiploid Apoptotic Cells For the quantitative analysis of apoptosis, cells (3 × 10 5 /well) were cultured in 6-well plates and treated as shown in the figure legends. Cell cycle analysis (5 × 10 3 cells/sample) was carried out in a FACSCalibur cytometer with Cell Quest software, Version 5.1 (Becton Dickinson, Mountain View, CA, USA), according to published procedures [35]. 2.5. Analysis of Cell Viability HeLa cells (3 × 10 5 /well) were cultured in 6-well plates and treated as shown in the figure legends. Cell viability was assessed after washing the cultures with phosphatebuffered saline (PBS)/0.1% bovine serum albumin and incubation for 15 min in the same buffer containing propidium iodide (2 µ g/mL) in the dark. The uptake of propidium iodide was determined via flow cytometry with the Cell Quest Software. 2.6. Western Blotting of Proteins After washing the cultures with PBS, cells (3 × 10 5 ) were lysed in buffer containing 10 mM Na2HPO4, 3% SDS and 10% Glycerol. Once the protein concentration of lysates was measured with the Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA), Laemmli sample buffer was added and proteins were resolved through electrophoresis on SDSpolyacrylamide minigels. Proteins were identified via Western blotting as described [ 35 ], using GAPDH as a protein loading control. 2.7. Real-Time qPCR Total RNA (1 µ g) was isolated with PRImeZOL reagent (Canvax Biotech, Córdoba, Spain) and reverse-transcribed using an iScript cDNA synthesis kit (Bio-Rad, ref. 1708891). Real-time PCRs were carried out using SYBR green reagent (Bio-Rad, ref. 1725124). Alternatively, M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used to reverse-transcribe total RNA, and mRNA expression was assessed via qPCR with primers and probes from Applied Biosystems (Waltham, MA, USA), normalized to hypoxanthineguanine phosphoribosyltransferase levels. mRNA levels were assessed on the ABI Prism7500 sequence detection system. Primers and probes for real-time qPCR: Primers for SYBR green analysis: GAPDH Forward: 50-ATGGGGAAGGTGAAGGTCG-30 Reverse: 50-GGGTCATTGATGGCAACAATATC-30 FLIPLForward:50-CCTAGGAATCTGCGTGATAATCGA-30 Reverse: 50-TGGGATATACCATGCATACTGAGATG-30 FLIPSForward: 50-GGGCCGAGGCAAGATAAGCAAGG-30 Reverse: 50-TCAGGACAATGGGCATAGGGTGT-30 CTGF Forward: 50-AGCTGACCTGGAAGAGAACA-30 Reverse: 50-CAGGCACAGGTCTTGATGAA-30 Taqman primers and probes: CYR61 Hs00155479_m1 HPRT1 Hs01003267_m1 2.8. RNA Interference siRNAs and non-targeting oligonucleotide were purchased from Sigma Aldrich (St. Louis, MO, USA). Dharmafect-1 (Dharmacon) was used for siRNA transfection, as described by the manufacturer. After 6 h, the transfection medium was replaced with regular medium and cells were further incubated for 48 h before analysis. The transfection of siRNAs in HCT116 cells was performed using jetPRIME (Polyplus transfection reagent), according to manufacturer guidelines. The transfection medium was removed, and cells were cultured in regular medium for 48 h before further analysis. Cells 2023,12, 2370 4 of 19 siRNAs sequences: YAP#1: 50-GACAUCUUCUGGUCAGAGAdTdT-30 YAP#2: 50-CUGGUCAGAGAUACUUCUUdTdT-30 YAP#3: 50-GGUGAUACUAUCAACCAAAdTdT-30 TAZ#1: 50-ACGUUGACUUAGGAACUUUdTdT-3’ TAZ#2: 5´-AGGUACUUCCUCAAUCACAdTdT-3´ cFLIP: 50-GGGACCUUCUGGAUAUUUUdTdT-30 Non-targeting control siRNA (Scr) 50-CUUUGGGUGAUCUACGUUAdTdT-30 YAP/TAZ pairs of siRNAs: siY/T#1: siYAP#1 + siTAZ#1 siY/T#2: siYAP#2 + siTAZ#2 siY/T#3: siYAP#3 + siTAZ#2 2.9. Retroviral and Lentiviral Vectors pQCXIH-Myc-YAP-5SA was a gift from Kunliang Guan (Addgene plasmid # 33093; http://n2t.net/addgene:33093; accessed on 16 September 2019; RRID:Addgene_33093), FUW-tetO-wtYAP was a gift from Stefano Piccolo (Addgene plasmid # 84009; http://n2t.net/addgene:84009; accessed on 17 September 2019; RRID:Addgene_84009), FUW-tetO-EGFP was a gift from Stefano Piccolo (Addgene plasmid # 84041; http://n2t.net/addgene:84041; accessed on 17 September 2019; RRID:Addgene_84041), FUdeltaGW-rtTA was a gift from Konrad Hochedlinger (Addgene plasmid # 19780; http://n2t.net/addgene:19780; accessed on 17 September 2019; RRID:Addgene_19780) and pInducer20 EGFP-TEADi was a gift from Ramiro Iglesias-Bartolome (Addgene plasmid # 140145; http://n2t.net/addgene:140145; accessed on 4 September 2020; RRID:Addgene_140145). All these vectors for stable gene expression were obtained from the Addgene plasmid repository (Watertown, MA, USA). The pBabe puro vector has been described previously [ 35 ]. To perform silencing experiments, shRNAs against caspase-8 and TRAILR2, in a pSUPER vector (OligoEngine, Seattle, WA, USA), were digested and cloned between EcoR1 and Cla1 into a H1 promoter-driven GFP-encoding pLVTHM lentiviral vector [36]. shRNAs sequences: TRAIL-R2: 50-GATCCCCGACCCTTGTGCTCGTTGTCTTCAAGAGA GACAACGAGCACAAGGGTCTTTTTTA-30 Caspase-8: 50-GATCCCCGGAGCTGCTCTTCCGAATTTTCAAGAG AAATTCGGAAGAGCAGCTCCTTTTTA-30 Scrambled (Scr): 50-GATCCCCCTTTGGGTGATCTACGTTATTCAAGAGA TAACGTAGATCACCCAAAGTTTTTA-30 To produce lentiviruses and retroviruses, HEK293-T cells were transfected with the corresponding vectors using the calcium phosphate method. After 48 h, lentivirusor retrovirus-containing supernatants were collected, and viruses were concentrated via ultracentrifugation. 