Modeling of Intracellular Taurine Levels Associated with Ovarian Cancer Reveals Activation of p53, ERK, mTOR and DNA-Damage-Sensing-Dependent Cell Protection

Taurine, a non-proteogenic amino acid and commonly used nutritional supplement, can protect various tissues from degeneration associated with the action of the DNA-damaging chemotherapeutic agent cisplatin. Whether and how taurine protects human ovarian cancer (OC) cells from DNA damage caused by cisplatin is not well understood. We found that OC ascites-derived cells contained significantly more intracellular taurine than cell culture-modeled OC. In culture, elevation of intracellular taurine concentration to OC ascites-cell-associated levels suppressed proliferation of various OC cell lines and patient-derived organoids, reduced glycolysis, and induced cell protection from cisplatin. Taurine cell protection was associated with decreased DNA damage in response to cisplatin. A combination of RNA sequencing, reverse-phase protein arrays, live-cell microscopy, flow cytometry, and biochemical validation experiments provided evidence for taurine-mediated induction of mutant or wild-type p53 binding to DNA, activation of p53 effectors involved in negative regulation of the cell cycle (p21), and glycolysis (TIGAR). Paradoxically, taurine’s suppression of cell proliferation was associated with activation of pro-mitogenic signal transduction including ERK, mTOR, and increased mRNA expression of major DNA damage-sensing molecules such as DNAPK, ATM and ATR. While inhibition of ERK or p53 did not interfere with taurine’s ability to protect cells from cisplatin, suppression of mTOR with Torin2, a clinically relevant inhibitor that also targets DNAPK and ATM/ATR, broke taurine’s cell protection. Our studies implicate that elevation of intracellular taurine could suppress cell growth and metabolism, and activate cell protective mechanisms involving mTOR and DNA damage-sensing signal transduction.


Introduction
Taurine is a non-proteogenic sulfonic amino acid implicated in tissue regeneration [1].Taurine's levels drop significantly with aging [2], and human clinical studies have demonstrated that taurine supplementation can treat congestive heart failure [3], retinal degeneration, and cardiomyopathy [1].Studies using various animal models showed that taurine improved cognitive functions [4], glucose metabolism [5], and motor functions [6] and protected various tissues from degeneration associated with cisplatin treatment [7,8].At the molecular level, taurine's cell protection has been linked to decreased DNA damage and chromatin remodeling [2].These studies established a possible role for taurine in managing age-related or treatment-induced morbidities linked to DNA damage.
The tumor suppressor p53 protein, a product of the TP53 gene, is a transcription factor that activates a plethora of signal transduction mechanisms involved in cell cycle arrest, regulation of metabolism, DNA damage resolution, and cell survival under stress [9][10][11].During DNA damage, p53 is rapidly stabilized through the ATM/ATR-dependent negative regulation of E3 ubiquitin ligase MDM2 [12] and phosphorylation by DNAPK [13].Stabilized p53 can suppress the cell cycle, trigger DNA repair, and rewire metabolism to support cell regeneration [14,15].TP53 mutations are common in OC [16], and studies have linked loss-of-p53 functions with increased carcinoma cell proliferation and cancer progression [17].Paradoxically, however, in humans, the prevalence of TP53 mutations among non-malignant epithelial cells can be high [18], suggesting the existence of factors that support p53-growth-suppressing activities despite the mutations.
The target of rapamycin (TOR) (including its mammalian ortholog, mTOR) is a serine/threonine kinase that is essential for cell proliferation, cell survival, and autophagy [19].mTOR phosphorylation of ribosomal-6-kinase leads to activation of ribosomal protein S6 and translation initiation factor eIF4B [20], which ultimately regulates protein translation [21].mTOR can be activated by signal transduction pathways downstream of Ras [22], and chemical biology approaches have been used to target mTOR in cancers [23].For instance, inhibition of mTOR with the rapamycin derivative everolimus increases cisplatin sensitivity of cancer cells upon p53 pathway restoration [24], implying that a combination of mTOR inhibition and reactivation of wild-type functions in mutant p53 proteins might provide an additional strategy for increasing the sensitivity of cancer cells to standard forms of therapy, such as cisplatin.
In this study, we report levels of intracellular pools of the free amino acid taurine in ascites cells derived from OC patients not previously administered chemotherapeutic treatment.Mimicking intracellular taurine levels in cultured cell lines and supplementing ovarian patient-derived organoids with taurine revealed a taurine-mediated increase in cell size, inhibition of proliferation, suppression of glycolysis, and cell protection from the DNA damage agent cisplatin.Combined RNA sequencing, reverse-phase protein array (RPPA), Western blotting, and flow cytometry analysis of taurine-supplemented cell cultures underscored p53, ERK, mTOR/DNAPK/ATM/ATR pathway activation and suppression of cisplatin-induced DNA damage (as measured by γH2AX and DNAPK phosphorylation).Inhibition of mTOR/DNAPK/ATM/ATR (Torin2), but not MEK (PD03255901), S6K1 (PF-4708671) or p53 expression (shRNA) induced the death of taurine-supplemented cultures in the presence of cisplatin.Our data support the idea that elevation of the intracellular freeamino acid taurine can activate growth-suppressing and cell-protecting signal transduction pathways involving p53 and mTOR/DNAPK/ATM/ATR.

