Various Mechanisms Involve the Nuclear Factor (Erythroid-Derived 2)-Like (NRF2) to Achieve Cytoprotection in Long-Term Cisplatin-Treated Urothelial Carcinoma Cell Lines

Therapeutic efficacy of cisplatin-based chemotherapy for advanced-stage urothelial carcinoma (UC) is limited by drug resistance. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway is a major regulator of cytoprotective responses. We investigated its involvement in cisplatin resistance in long-term cisplatin treated UC cell lines (LTTs). Expression of NRF2 pathway components and targets was evaluated by qRT-PCR and western blotting in LTT sublines from four different parental cells. NRF2 transcriptional activity was determined by reporter assays and total glutathione (GSH) was quantified enzymatically. Effects of siRNA-mediated NRF2 knockdown on chemosensitivity were analysed by viability assays, γH2AX immunofluorescence, and flow cytometry. Increased expression of NRF2, its positive regulator p62/SQSTM1, and elevated NRF2 activity was observed in 3/4 LTTs, which correlated with KEAP1 expression. Expression of cytoprotective enzymes and GSH concentration were upregulated in some LTTs. NRF2 knockdown resulted in downregulation of cytoprotective enzymes and resensitised 3/4 LTTs towards cisplatin as demonstrated by reduced IC50 values, increased γH2AX foci formation, and elevated number of apoptotic cells. In conclusion, while LTT lines displayed diversity in NRF2 activation, NRF2 signalling contributed to cisplatin resistance in LTT lines, albeit in diverse ways. Accordingly, inhibition of NRF2 can be used to resensitise UC cells to cisplatin, but responses in patients may likewise be variable.


Introduction
Bladder cancer is the 9th most common tumour world-wide and the most common cancer of the urinary tract [1]. About 90% of bladder cancers in industrialised countries are urothelial (EMT) and canonical WNT pathway target genes [44,45]. In the present study, we investigated NRF2 and its related pathways in these LTT cells.

Increased Expression and Transcriptional Activity of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) in Long-Term Cisplatin Treated Cell Lines (LTTs)
We first evaluated expression of NRF2 and its regulators across a panel of UCCs covering the heterogeneity of the disease and then, in particular, compared the four long-term cisplatin treated LTTs with their parental cell lines. Across the UCCs, NRF2 and KEAP1 protein expression were heterogeneous and tended towards the expected inverse pattern, whereas p62/SQSTM1 expression was more uniform ( Figure S1a). NRF2 protein levels were significantly increased in three out of four LTT cell lines, except in T-24-LTT, compared to the parental cell lines (Figure 1a). KEAP1 protein was only diminished in J82-LTT, which also overexpressed p62/SQSTM1. In T-24-LTT, conversely, KEAP1 was increased and p62/SQSTM1 was diminished (Figure 1a). Elevation of NRF2 protein in the three LTTs was constitutive and not due to induction by culturing with cisplatin, as shown by comparing cisplatin treated LTTs and LTTs without treatment for 10 days with their parental cell lines with or without cisplatin treatment ( Figure S1b).
NRF2 and KEAP1 mRNA expression were both significantly decreased in three LTTs compared to their parental cell lines, whereas p62/SQSTM1 mRNA was upregulated. T-24-LTT was again the exception with no significant difference in NRF2 or p62/SQSTM1 mRNA, but increased KEAP1 mRNA expression ( Figure S1c).
NRF2 transcriptional activity was measured by a reporter assay following transfection with an ARE-luciferase expression construct. Compared to the untreated control, ARE-dependent luciferase activity was increased in RT-112-LTT, J82-LTT, and 253J-LTT, but was significantly decreased in T-24-LTT. Co-transfection with an NRF2 expression construct significantly increased luciferase activity in all four LTTs as well as in the parental lines; however, in T-24-LTT the inducible NRF2 luciferase activity remained lower compared to the parental cell line (Figure 1b). Remarkably, basal and inducible luciferase activity in RT-112-LTT was 100-and 1000-fold increased, respectively, in comparison to their parental cell line. These differences among LTT lines could relate to the differences in the cisplatin end concentrations they tolerated, as described in the Material and Methods. Immunofluorescence staining for NRF2 revealed increased expression in RT-112-LTT and 253J-LTT and nuclear staining in three out of four LTTs, with T-24-LTT being the only exception ( Figure 1c).
As a possible cause of enhanced NRF2 expression and transcriptional activity, we searched for mutations in the NFE2L2 and KEAP1 genes. According to the TCGA data on UC [46,47], missense mutations in NRF2/NFE2L2 are usually located in exon 2 and were detected in 14 (11%) of 126 sequenced UC. KEAP1 was altered in 10 (7%) of 126 sequenced cases/patients, with 4 missense and 1 truncating mutation ( Figure S2a). Deletion of NFE2L2 exon 2 represents an alternative mechanism for activation of NRF2 in a subset of squamous lung and head-and-neck cancers [48]. Somatic mutations of the KEAP1 gene, especially in the BTB domain encoded by exon 2 have been identified in several solid cancers, e.g., NSCLC and gastric adenocarcinoma [49,50]. Exons 2 of NFE2L2 and KEAP1 were detectable in all four UCCs and their LTT sublines ( Figure S2b). Sanger sequencing revealed no changes in NFE2L2 exon 2 (data not shown), but a non-silent missense mutation in KEAP1 exon 2, namely c. 334A>T (T112S), in RT-112-LTT, which was not present in the parental cell line ( Figure S2c). Interestingly, an independently obtained RT-112 cisplatin-resistant cell line, RT-112-cp [51] contained the same mutation ( Figure S2d,e). Despite introducing a serine residue this previously unreported mutation is not predicted to change phosphorylation by in silico analysis [52], but according to the KEAP1 crystal structure (data not shown, PDB ID 5NLB [53,54]), the amino acid change could lead to a conformational change impairing KEAP1 binding to Cullin-3 and thereby interfere with NRF2 degradation. It remains to be elucidated why this mutation occurs preferentially during treatment of RT-112. Two additional cisplatin-resistant cell lines derived by pulse-treatment, RT-112-R and J82-R [45] contained no alternations in exons 2 of NFE2L2 and KEAP1 ( Figure S2d). degradation. It remains to be elucidated why this mutation occurs preferentially during treatment of RT-112. Two additional cisplatin-resistant cell lines derived by pulse-treatment, RT-112-R and J82-R [45] contained no alternations in exons 2 of NFE2L2 and KEAP1 ( Figure S2d).