2.10. Generation of A549 Cell Lines A549 cell lines were obtained after infection with lentiviruses or retroviruses, and selection was performed in medium with the corresponding antibiotic: puromycin (1.5 µg/mL) , hygromycin (200 µ g/mL) or G-418 (1 mg/mL). The infection of tumor cells with GFPexpressing lentiviruses was determined via flow cytometry. 2.11. Immunofluorescence Cells were grown on coverslips and fixed for 10 min in 4% paraformaldehyde at room temperature. Following permeabilization with Triton X-100 (0.5%), cells on coverslips were incubated at room temperature for 1 h with primary antibodies diluted in blocking buffer. After washing with 0.1% PBS-Tween, cells were stained for 1 h with fluorescently labelled Cells 2023,12, 2370 5 of 19 secondary antibody. Images were acquired with an Axio Imager 2 Zeiss microscope (Zeiss, Oberkochen, Germany). The nuclear localization of YAP was determined by calculating the ratio between nuclear YAP fluorescence and fluorescence in the cytoplasmic region immediately adjacent. Co-staining the nucleus with DAPI was used as a criteria to differentiate nuclear and cytoplasmic regions. Images analysis was performed using ImageJ software, Version 1.0. 2.12. Assessment of Luciferase Activity YAP/TAZ-responsive reporter 8xGTII–luciferase plasmid (a gift from Stefano Piccolo, Addgene plasmid #34615; http://n2t.net/addgene:34615; accessed on 16 December 2020; RRID: Addgene_34615) was transfected in A549 cells together with a Renilla plasmid to normalize for transfection efficiency. Six hours after transfection, cells were treated with doxycycline (1 µ g/mL, A549-iTEAD cells) or XAV939 (5 µ M, A549 cells) during 24 h before collection. Luciferase activity in cell lysates was assessed using the DualLuciferase Reporter Assay System (Promega, Madison, WI, USA). Samples were analyzed in a Varioskan Flash microplate reader (Thermo Electron Corporation, Waltham, MA, USA). Every experimental condition was performed in duplicate. 2.13. Quantitative Analysis of Caspase-8 Activity Cells growing in 6-well plates were treated as indicated in the figure legend. Following collection, cells were washed with PBS and resuspended in regular medium. After 90 minincubation, caspase-8 activity was assessed using the Caspase-Glo ®® 8 Assay (Promega, Madison, WI, USA) by measuring the luminescence intensity in a Varioskan Flash microplate reader. Every experimental condition was performed in duplicate. 2.14. Global Protein Synthesis Determination by the SUnSET Method General protein synthesis was assessed by the non-isotopic SUnSET assay as previously described [ 37 ]. Cells were pulse-labelled with puromycin and proteins in cell lysates were separated by denaturing electrophoresis. Rate of protein synthesis was determined via Western blot with an anti-puromycin antibody (clone 12d10, MABE343, Merck/Sigma Aldrich). 2.15. Statistics Results are presented as the mean ± SD. Experiments were performed independently at least three times. GraphPAD Prism 8 software was used to carry out the statistical analysis (GraphPad Software, San Diego, CA, USA). The statistical tests used are indicated in the figure legends. p< 0.05 was considered significant. * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001. 3. Results 3.1. Control of TRAIL Sensitivity by the Mevalonate Pathway in Cancer Cells Simvastatin, a mevalonate pathway inhibitor widely used in the treatment of cardiovascular disease, also shows inhibitory effects on various types of cancers [ 38 ]. Interestingly, different studies have described that inhibiting the mevalonate pathway with statins sensitizes tumor cells to apoptosis induction by death ligand TRAIL [ 18 – 20 ]. However, the signaling mechanism involved in this synergistic action remains poorly understood. To investigate the molecular mechanism responsible for the sensitization to TRAIL in tumor cells treated with statins, different tumor cell lines were incubated for 24 h with simvastatin followed by treatment with TRAIL for a further 24 h-period. In these cell lines, simvastatin significantly sensitized the cells to TRAIL-induced apoptosis (Figure 1A). Statins inhibit the enzymatic activity of HMG-CoA reductase, thus reducing the levels of the downstream metabolites of the mevalonate pathway [ 11 ]. To validate the on-target effects of simvastatin increasing the sensitivity of tumor cells to TRAIL, we carried out the rescue experiments via the supplementation of simvastatin-treated tumor cell cultures with mevalonate. Our Cells 2023,12, 2370 6 of 19 results clearly indicated that simvastatin-mediated sensitization to TRAIL was abolished when