Patient Ascites
For a study from 2021 to 2022, we enrolled Caucasian patients with histologically confirmed high-grade serous OC and ascites in the Department of Gynecologic Oncology, Pozna ń University of Medical Sciences.Ovarian tumors were staged according to the FIGO (International Federation of Gynecology and Obstetrics) system.Ascites fluid cell samples were collected from patients (i) at laparoscopy before starting neoadjuvant chemotherapy.Ascites fluid (10 mL) was centrifuged within 2 h after collection in a falcon tube at 1100× g for 10 min at room temperature to separate a cell pellet.The supernatant and malignant cell pellets were stored at −80 • C and analyzed collectively.

Mass-Spectrometry-Based Quantification of Taurine Levels in Cell Lysates
The panel of amino acids was quantified based on aTRAQ kit for amino acid analysis (SCIEX, Framingham, MA, USA) and liquid chromatography coupled to a triple quadrupole tandem mass spectrometry technique.The samples (cell lysates) were thawed at room temperature, and 40 µL of a matrix was transferred to a 0.5 mL Eppendorf tube.Then, 10 µL of sulfosalicylic acid was added to precipitate the proteins, and the vial contents were mixed and centrifuged.Subsequently, 10 µL of supernatant was transferred to a clean tube, and 40 µL of borate buffer was added, mixed, and centrifuged.In the next step, 10 µL of the obtained mixture was transferred to a clean tube and mixed with 5 µL of aminolabeling reagent (aTRAQ Reagent ∆8 ® , Framingham, MA, USA).After centrifugation, samples were incubated for 30 min at room temperature.The incubation was followed by the addition of 5 µL of hydroxylamine solution, mixing and centrifugation.Then, the samples were incubated for 15 min at room temperature.In the next step, 32 µL of freshly prepared internal standards solution was added, mixed up, and centrifuged.The contents of the tubes were concentrated (temperature 50 • C for about 15 min) to a volume of about 20 µL using a vacuum concentrator (miVac Duo, Genevac, Stone Ridge, NY, USA).In the last step, 20 µL of ultrapure water was added to each vial and mixed.The contents of the tubes were transferred to amber-glass autosampler vials with inserts.Samples were analyzed in random order by chromatographic separation followed by tandem mass spectrometry detection LC-MS/MS.The analytes were separated on a Sciex C18 column (4.6 mm × 150 mm, 5 µm) maintained at 50 • C using a 1260 Infinity HPLC instrument (Agilent Technologies, Santa Clara, CA, USA).A gradient flow of the mobile phase was applied.The mobile phase consisted of 0.1% formic acid (FA) and 0.01% heptafluorobutyric acid (HFBA) in water (phase A) and 0.1% FA and 0.01% HFBA in methanol (phase B) maintained at a flow rate of 800 µL/min.Total runtime was 18 min per sample, with injection volume equal to 2 µL.Detection and quantitation of analytes were performed by means of a quadrupole tandem mass spectrometer with an electrospray ionization (ESI) TurboV ion source operated in positive ion mode.All results were generated in a scheduled multiple-reaction monitoring mode.Raw data from amino acid assays were acquired and analyzed using Analyst software version 1.6.3(Sciex, Framingham, MA, USA).The method validation and sample preparation have been described in detail previously [26].