Induction of Cytoprotective Enzymes and Elevated Glutathione (GSH) Levels in LTTs
Expression of NRF2 target genes was quantified by qRT-PCR (Figure 2a-d, Table S1). Significantly increased expression of the cytoprotective enzyme genes GSR, NQO1, GPX1, GPX2, GSTM1, and GSTP1 was observed in RT-112-LTT compared to their parental cells (Figure 2a) and, except for GSTM1, also in T-24-LTT ( Figure 2d). J82-LTT expressed higher GSR mRNA compared to its parental cell line (Figure 2b). Cytoprotective enzymes mRNAs remained mostly unchanged in 253J-LTT compared to its parental cell line (Figure 2c).
Among GSH biosynthetic genes, GCLM mRNA was elevated in three out of four LTTs, albeit not in J82-LTT, whereas GCLC mRNA expression was generally unchanged (Figure 2e). The mRNA of the SLC3A2 transporter was significantly elevated in RT-112-LTT, J82-LTT, and T-24-LTT compared to their parental cell lines. SLC7A11 mRNA expression was significantly increased in RT- As a loading control, α-Tubulin was stained. (b) Luciferase reporter activity was determined 72 h after transfection with pGL3-8xARE for basal NRF2 activity and in combination with NC16 pCDNA3.1 FLAG NRF2 for inducible NRF2 activity. Values represent the mean ± SD of biological triplicates. p < 0.05 * in basal vs. inducible NRF2 activity. (c) Immunofluorescence staining for NRF2 (green) in LTTs and their parental cell lines. DAPI staining (blue) was used to visualise nuclei. Scale bars = 50 µm.