cells were treated with the statin in the presence of mevalonate, prior to the addition of TRAIL (Figure 1A). These results were confirmed in experiments performed with cerivastatin, another inhibitor of HMG-CoA reductase (Figure 1B). Cells 2023, 12, x FOR PEER REVIEW 6 of 19 in tumor cells treated with statins, different tumor cell lines were incubated for 24 h with simvastatin followed by treatment with TRAIL for a further 24 h-period. In these cell lines, simvastatin significantly sensitized the cells to TRAIL-induced apoptosis (Figure 1A). Statins inhibit the enzymatic activity of HMG-CoA reductase, thus reducing the levels of the downstream metabolites of the mevalonate pathway [11]. To validate the on-target effects of simvastatin increasing the sensitivity of tumor cells to TRAIL, we carried out the rescue experiments via the supplementation of simvastatin-treated tumor cell cultures with mevalonate. Our results clearly indicated that simvastatin-mediated sensitization to TRAIL was abolished when cells were treated with the statin in the presence of mevalonate, prior to the addition of TRAIL (Figure 1A). These results were confirmed in experiments performed with cerivastatin, another inhibitor of HMG-CoA reductase (Figure 1B). Figure 1. Inhibition of the mevalonate pathway by statins sensitizes cancer cells to TRAIL. Apoptosis in cancer cells treated with (A) simvastatin (Simv, 5 µM) or (B) cerivastatin (Ceriv, 1 µM), in the presence or absence of mevalonate (Mev, 0.5 mM) during 24 h prior to incubation with or without Figure 1. Inhibition of the mevalonate pathway by statins sensitizes cancer cells to TRAIL. Apoptosis in cancer cells treated with ( A ) simvastatin (Simv, 5 µ M) or ( B ) cerivastatin (Ceriv, 1 µ M), in the presence or absence of mevalonate (Mev, 0.5 mM) during 24 h prior to incubation with or without TRAIL. Cell death was determined via the quantification of hypodiploid cells through flow cytometry (5 × 10 3 cells/sample) (A549, MDA-MB468, HCT116). In HeLa cells, cell viability was assessed via propidium iodide uptake. Data show the mean ± SD of at least three independent experiments. *p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001. Two-way ANOVA with Tukey ´ s multi-comparisons test. Collectively, our results demonstrate that the sensitivity of these tumor cells to the induction of the apoptotic pathway by TRAIL significantly depends on the levels of metabo- Cells 2023,12, 2370 7 of 19 lites of the mevalonate pathway downstream of the activation of HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway [11]. 3.2. Metabolic Control of YAP/TAZ Subcellular Localization and Activity by the Mevalonate Pathway in Cancer Cells Several studies have shown that the mevalonate pathway could be involved in the regulation of YAP/TAZ subcellular localization and activity in cancer cells [ 31 – 33 ]. Given the important role of YAP/TAZ in cancer cell survival [ 25 , 27 ], we examined whether YAP/TAZ may function as downstream effectors of the mevalonate pathway to promote resistance to TRAIL. First, we monitored by immunofluorescence the effect of simvastatin on the subcellular localization of YAP/TAZ in both the human lung adenocarcinoma cell line A549 and the triple-negative breast cancer cell line MDA-MB468 (Figure 2A). Interestingly, simvastatin treatment induced a significant cytoplasmic re-localization of YAP/TAZ in both cell lines (Figure 2A). Similar results were obtained by treating these cells with cerivastatin (Figure 2B). Importantly, the cytoplasmic re-localization of YAP/TAZ was not observed in cells treated with statins when mevalonate was added to the culture medium (Figure 2A,B). Moreover, we evaluated the effect of inhibiting the mevalonate pathway on CYR61 gene expression, a downstream target of the YAP/TAZ/TEAD transcriptional system [ 39 ]. Data shown in Figure 2C indicate that CYR61 gene expression was markedly impeded by simvastatin treatment and that mevalonate addition completely prevented the inhibition of YAP/TAZ/TEAD activity by the statin. 3.3. Inhibition of YAP/TAZ Signaling Mediates Statin-Induced Sensitization to TRAIL The mevalonate pathway is known to be involved in the regulation of YAP/TAZ phosphorylation and activity independently of LATS1/2 kinases [ 33 ]. To determine whether the decreased YAP/TAZ signaling observed in cancer cells treated with statins plays a role in the sensitization to TRAIL-induced apoptosis, we first generated A549 cells expressing an activated form of YAP (YAP5SA), lacking inhibitory phosphorylation sites. As shown in Figure 3A, the expression of YAP5SA in A549 cells was sufficient to prevent simvastatininduced sensitization to TRAIL. Cells 2023, 12, x FOR PEER REVIEW 7 of 19 TRAIL. Cell death was determined via the quantification of hypodiploid cells through flow cytometry (5 × 103 cells/sample) (A549, MDA-MB468, HCT116). In HeLa cells, cell viability was assessed via propidium iodide uptake. Data show the mean ± SD of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Two-way ANOVA with Tukeys multi-comparisons test. Collectively, our results demonstrate that the sensitivity of these tumor cells to the induction of the apoptotic pathway by TRAIL significantly depends on the levels of metabolites of the mevalonate pathway downstream of the activation of HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway [11]. 