Quantification of Taurine Levels in Tissue Culture Cell Lysates Using a Commercial Kit
To determine intracellular taurine concentrations, a taurine assay kit (Cell Biolabs, Inc., San Diego, CA, USA) was used.Cells were treated with taurine for the indicated amount of time, washed three times with PBS, and lysed in ice-cold RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) in order to extract intracellular contents; protein content was determined using a BCA assay.Then, 25 µg of protein was loaded into a 96-well plate in triplicate for each sample.Taurine concentration was determined using the taurine assay kit following the manufacturer's instructions, and absorbance values were read at 405 nm using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).Absorbance values were then background-subtracted and normalized to the average absorbance value of the control group.Values were reported as fold change in intracellular taurine concentration.
The medium ± taurine was fully changed every 2-3 days.For organoid collection and plating, 20-50 µm organoids were extracted from the Cultrex BME droplet using cold cell recovery solution containing 10 mg/mL DNase I at 4 • C for 30 min.The dissolved Cultrex BME containing intact organoids was then collected into 2-3 × volume of cold advanced DMEM/F12 containing 0.1% BSA (Sigma-Aldrich) and 10 mg/mL DNase I and centrifuged at 200-250× g for 5 min.The organoid solution is resuspended in cold ovarian tumor organoid medium containing 10% Cultrex BME with or without taurine.The organoid solution was counted prior to plating at 500 organoids/well in 384-well plate format.

Organoid Metrics
Bright-field images were acquired and analyzed using a Perkin Elmer Opera Phenix High-Content Screening System and Harmony high-content analysis software (4.9).The area of each organoid was measured with an automated quantification pipeline set up to identify well-defined organoid structures in the same focal plane of one single z-plane after selection based on image texture, single structure size, and morphology.

Organoid ATP Assay
CellTiter-Glo ® 3D Cell Viability Assay (Promega, Madison, WI, USA) was performed on patient-derived organoids (PDOs) to measure ATP in metabolically active cells at the specified experimental endpoint following the manufacturer's instructions.Briefly, CellTiter-Glo ® 3D Reagent was added to each well at 1:1 ratio.Plates were shaken for 25 min, and luminescence was measured with a CLARIOstar ® Plus reader (BMG LABTECH, Ortenberg, Germany).

Cell Proliferation Analysis
Cell proliferation was determined using an automated cell counter or by live cell imaging.To assess proliferation using an automated cell counter, cells were first seeded into a 12-well plate.The following day, three wells per condition were treated as indicated, and another three wells were trypsinized and counted using a Luna II Automated Cell Counter (Logos Biosystems, Annandale, VA, USA).After 72 h of treatment, cells were once again harvested and counted, and cell numbers were normalized to the average value of the initial count and reported as fold change in cell number.
To determine proliferation by live cell imaging, GFP-expressing FNE-m-p53 cells were seeded in a 6-well plate and captured over time using a BioTek Lionheart FX Automated Microscope (BTLFX, Biotek, Winooski, VT, USA).Images were analyzed using the ImageJ (National Institutes of Health, Bethesda, MD, USA) TrackMate plugin.