Induction of Cytoprotective Enzymes and Elevated Glutathione (GSH) Levels in LTTs
Expression of NRF2 target genes was quantified by qRT-PCR (Figure 2a-d, Table S1). Significantly increased expression of the cytoprotective enzyme genes GSR, NQO1, GPX1, GPX2, GSTM1, and GSTP1 was observed in RT-112-LTT compared to their parental cells ( Figure 2a) and, except for GSTM1, also in T-24-LTT ( Figure 2d). J82-LTT expressed higher GSR mRNA compared to its parental cell line ( Figure 2b). Cytoprotective enzymes mRNAs remained mostly unchanged in 253J-LTT compared to its parental cell line (Figure 2c).  Among GSH biosynthetic genes, GCLM mRNA was elevated in three out of four LTTs, albeit not in J82-LTT, whereas GCLC mRNA expression was generally unchanged (Figure 2e). The mRNA of the SLC3A2 transporter was significantly elevated in RT-112-LTT, J82-LTT, and T-24-LTT compared to their parental cell lines. SLC7A11 mRNA expression was significantly increased in RT-112-LTT and T-24-LTT, but remained unchanged or were even significantly decreased in 253J-LTT and J82-LTT, respectively, compared to their parental cell lines ( Figure 2e, Table S1).
Total GSH was significantly increased in J82-LTT and T-24-LTT, but not in RT-112-LTT and 253J-LTT ( Figure 2f). Interestingly, total GSH increased significantly after short-term cisplatin treatment (STT) of parental cell lines (Figure 2e), without changes in the NRF2 protein level (compare Figure S1b). Moreover, compared to their parental cell lines, significantly lower intracellular ROS concentrations were detected after cisplatin treatment in J82-LTT, 253J-LTT and T-24-LTT, but not in RT-112-LTT, where significantly higher levels were observed after cisplatin treatment (Figures 2g and S3).

NRF2 as A Target to Sensitise Cisplatin-Resistant Urothelial Carcinoma Cell Lines (UCCs)
Efficient knockdown of NRF2 mRNA and protein was achieved in all four LTTs (Figure 3a

Relation of Increased NRF2 Expression to Hippo and Nuclear Factor Kappa B (NF-κB) Pathways in LTTs
To study crosstalk between the NRF2, Hippo, and NF-κB pathways, we first assessed YAP1, IKKα/CHUK, and NF-κB p65/RELA protein expression across an UC cell line panel. With the exception of 5637, IKKα and NF-κB p65 protein expression were very similar among the tested UCCs, whereas YAP1 protein expression was heterogeneous. Notably, J82 and T-24 were among the cell lines with high expression ( Figure S1a). In most LTTs, YAP1 mRNA and protein expression were unchanged compared to the parental cell lines, except for a significant decrease in T-24-LTT (Figure 5a,b, Table S1). Further, IKKα and NF-κB p65 mRNA expression was significantly decreased in J82-LTT and 253J-LTT compared to their parental cell lines ( Figure 5c, Table S1). IKKα and NF-κB p65 mRNA expression were accordingly decreased in 253J-LTT. However, both IKKα and NF-κB p65 proteins appeared unchanged in J82-LTT ( Figure 5d). By immunofluorescence staining, NF-κB p65 was exclusively cytoplasmically localised in all four UCCs and their LTT sublines (Figure 5e).

Discussion
This study revealed NRF2 as an important factor in each of four independent long-term cisplatin treated (LTT) urothelial carcinoma cell lines (UCCs), fitting the established notion of NRF2 as a generally important factor in the development of chemoresistance and as a rational target to restore chemosensitivity [39]. Upon closer analysis, however, the cell lines differed in the extent of NRF2 expression and activation, its upstream regulation, and the expression of its downstream targets. Thus, our study of four different cell lines from the same tumour entity highlights the diversity in the modes of NRF2 activation and the ensuing gene expression changes. We consider it likely that a similar degree of diversity in NRF2 activation will occur in patients during cancer treatment with cisplatin-containing regimens, especially in cancer types with pronounced heterogeneity like UC. Therefore, while our data, on the one hand, support the notion of NRF2 as an important factor in chemoresistance, they suggest, on the other hand, that targeting NRF2 may not lead to a uniform