3.2. Metabolic Control of YAP/TAZ Subcellular Localization and Activity by the Mevalonate Pathway in Cancer Cells Several studies have shown that the mevalonate pathway could be involved in the regulation of YAP/TAZ subcellular localization and activity in cancer cells [31–33]. Given the important role of YAP/TAZ in cancer cell survival [25,27], we examined whether YAP/TAZ may function as downstream effectors of the mevalonate pathway to promote resistance to TRAIL. First, we monitored by immunofluorescence the effect of simvastatin on the subcellular localization of YAP/TAZ in both the human lung adenocarcinoma cell line A549 and the triple-negative breast cancer cell line MDA-MB468 (Figure 2A). Interestingly, simvastatin treatment induced a significant cytoplasmic re-localization of YAP/TAZ in both cell lines (Figure 2A). Similar results were obtained by treating these cells with cerivastatin (Figure 2B). Importantly, the cytoplasmic re-localization of YAP/TAZ was not observed in cells treated with statins when mevalonate was added to the culture medium (Figure 2A,B). Moreover, we evaluated the effect of inhibiting the mevalonate pathway on CYR61 gene expression, a downstream target of the YAP/TAZ/TEAD transcriptional system [39]. Data shown in Figure 2C indicate that CYR61 gene expression was markedly impeded by simvastatin treatment and that mevalonate addition completely prevented the inhibition of YAP/TAZ/TEAD activity by the statin. Figure 2. Cont. Cells 2023,12, 2370 8 of 19 Cells 2023, 12, x FOR PEER REVIEW 8 of 19 Figure 2. Nuclear localization and activity of YAP/TAZ are regulated by the mevalonate pathway in cancer cells. (A) Representative images of YAP/TAZ immunofluorescence (left panel) in cells treated with dimethylsulphoxide (NT) or simvastatin (Simv) alone or with mevalonate (Mev) for 24 h. Quantification of nuclear/cytosolic YAP/TAZ (right panel) from three independent experiments. (B) Representative images of YAP/TAZ immunofluorescence (left panel) in cells treated with dimethylsulphoxide (NT) or cerivastatin (Ceriv) alone or with mevalonate (Mev) for 24 h. Quantification of nuclear/cytosolic YAP/TAZ (right panel) from three independent experiments. At least 100 cells were analyzed in every experiment. Scale bars, 25 µm. (C) A549 cells were cultured with dimethylsulphoxide (NT) or simvastatin (Simv) alone or with mevalonate (Mev) for 24 h, and CYR61 mRNA levels were assessed using RT-qPCR. Data show the mean ± SD of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Two-way ANOVA with Tukeys multi-comparisons test. 3.3. Inhibition of YAP/TAZ Signaling Mediates Statin-Induced Sensitization to TRAIL The mevalonate pathway is known to be involved in the regulation of YAP/TAZ phosphorylation and activity independently of LATS1/2 kinases [33]. To determine whether the decreased YAP/TAZ signaling observed in cancer cells treated with statins plays a role in the sensitization to TRAIL-induced apoptosis, we first generated A549 cells expressing an activated form of YAP (YAP5SA), lacking inhibitory phosphorylation sites. As shown in Figure 3A, the expression of YAP5SA in A549 cells was sufficient to prevent simvastatin-induced sensitization to TRAIL. To further demonstrate the role of YAP/TAZ proteins in the control of TRAIL sensitivity in cancer cells, we performed the simultaneous knockdown of YAP and TAZ proteins in the various cancer cell lines using two independent sets (siY/T#1 and siY/T#2) of Figure 2. Nuclear localization and activity of YAP/TAZ are regulated by the mevalonate pathway in cancer cells. ( A ) Representative images of YAP/TAZ immunofluorescence (left panel) in cells treated with dimethylsulphoxide (NT) or simvastatin (Simv) alone or with mevalonate (Mev) for 24 h. Quantification of nuclear/cytosolic YAP/TAZ (right panel) from three independent experiments. ( B ) Representative images of YAP/TAZ immunofluorescence (left panel) in cells treated with dimethylsulphoxide (NT) or cerivastatin (Ceriv) alone or with mevalonate (Mev) for 24 h. Quantification of nuclear/cytosolic YAP/TAZ (right panel) from three independent experiments. At least 100 cells were analyzed in every experiment. Scale bars, 25 µ m. ( C ) A549 cells were cultured with dimethylsulphoxide (NT) or simvastatin (Simv) alone or with mevalonate (Mev) for 24 h, and CYR61 mRNA levels were assessed using RT-qPCR. Data show the mean ± SD of at least three independent experiments. * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001. Two-way