Cell Volume and Adhesion Area Quantification
GFP-expressing FNE-m-p53 cells were seeded in a 6-well plate and incubated overnight.The following day, cells were treated as indicated and incubated for 72 h.Cells were then trypsinized and seeded into a low-adhesion 96-well round bottom plate at a density of 100 cells per well.The plate was then centrifuged at 900 rpm for 5 min, and cell clusters were immediately imaged using a BTLFX.Images were captured using a 10×objective in the GFP-and phase-contrast channels.To determine cell volume, GFP images were processed in FIJI (Version 2.14) using an in-house macro.Briefly, images were backgroundsubtracted, and an automated threshold was applied.Images were then converted to binary and a watershed was applied to separate adjacent cells.The area of each particle (cell) was then measured.From this value, the radius of each cell was determined, and volume was estimated from the equation for the volume of a sphere (V = 4/3 π r 3 ).Between 15-20 wells were analyzed per condition, and the average volume of a cell per well was reported.
To quantify cell adhesion area, cells were seeded and treated as described for the proliferation assay.At the endpoint, images were captured using a BTLFX with a 10X objective in the GFP-and phase-contrast channels.Then, as described for the fluorescence microscopy cell proliferation assay, images were processed in FIJI with an in-house macro, and the area of each particle (cell) was reported as the cell adhesion area.

Basement Membrane Reconstitution and Spheroid Size Quantification
To examine the effects of taurine on reconstituted basement membrane organotypic structures, FNE-m-p53, Hey-A8, and OV90 cells were reconstituted in Matrigel ® Growth Factor Reduced Basement Membrane Matrix (MG) (Corning Corning NY, USA), as previously described [28].A total of 100 cells were seeded per well in a 96-well ultra-lowadhesion Nunclon Sphera™ plate, centrifuged at 900 rpm for 5 min, and incubated overnight.The following day, on ice, MG was added to cell culture media to a concentration of 4% v/v.MG solution was then added to each well containing 100 µL of clustered spheroids to a final volume of 200 µL and an MG concentration of 2% v/v.Zprojections of organotypic structures were captured at the indicated time points using a BTLFX using the 4X objective in the bright-field and GFP channels.Z-projections were processed into focused stacks using Gen5 software (Version 3.11), and the area of each structure was measured using FIJI (Version 2.14).

RNA Isolation and Targeted RT-qPCR Analysis
Total RNA was isolated using Quick-RNA miniprep Kit (Zymo Reasearch, Irvine, CA, USA) and reverse-transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA) following the manufacturer's instructions.To analyze mRNA expression, 10 ng of cDNA was added to a 10 µL reaction mixture containing Power SYBR Green Master Mix (Applied Biosystems) and primers at a concentration of 330 nM designed to detect the transcript of interest.mRNA levels were quantified using a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and mRNA expression was normalized to a housekeeping gene.