Discussion
This study revealed NRF2 as an important factor in each of four independent long-term cisplatin treated (LTT) urothelial carcinoma cell lines (UCCs), fitting the established notion of NRF2 as a generally important factor in the development of chemoresistance and as a rational target to restore chemosensitivity [39]. Upon closer analysis, however, the cell lines differed in the extent of NRF2 expression and activation, its upstream regulation, and the expression of its downstream targets. Thus, our study of four different cell lines from the same tumour entity highlights the diversity in the modes of NRF2 activation and the ensuing gene expression changes. We consider it likely that a similar degree of diversity in NRF2 activation will occur in patients during cancer treatment with cisplatin-containing regimens, especially in cancer types with pronounced heterogeneity like UC. Therefore, while our data, on the one hand, support the notion of NRF2 as an important factor in chemoresistance, they suggest, on the other hand, that targeting NRF2 may not lead to a uniform response and NRF2 activation needs to be considered in a broader perspective, also including interfering and crosstalk pathways.
In detail, the three resistant cell lines RT-112-LTT, J82-LTT, and 253J-LTT expressed more NRF2 protein, which was localised in the nucleus, and was associated with basal and inducible NRF2 activity in reporter assays. NRF2 protein expression was unchanged in a fourth resistant cell line, T-24-LTT, compared to parental T-24 cells. SiRNA-mediated knockdown of NRF2 decreased IC 50 values for cisplatin in RT-112-LTT, 253J-LTT, and T-24-LTT and this sensitisation was paralleled by increased γH2AX foci formation, in line with the presumed functions of NRF2. Unexpectedly, only a minor sensitisation was achieved in J82-LTT by NRF2 knockdown, which, also unexpectedly, instead induced apoptosis in T-24-LTT even in the absence of treatment (see detailed discussion below). Concordantly with our results, NRF2 knockdown sensitised the lung carcinoma cell line A549 to cisplatin, whereas its stable overexpression enhanced cisplatin resistance of breast adenocarcinoma and neuroblastoma cells MDA-MB-231 and SH-SY5Y [55]. Similarly, inhibition of KEAP1 stabilised NRF2 expression in SCC stem cells and rendered them resistant to cisplatin [30].
As a possible cause of increased NRF2 expression, we found elevated p62/SQSTM1 protein and mRNA expression in RT-112-LTT, J82-LTT, and 253J-LTT. Analogously, cisplatin-resistant ovarian cancer SKOV3 cells expressed more p62/SQSTM1 and were resensitised upon knockdown of p62/SQSTM1 [56,57]. The deletion of NFE2L2 exon 2 represents an alternative mechanism for activation of NRF2 in squamous carcinomas [48]. In hepatocarcinogenesis most NRF2 mutations are located in the regions coding for the DLG or ETGE motives, which bind to the Kelch domain in KEAP1 [58,59]. However, exon 2 was present and unchanged in all four UC LTT. Instead, we detected a mutation near the BTB domain of KEAP1, c. 334A>T (T112S), in RT-112-LTT, which, despite the newly introduced serine, likely does not affect protein phosphorylation. Nevertheless, the T112 side chain interacts with the neighbouring amino acid backbone to stabilise a protein loop, and its replacement by a serine could impair interaction with Cullin 3 [60]. In their cisplatin-resistant RT-112 subline, Hayden et al. [19] observed loss of KEAP1 expression, but its cause was not elucidated. In other cancer types, KEAP1 mutations in the first Kelch domain (e.g., G333C in A549 cells) and in the intervention region (IVR, D236H in H460 cells) modified NRF2 signalling and influenced platinum sensitivity [50]. Of note, in addition to p62/SQSTM1, other negative regulators of the NRF2-KEAP interaction, such as Gankyrin (PSMD10, 26S proteasome non-ATPase regulatory subunit 10) [61,62] could be involved and may deserve further investigation. In theory, KEAP1-mediated control of NRF2 might also be disrupted by the cyclin inhibitor p21 Cip1/Waf1 , which competes with oxidised KEAP1 for binding to the NRF2 DLG motif to enhance the stability of the transcription factor [21,[63][64][65][66]. In the current study, p21 was not differentially expressed between parental and LTT cell lines (data not shown) and thus appeared unrelated to NRF2 expression and activity in LTTs. Moreover, some UCCs, including RT-112, do not express functional p21 because of frameshift mutations.
Many cytoprotective enzymes inducible by NRF2 [67] were upregulated in RT-112-LTT compared to its parental cell line, similar to the observations by Hayden et al. in their independently derived cisplatin-resistant RT-112 line [19]. Similar results were obtained for T-24-LTT even though these cells did not display increased NRF2 protein expression. In J82-LTT and 253J-LTT most NRF2 targets were not significantly induced. In most LTTs, though, NRF2 knockdown diminished mRNA expression of downstream cytoprotective enzymes, such as GSR, NQO1, HMOX1, and GSTP1, as well as the NRF2 target p62/SQSTM1. Increased glutathione availability with consequently decreased intracellular ROS formation is another potential consequence of NRF2 activation that protects against cisplatin by forming extrudable conjugates and by ameliorating oxidative stress. For instance, higher GSH levels were observed in cisplatin-resistant A2780 ovarian carcinoma cells and the U373MG glioblastoma cell line [68,69]. Another cisplatin-resistant ovarian carcinoma cell line containing high levels of GSH could be sensitised by NRF2 inhibition [70]. In our study, elevated GSH content and decreased intracellular ROS production was found in J82-LTT and especially in 253J-LTT and T-24-LTT, compared to their respective parental lines. Elevated GSH corresponded with decreased oxidative stress following cisplatin treatment. Surprisingly, total GSH levels remained unchanged and intracellular oxidative stress was increased in RT-112-LTT compared to its parental cell line. Conceivably, GSH is efficiently conjugated with cisplatin in RT-112-LTT and exported by the overexpressed multidrug resistance-associated protein 2 (MRP2), resulting in a decreased steady-state GSH content. Key determinants of glutathione biosynthetic capacity are the cystine-glutamate transporters SLC7A11 and SLC3A2, both of which have previously been associated with cisplatin resistance [37,38]. These were upregulated in RT-112-LTT and T-24-LTT. Specifically, SLC7A11 overexpression as a consequence of diminished expression of its negative regulator miRNA-27a has been identified as a mechanism of cisplatin resistance in derivatives of the EJ UC cell line [71]. In that study, the combination of low miRNA-27a and high SLC7A11 was also observed in cancer tissues, but in a relatively low fraction of cases. This finding thus further illustrates the diversity of mechanisms involved in NRF2 action in UC, in cell lines as well as in tumours.