ANOVA with Tukey ´ s multi-comparisons test. Cells 2023, 12, x FOR PEER REVIEW 9 of 19 previously validated YAP and TAZ siRNAs [40]. As shown in Figure 3B, YAP/TAZ knockdown significantly sensitized cancer cells to TRAIL-induced apoptosis. Importantly, sensitization to TRAIL by YAP/TAZ siRNAs was not observed in cells expressing a siRNAinsensitive form of wild-type YAP (Figure 3C). Figure 3. Cont. Cells 2023,12, 2370 9 of 19 Cells 2023, 12, x FOR PEER REVIEW 9 of 19 previously validated YAP and TAZ siRNAs [40]. As shown in Figure 3B, YAP/TAZ knockdown significantly sensitized cancer cells to TRAIL-induced apoptosis. Importantly, sensitization to TRAIL by YAP/TAZ siRNAs was not observed in cells expressing a siRNAinsensitive form of wild-type YAP (Figure 3C). Figure 3. Role of YAP/TAZ in the control of TRAIL sensitivity via statins. ( A ) A549-pBabe or A549-YAP5SA cells were treated for 24 h with the indicated doses of simvastatin prior to incubation in the presence or absence of TRAIL for 24h. Cell death was analyzed by subG1 quantification (5 × 10 3 cells/sample). Data are normalized to NT. ( B ) Cells were transfected with two different siRNA oligonucleotides pairs targeting both YAP and TAZ, as described in the Materials and Methods section. A scrambled RNA was also used as non-targeting control oligonucleotide. After 30 h, cells were treated with TRAIL for 24 h. Cell death was analyzed via subG1 quantification. ( C ) A549EGFP or A549-wtYAP cells were transfected with siRNAs against both YAP and TAZ (YAP/TAZ#1). After 6 h , cells were treated with doxycycline (1 µ g/mL). At 30 h post-transfection, cells were treated with TRAIL for 24 h and apoptotic cell death was then determined. Protein knockdown was confirmed via immunoblot analysis. Data show the mean ± SD of at least three independent experiments. *p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001. Two-way ANOVA with Tukey ´ s multi-comparisons test. To further demonstrate the role of YAP/TAZ proteins in the control of TRAIL sensitivity in cancer cells, we performed the simultaneous knockdown of YAP and TAZ proteins in the various cancer cell lines using two independent sets (siY/T#1 and siY/T#2) of previously validated YAP and TAZ siRNAs [ 40 ]. As shown in Figure 3B, YAP/TAZ knockdown significantly sensitized cancer cells to TRAIL-induced apoptosis. Importantly, sensitization to TRAIL by YAP/TAZ siRNAs was not observed in cells expressing a siRNA-insensitive form of wild-type YAP (Figure 3C). Cells 2023,12, 2370 16 of 19 that overcomes the resistance of tumor cells to TRAIL. However, preclinical work should be performed to confirm this hypothesis before its application as an antitumor therapy in humans could be considered. Author Contributions: Conceptualization, A.L.-R. and R.Y.; formal analysis, Y.E.Y., R.M.-M. and R.Y.; funding acquisition, A.L.-R.; investigation, Y.E.Y., R.M.-M. and R.Y.; methodology, Y.E.Y., R.M.-M. and R.Y.; project administration, R.Y.; supervision, A.L.-R. and R.Y.; validation, Y.E.Y., R.M.-M. and R.Y.; visualization, Y.E.Y. and R.Y.; writing—original draft, R.Y.; writing—review and editing, Y.E.Y., R.M.-M., A.L.-R. and R.Y. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grants from Ministerio de Ciencia e Innovación (PGC2018093960-B-I00 and PID2021-122226NB-I00), Junta de Andalucía (PY20-00754) and the European Community through the regional development funding program (FEDER) to ALR. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data generated or analyzed during this study are included in the main text. Acknowledgments: We are grateful to C. Palacios for the helpful discussions. We thank F.J. FernándezFarrán for excellent technical assistance. The core facilities of the Andalusian Centre of Molecular Biology and Regenerative Medicine are acknowledged for their technical support. Conflicts of Interest: The authors disclose no potential conflict of interest. References 1. Walczak, H.; Miller, R.E.; Ariail, K.; Gliniak, B.; Griffith, T.S.; Kubin, M.; Chin, W.; Jones, J.; Woodward, A.; Le, T.; et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat. Med. 1999,5, 157–163. [CrossRef] 2. Ashkenazi, A.; Pai, R.C.; Fong, S.; Leung, S.; Lawrence, D.A.; Marsters, S.A.; Blackie, C.; Chang, L.; McMurtrey, A.E.; Hebert, A.; et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J. Clin. Investig. 1999 ,104, 155–162. [CrossRef] [PubMed] 3. Sprick, M.R.; Weigand, M.A.; Rieser, E.; Rauch, C.T.; Juo, P.; Blenis, J.; Krammer, P.H.; Walczak, H. FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity 2000 , 12, 599–609 . [CrossRef] [PubMed] 4. Bodmer, J.L.; Holler, N.; Reynard, S.; Vinciguerra, P.; Schneider, P.; Juo, P.; Blenis, J.; Tschopp, J. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat. Cell Biol. 2000,2, 241–243. [CrossRef] [PubMed] 5. Irmler, M.; Thome, M.; Hahne, M.; Schneider, P.; Hofmann, K.; Steiner, V.; Bodmer, J.L.; Schroter, M.; Burns, K.; Mattmann, C.; et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997,388, 190–195. [CrossRef] [PubMed] 6. Hietakangas, V.; Poukkula, M.; Heiskanen, K.M.; Karvinen, J.T.; Sistonen, L.; Eriksson, J.E. Erythroid differentiation sensitizes K562 leukemia cells to TRAIL-induced apoptosis by downregulation of c-FLIP. Mol. Cell Biol. 2003,23, 1278–1291. [CrossRef] 7. Kim, Y.; Suh, N.; Sporn, M.; Reed, J.C. An inducible pathway for degradation of FLIP protein sensitizes tumor cells to TRAILinduced apoptosis. J. Biol. Chem. 2002,277, 22320–22329. [CrossRef] 8. Mauro-Lizcano, M.; Lopez-Rivas, A. Glutamine metabolism regulates FLIP expression and sensitivity to TRAIL in triple-negative breast cancer cells. Cell Death Dis. 2018,9, 205. [CrossRef] 9. Ortiz-Ferron, G.; Yerbes, R.; Eramo, A.; Lopez-Perez, A.I.; De Maria, R.; Lopez-Rivas, A. Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism. Cell Res. 2008,18, 664–676. [CrossRef] 10. Palacios, C.; Yerbes, R.; Lopez-Rivas, A. Flavopiridol induces cellular FLICE-inhibitory protein degradation by the proteasome and promotes TRAIL-induced early signaling and apoptosis in breast tumor cells. Cancer Res. 2006,66, 8858–8869. [CrossRef] 11. Goldstein, J.L.; Brown, M.S. Regulation of the mevalonate pathway. Nature 1990,343, 425–430. [CrossRef] 12. Feussner, G. HMG CoA reductase inhibitors. Curr. Opin. Lipidol. 1994,5, 59–68. [CrossRef] [PubMed] 13. Grundy, S.M. HMG-CoA reductase inhibitors for treatment of hypercholesterolemia. N. Engl. J. Med. 1988 ,319, 24–33. [PubMed] 14. Swanson, K.M.; Hohl, R.J. Anti-cancer therapy: Targeting the mevalonate pathway. Curr. Cancer Drug Targets 2006 ,6, 15–37. [CrossRef] [PubMed] 15. Edwards, P.A.; Ericsson, J. Sterols and isoprenoids: Signaling molecules derived from the cholesterol biosynthetic pathway. Annu. Rev. Biochem. 1999,68, 157–185. [CrossRef] [PubMed] 16. Jiang, W.; Hu, J.W.; He, X.R.; Jin, W.L.; He, X.Y. Statins: A repurposed drug to fight cancer. J. Exp. Clin. Cancer Res. 2021 ,40, 241. [CrossRef] [PubMed] 17. Nielsen, S.F.; Nordestgaard, B.G.; Bojesen, S.E. Statin use and reduced cancer-related mortality. N. Engl. J. Med. 2012 , 367, 1792–1802. [CrossRef] Cells 2023,12, 2370 17 of 19 18. Ivanov, V.N.; Hei, T.K. Regulation of apoptosis in human melanoma and neuroblastoma cells by statins, sodium arsenite and TRAIL: A role of combined treatment versus monotherapy. Apoptosis 2011,16, 1268–1284. [CrossRef] 19. Jin, Z.; Dicker, D.T.; El-Deiry, W.S. Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL. Cell Cycle 2002,1, 82–89. [CrossRef] 20. Liu, P.C.; Lu, G.; Deng, Y.; Wang, C.D.; Su, X.W.; Zhou, J.Y.; Chan, T.M.; Hu, X.; Poon, W.S. Inhibition of NF-kappaB Pathway and Modulation of MAPK Signaling Pathways in Glioblastoma and Implications for Lovastatin and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) Combination Therapy. PLoS ONE 2017,12, e0171157. 21. Zanconato, F.; Cordenonsi, M.; Piccolo, S. YAP/TAZ at the Roots of Cancer. Cancer Cell 2016,29, 783–803. [CrossRef] [PubMed] 22. Mo, J.S.; Park, H.W.; Guan, K.L. The Hippo signaling pathway in stem cell biology and cancer. EMBO Rep. 2014 ,15, 642–656. [CrossRef] [PubMed] 23. Meng, Z.; Moroishi, T.; Guan, K.L. Mechanisms of Hippo pathway regulation. Genes Dev. 2016,30, 1–17. [CrossRef] [PubMed] 24. Urtasun, R.; Latasa, M.U.; Demartis, M.I.; Balzani, S.; Goni, S.; Garcia-Irigoyen, O.; Elizalde, M.; Azcona, M.; Pascale, R.M.; Feo, F.; et al. Connective tissue growth factor autocriny in human hepatocellular carcinoma: Oncogenic role and regulation by epidermal growth factor receptor/yes-associated protein-mediated activation. Hepatology 2011,54, 2149–2158. [CrossRef] 25. Diep, C.H.; Zucker, K.M.; Hostetter, G.; Watanabe, A.; Hu, C.; Munoz, R.M.; Von Hoff, D.D.; Han, H. Down-regulation of Yes Associated Protein 1 expression reduces cell proliferation and clonogenicity of pancreatic cancer cells. PLoS ONE 2012 ,7, e32783. [CrossRef] 26. Kim, M.; Kim, T.; Johnson, R.L.; Lim, D.S. Transcriptional co-repressor function of the hippo pathway transducers YAP and TAZ. Cell Rep. 2015,11, 270–282. [CrossRef] 27. Ma, K.; Xu, Q.; Wang, S.; Zhang, W.; Liu, M.; Liang, S.; Zhu, H.; Xu, N. Nuclear accumulation of Yes-Associated Protein (YAP) maintains the survival of doxorubicin-induced senescent cells by promoting survivin expression. Cancer Lett. 2016 ,375, 84–91. [CrossRef] 28. Strano, S.; Monti, O.; Pediconi, N.; Baccarini, A.; Fontemaggi, G.; Lapi, E.; Mantovani, F.; Damalas, A.; Citro, G.; Sacchi, A.; et al. The transcriptional coactivator Yes-associated protein drives p73 gene-target specificity in response to DNA Damage. Mol. Cell 2005,18, 447–459. [CrossRef] 29. Lapi, E.; Di Agostino, S.; Donzelli, S.; Gal, H.; Domany, E.; Rechavi, G.; Pandolfi, P.P.; Givol, D.; Strano, S.; Lu, X.; et al. PML, YAP, and p73 are components of a proapoptotic autoregulatory feedback loop. Mol. Cell 2008,32, 803–814. [CrossRef] 