The diversity of NRF2 regulation is further illustrated by the J82-LTT line, which presented high basal NRF2 protein expression and enhanced reporter gene activity. Nevertheless, efficient siRNA-mediated knockdown of NRF2 had minimal effects on cisplatin sensitivity in this resistant subline. Indeed, expression of cytoprotective enzymes and of genes involved in GSH transport and biosynthesis was largely unchanged in J82-LTT compared to its parental line. As nuclear immunofluorescence staining of NRF2 was not as prominent in J82-LTT as in RT-112-LTT and 253J-LTT, we suspect that NRF2 might be prevented from activating its target genes in a KEAP1 independent manner. An established mechanism that might explain this constellation involves phosphorylation of nuclear NRF2 by GSK3β and recognition of phosphorylated NRF2 by β-TrCP in the ubiquitin E3 ligase complex SCF (Skp1-Cullin-F-box), leading to its poly-ubiquitination and degradation [72][73][74][75]. Alternatively, negative regulators of the NRF2-MafK interaction like BACH1 [18] might account for the phenotype of this cell line.
Whereas NRF2 protein was increased in all other LTTs, it was unchanged in T-24-LTT compared to its parental cell line. Basal NRF2 activity was decreased, but luciferase activity was inducible, albeit to a lesser extent than in its parental cell line. Expression of p62/SQSTM1 remained essentially unchanged, but KEAP1 protein and mRNA were upregulated. However, the low amount of NRF2 present proved essential in this cell line, as siNRF2 knockdown not only sensitised T-24-LTT to cisplatin, but also induced apoptosis and decreased clonogenicity in the absence of cisplatin especially in this cell line. Suppression of cell proliferation by complete siNRF2-mediated knockdown has also been observed in the cholangiocarcinoma cell lines KKU-156 and KKU-100 and was similarly enhanced by cisplatin treatment [76]. The sensitivity of T-24-LTT to NRF2 knockdown may of course relate to the downregulation of cytoprotective and GSH biosynthetic enzymes by the treatment. Strikingly, many of these factors were elevated in T-24-LTT compared to the parental cell line, even though NRF2 expression and activity were not increased. This observation suggests that NRF2 cooperates with another pathway acting on its target genes in this cell line. A good candidate is the aryl hydrocarbon (AhR) pathway, which exhibits crosstalk with NRF2 signalling at several levels [16]. AhR is kept inactive in the cytoplasm by binding to a complex of Hsp90, XAP2, and p23 protein. Upon activation, AhR translocates into the nucleus where it heterodimerises with the aryl hydrocarbon receptor nuclear translocator (ARNT) at xenobiotic response elements (XREs) to activate transcription [75,[77][78][79]. Many NRF2 targets also contain XREs [80,81].
Two other potentially interacting pathways, the Hippo and NF-κB pathways [11,12], were not altered in the LTTs. The major crosstalk between NRF2 and NF-κB pathways occurs through competition between the NF-κB p65 subunit and NRF2 for their common co-activator CBP. Following its activation by phosphorylation at Ser276, p65 suppresses transcription of ARE-dependent genes by depriving NRF2 of CBP [21,25,72]. Another factor mediating this crosstalk is p62/SQSTM1, which binds tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) via its TRAF6 binding domain (TB). Activation of TRAF6 leads to the phosphorylation of IKKβ, which phosphorylates IκB resulting in its ubiquitination and thereby the release of the NF-κB dimer consisting of p50 and p65 (Rel-A), which can then enter the nucleus for transcriptional regulation [26,28,82,83]. Moreover, the emerging role of the Hippo pathway has been described in cisplatin-resistant UC patient-derived xenograft models. There, YAP1 knockdown in T-24 cells increased sensitivity towards cisplatin by increasing DNA damage accumulation leading to apoptosis [13]. In our study, we did not observe a correlation between increased NRF2 activity and YAP1 protein expression.
In conclusion, our results indicate that NRF2-in different ways and to different extents-is a key player in the development of cisplatin resistance in UCCs and may constitute a reasonable target to combat chemoresistance in UC. Nevertheless, the diversity in NRF2 activation among the four tested LTTs highlights the complexity of cisplatin resistance even within one tumour entity and predicts that responses to NRF2 inhibition may likewise be highly variable in patients.  [44]. Reporter plasmids NC16 pCDNA3.1 FLAG NRF2, a gift from Randall Moon (Addgene plasmid #36971, Cambridge, MA, USA) [84], and GL3-8xARE, kindly provided by R. Wolf (Dundee, UK) [85], were transfected using X-tremeGENE9 DNA Transfection Reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions. For siRNA-mediated knockdown, UCCs and LTTs were transfected with 10 nmol/L siNRF2 or a non-targeting control (#L-003755-00 and #D-001810-10-05, both Dharmacon, Lafayette, CO, USA) using Lipofectamine RNAiMAX Reagent (Thermo Fisher, Waltham, MA, USA) according to the manufacturer's protocol. For colony formation assay cells were fixed in methanol before staining with Giemsa. Quantification of colonies was performed with ImageJ software 1.51k (National Institute of Mental Health, Bethesda, MD, USA) and a cell counter plugin.

Molecular Analyses
RNA isolation, cDNA synthesis, and quantitative real-time PCR were performed as previously described [44]. qRT-PCR was conducted using self-designed primers on the Lightcycler 96 system (Roche, Basel, Switzerland) (Table S2). For normalisation the housekeeping gene SDHA was used. DNA was extracted using the Blood & Cell Culture DNA Midi Kit according to the manufacturer's protocol (Qiagen, Hilden, Germany).

Mutation Analysis by Sequencing
For NFE2L2 and KEAP1 mutation analysis 468 and 842 bp amplicons containing exon 2, respectively, were amplified following PCR from genomic DNA. Primer sequences are detailed in Table S3. PCR products were purified using the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA) and analysed by Sanger sequencing. The interaction between the BTB domain of KEAP1 and Cullin 3 (PDB ID 5NLB [53,54]) was analysed by using PyMOL software version 1.5.0.4 (Schrödinger, Portland, OR, USA). The Protein Kinase Identification Server (PKIS) was used to predict changes in phosphorylation at the mutated site [52].

Measurement of Cell Viability
Cell viability was measured in quadruplicates by means of MTT assay (Sigma-Aldrich, St. Louis, MO, USA) and CellTiter-Glo assay (Promega, Fitchburg, WI, USA).

GSH Assay
Total glutathione was measured enzymatically by the method of Tietze [89][90][91] and normalised to cellular protein measured by the bicinchoninic acid-based method (Pierce, Thermo Fisher Scientific).

Use of the cBioPortal Data Base
NRF2/NFE2L2 and KEAP1 mutations in 413 bladder urothelial carcinoma samples (TCGA) were analysed using the cBioPortal for Cancer Genomics [46,47].

Conflicts of Interest:
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.