30. Xiao, Q.; Qian, Z.; Zhang, W.; Liu, J.; Hu, E.; Zhang, J.; Li, M.; Wang, J.; Kong, F.; Li, Y.; et al. Depletion of CABYR-a/b sensitizes lung cancer cells to TRAIL-induced apoptosis through YAP/p73-mediated DR5 upregulation. Oncotarget 2016 ,7, 9513–9524. [CrossRef] 31. Santinon, G.; Pocaterra, A.; Dupont, S. Control of YAP/TAZ Activity by Metabolic and Nutrient-Sensing Pathways. Trends Cell Biol. 2016,26, 289–299. [CrossRef] [PubMed] 32. Wang, Z.; Wu, Y.; Wang, H.; Zhang, Y.; Mei, L.; Fang, X.; Zhang, X.; Zhang, F.; Chen, H.; Liu, Y.; et al. Interplay of mevalonate and Hippo pathways regulates RHAMM transcription via YAP to modulate breast cancer cell motility. Proc. Natl. Acad. Sci. USA 2014 , 111, E89–E98. [CrossRef] [PubMed] 33. Sorrentino, G.; Ruggeri, N.; Specchia, V.; Cordenonsi, M.; Mano, M.; Dupont, S.; Manfrin, A.; Ingallina, E.; Sommaggio, R.; Piazza, S.; et al. Metabolic control of YAP and TAZ by the mevalonate pathway. Nat. Cell Biol. 2014,16, 357–366. [CrossRef] 34. Harper, N.; Farrow, S.N.; Kaptein, A.; Cohen, G.M.; MacFarlane, M. Modulation of tumor necrosis factor apoptosis-inducing ligandinduced NF-kappa B activation by inhibition of apical caspases. J. Biol. Chem. 2001 ,276, 34743–34752. [CrossRef] [PubMed] 35. Yerbes, R.; Palacios, C.; Reginato, M.J.; Lopez-Rivas, A. Cellular FLIP(L) plays a survival role and regulates morphogenesis in breast epithelial cells. Biochim. Biophys. Acta 2011,1813, 168–178. [CrossRef] 36. Wiznerowicz, M.; Trono, D. Conditional suppression of cellular genes: Lentivirus vector-mediated drug-inducible RNA interference. J. Virol. 2003,77, 8957–8961. [CrossRef] 37. Schmidt, E.K.; Clavarino, G.; Ceppi, M.; Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 2009,6, 275–277. [CrossRef] 38. Duarte, J.A.; de Barros, A.L.B.; Leite, E.A. The potential use of simvastatin for cancer treatment: A review. Biomed. Pharmacother. 2021,141, 111858. [CrossRef] 39. Zhao, B.; Ye, X.; Yu, J.; Li, L.; Li, W.; Li, S.; Yu, J.; Lin, J.D.; Wang, C.Y.; Chinnaiyan, A.M.; et al. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 2008,22, 1962–1971. [CrossRef] 40. Aragona, M.; Panciera, T.; Manfrin, A.; Giulitti, S.; Michielin, F.; Elvassore, N.; Dupont, S.; Piccolo, S. A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 2013,154, 1047–1059. [CrossRef] 41. Zanconato, F.; Forcato, M.; Battilana, G.; Azzolin, L.; Quaranta, E.; Bodega, B.; Rosato, A.; Bicciato, S.; Cordenonsi, M.; Piccolo, S. Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nat. Cell Biol. 2015 , 17, 1218–1227. [CrossRef] [PubMed] 42. Stein, C.; Bardet, A.F.; Roma, G.; Bergling, S.; Clay, I.; Ruchti, A.; Agarinis, C.; Schmelzle, T.; Bouwmeester, T.; Schubeler, D.; et al. YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers. PLoS Genet. 2015 ,11, e1005465. [CrossRef] [PubMed] Cells 2023,12, 2370 18 of 19 43. Troilo, A.; Benson, E.K.; Esposito, D.; Garibsingh, R.A.; Reddy, E.P.; Mungamuri, S.K.; Aaronson, S.A. Angiomotin stabilization by tankyrase inhibitors antagonizes constitutive TEAD-dependent transcription and proliferation of human tumor cells with Hippo pathway core component mutations. Oncotarget 2016,7, 28765–28782. [CrossRef] [PubMed] 44. Yuan, Y.; Park, J.; Feng, A.; Awasthi, P.; Wang, Z.; Chen, Q.; Iglesias-Bartolome, R. YAP1/TAZ-TEAD transcriptional networks maintain skin homeostasis by regulating cell proliferation and limiting KLF4 activity. Nat. Commun. 2020 ,11, 1472. [CrossRef] [PubMed] 45. Sheridan, J.P.; Marsters, S.A.; Pitti, R.M.; Gurney, A.; Skubatch, M.; Baldwin, D.; Ramakrishnan, L.; Gray, C.L.; Baker, K.; Wood, W.I.; et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997 ,277, 818–821. [CrossRef] 46. Martin-Perez, R.; Palacios, C.; Yerbes, R.; Cano-Gonzalez, A.; Iglesias-Serret, D.; Gil, J.; Reginato, M.J.; Lopez-Rivas, A. Activated ERBB2/HER2 licenses sensitivity to apoptosis upon endoplasmic reticulum stress through a PERK-dependent pathway. Cancer Res. 2014,74, 1766–1777. [CrossRef] 47. Caro-Maldonado, A.; Tait, S.W.; Ramirez-Peinado, S.; Ricci, J.E.; Fabregat, I.; Green, D.R.; Munoz-Pinedo, C. Glucose deprivation induces an atypical form of apoptosis mediated by caspase-8 in Bax-, Bak-deficient cells. Cell Death Differ. 2010 ,17, 1335–1344. [CrossRef] 48. Lu, M.; Lawrence, D.A.; Marsters, S.; Acosta-Alvear, D.; Kimmig, P.; Mendez, A.S.; Paton, A.W.; Paton, J.C.; Walter, P.; Ashkenazi, A. Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis. Science 2014 ,345, 98–101. [CrossRef] 49. Martin-Perez, R.; Yerbes, R.; Mora-Molina, R.; Cano-Gonzalez, A.; Arribas, J.; Mazzone, M.; Lopez-Rivas, A.; Palacios, C. Oncogenic p95HER2/611CTF primes human breast epithelial cells for metabolic stress-induced down-regulation of FLIP and activation of TRAIL-R/Caspase-8-dependent apoptosis. Oncotarget 2017,8, 93688–93703. [CrossRef] 50. McComb, S.; Chan, P.K.; Guinot, A.; Hartmannsdottir, H.; Jenni, S.; Dobay, M.P.; Bourquin, J.P.; Bornhauser, B.C. Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7. Sci. Adv. 2019 ,5, eaau9433. [CrossRef] 51. Wilson, T.R.; McLaughlin, K.M.; McEwan, M.; Sakai, H.; Rogers, K.M.; Redmond, K.M.; Johnston, P.G.; Longley, D.B. c-FLIP: A key regulator of colorectal cancer cell death. Cancer Res. 2007,67, 5754–5762. [CrossRef] [PubMed] 52. Hughes, M.A.; Powley, I.R.; Jukes-Jones, R.; Horn, S.; Feoktistova, M.; Fairall, L.; Schwabe, J.W.; Leverkus, M.; Cain, K.; MacFarlane, M. Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate. Mol. Cell 2016,61, 834–849. [CrossRef] [PubMed] 53. Mora-Molina, R.; Stohr, D.; Rehm, M.; Lopez-Rivas, A. cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells. Cell Death Dis. 2022,13, 111. [CrossRef] [PubMed] 54. Yerbes, R.; Mora-Molina, R.; Fernandez-Farran, F.J.; Hiraldo, L.; Lopez-Rivas, A.; Palacios, C. Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells. Cell Death Dis. 2022 ,13, 906. [CrossRef] 55. Tuckow, A.P.; Jefferson, S.J.; Kimball, S.R.; Jefferson, L.S. Simvastatin represses protein synthesis in the muscle-derived C(2)C(1)(2) cell line with a concomitant reduction in eukaryotic initiation factor 2B expression. Am. J. Physiol. Endocrinol. Metab. 2011 , 300, E564–E570. [CrossRef] 56. Fukazawa, T.; Fujiwara, T.; Uno, F.; Teraishi, F.; Kadowaki, Y.; Itoshima, T.; Takata, Y.; Kagawa, S.; Roth, J.A.; Tschopp, J.; et al. Accelerated degradation of cellular FLIP protein through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancer cells. Oncogene 2001,20, 5225–5231. [CrossRef] 57. von Karstedt, S.; Montinaro, A.; Walczak, H. Exploring the TRAILs less travelled: TRAIL in cancer biology and therapy. Nat. Rev. Cancer 2017,17, 352–366. [CrossRef] 58. Lemke, J.; von Karstedt, S.; Zinngrebe, J.; Walczak, H. Getting TRAIL back on track for cancer therapy. Cell Death Differ. 2014 , 21, 1350–1364. [CrossRef] 59. Montinaro, A.; Walczak, H. Harnessing TRAIL-induced cell death for cancer therapy: A long walk with thrilling discoveries. Cell Death Differ. 2023,30, 237–249. [CrossRef] 60. Carr, R.M.; Qiao, G.; Qin, J.; Jayaraman, S.; Prabhakar, B.S.; Maker, A.V. Targeting the metabolic pathway of human colon cancer overcomes resistance to TRAIL-induced apoptosis. Cell Death Discov. 2016,2, 16067. [CrossRef] 61. Munoz-Pinedo, C.; Ruiz-Ruiz, C.; Ruiz de Almodovar, C.; Palacios, C.; Lopez-Rivas, A. Inhibition of glucose metabolism sensitizes tumor cells to death receptor-triggered apoptosis through enhancement of death-inducing signaling complex formation and apical procaspase-8 processing. J. Biol. Chem. 2003,278, 12759–12768. [CrossRef] [PubMed] 62. Humphreys, L.M.; Fox, J.P.; Higgins, C.A.; Majkut, J.; Sessler, T.; McLaughlin, K.; McCann, C.; Roberts, J.Z.; Crawford, N.T.; McDade, S.S.; et al. A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis. EMBO Rep. 2020,21, e49254. [CrossRef] [PubMed] 63. Sanchez-Perez, T.; Ortiz-Ferron, G.; Lopez-Rivas, A. Mitotic arrest and JNK-induced proteasomal degradation of FLIP and Mcl-1 are key events in the sensitization of breast tumor cells to TRAIL by antimicrotubule agents. Cell Death Differ. 2010 ,17, 883–894. [CrossRef] [PubMed] 64. Shirley, S.; Micheau, O. Targeting c-FLIP in cancer. Cancer Lett. 2013,332, 141–150. [CrossRef] Cells 2023,12, 2370 19 of 19 65. Hassanzadeh, A.; Farshdousti Hagh, M.; Alivand, M.R.; Akbari, A.A.M.; Shams Asenjan, K.; Saraei, R.; Solali, S. Down-regulation of intracellular anti-apoptotic proteins, particularly c-FLIP by therapeutic agents; the novel view to overcome resistance to TRAIL. J. Cell. Physiol. 2018,233, 6470–6485. [CrossRef] 66. Humphreys, L.; Espona-Fiedler, M.; Longley, D.B. FLIP as a therapeutic target in cancer. FEBS J. 2018 ,285, 4104–4123. [CrossRef] 67. Dey, A.; Varelas, X.; Guan, K.L. Targeting the Hippo pathway in cancer, fibrosis, wound healing and regenerative medicine. Nat. Rev. Drug Discov. 2020,19, 480–494. [CrossRef] 68. Liu, Y.; Chen, L.; Gong, Z.; Shen, L.; Kao, C.; Hock, J.M.; Sun, L.; Li, X. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells. Oncotarget 2015,6, 3055–3070. [CrossRef] 69. Guruswamy, S.; Rao, C.V. Synergistic effects of lovastatin and celecoxib on caveolin-1 and its down-stream signaling molecules: Implications for colon cancer prevention. Int. J. Oncol. 2009,35, 1037–1043. 70. De Miguel, D.; Gallego-Lleyda, A.; Martinez-Ara, M.; Plou, J.; Anel, A.; Martinez-Lostao, L. Double-Edged Lipid Nanoparticles Combining Liposome-Bound TRAIL and Encapsulated Doxorubicin Showing an Extraordinary Synergistic Pro-Apoptotic Potential. Cancers 2019,11, 1948. [CrossRef] 71. Pobbati, A.V.; Kumar, R.; Rubin, B.P.; Hong, W. Therapeutic targeting of TEAD transcription factors in cancer. Trends Biochem. Sci. 2023,48, 450–462. [CrossRef] [PubMed] 72. Vassilev, A.; Kaneko, K.J.; Shu, H.; Zhao, Y.; DePamphilis, M.L. TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm. Genes Dev. 2001,15, 1229–1241. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.