RNA Sequencing and Transcriptomic Analysis
RNA extraction was performed using an E.Z.N.A. ® total RNA kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer's protocol.RNA concentration and purity were assessed with a NanoDrop 2000 (ThermoFisher).RNA samples were stored in a −80 • C freezer and subsequently shipped on dry ice to Genewiz (Azenta US, South Plainfield, NJ, USA) for RNA sequencing.The RNA sequencing library preparation workflow started with PolyA-based mRNA enrichment, mRNA fragmentation, and random priming followed by first-and second-strand complementary DNA (cDNA) synthesis.Subsequently, end-repair with 5 ′ phosphorylation and adenine (dA)-tailing was carried out.Finally, adaptor ligation, PCR enrichment, and Illumina HiSeq 2500 sequencing with two 150-base-pair (bp) runs were performed, and sequence reads were mapped to the reference genome.The bioinformatics analysis workflow started with evaluation of sequence quality and trimming the reads by means of Trimmomatic v.0.36 software to remove possible adapter sequences and nucleotides of poor quality.STAR aligner v.2.5.2b software was used to map the trimmed reads to the Homo sapiens GRCh38 reference genome available on ENSEMBL.To extract gene hit counts, feature counts from the Subread package v.1.5.2. were used.The hit counts were reported based on the gene ID feature in the annotation file.Only reads that were within the exon regions were considered.The table generated from extracted gene hit counts was used for differential expression analysis.Using DESeq2, a comparison of gene-expression between the taurine-treated and control samples was performed.By using the Wald test, p-values and log2-fold changes were calculated.Genes with an adjusted p-value (Padj) of less than 0.05 and absolute log2-fold change more than 1 were counted as differentially expressed genes (DEGs).
Gene ontology analysis was applied on the statistically significant genes with GeneSCF v.1.1-p2software.The goa_human GO list was referenced to cluster the set of genes based on their biological functions and determine their statistical significance.Gene set enrichment analysis (GSEA) was performed, as described earlier [29], with GSEA_4.2.2.software, against selected gene sets from Molecular Signatures Database (MSigDB 7.5).GSEA was performed with the ranking metric set to Signal2noise and with the number of permutations set to 1000.Plots with a false discovery rate (FDR) of less than 0.25 were considered significant.

Flow Cytometry
DNA staining (cell cycle analysis) by flow cytometry was performed as follows.Cells were harvested by trypsinization, collected by centrifugation, and washed twice with PBS.Cell pellets were then fixed with ice-cold 70% ethanol for at least 30 min on ice.Cells were then washed twice with PBS and treated with PBS containing 100 µg/mL RNAse A (Sigma-Aldrich) and 2 µg/mL propidium iodide (PI) (Molecular Probes, Eugene, OR, USA) for 30 min at room temperature in the dark before analysis.Data acquisition was performed using an Attune NxT Flow Cytometer (ThermoFisher).Data were then analyzed using FlowJo software (Version 10.10.0,FlowJo, Ashland, CA, USA) To determine cell viability, the cell culture supernatant was collected, then adherent cells were trypsinized and centrifuged at 300× g for 5 min.Cells were washed twice with PBS containing 2% FBS.Cells were then incubated on ice with PBS containing 2% FBS and 2 µg/mL PI.Data were then acquired by flow cytometry analyzed using FlowJo, and viability was reported as the percentage of PI-positive cells.
To stain for phosphorylated γH2AX, cells were treated for 48 h and collected by trypsinization.Cells were washed and fixed in 4% paraformaldehyde (PFA) for 15 min, followed by permeabilization in ice-cold methanol for 30 min.Cells were stained with Phospho-Histone H2A.X primary antibody (Cell Signaling Technology #2577) at a dilution of 1:200 in 0.5% BSA dissolved in PBS for one hour at room temperature.Cells were then washed and stained with Alexa Fluor 568 secondary antibody (Invitrogen A-11011) at a dilution of 1:500.Cells were then analyzed by flow cytometry.

Lentivirus Production
To generate lentivirus, packaging plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) were mixed with a lentiviral plasmid containing a gene of interest or shRNA and incubated with Lipofectamine 3000 (Invitrogen) in serum-free Opti-MEM (Gibco) to generate liposomes containing plasmid DNA.The mixture was then added to HEK293T cells, and the medium was refreshed the following day.Cell culture supernatant containing viral particles was collected 48 h and 72 h after transfection.Cell lines were then transduced by adding viral particles to media containing polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 10 µg/mL and selected with an appropriate antibiotic 24 to 48 h after transduction in order to generate cells stably expressing transgene or shRNA.

Reverse-Phase protein Array
Adherent FNE-m-p53 cells were cultured in triplicate for 72 h in control media or with media supplemented with taurine.Cells were then washed three times on ice and then lysed in RPPA lysis buffer (1% Triton-X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl.1.5 MgCl 2 1 mM EGTA, 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1 mM Na 3 VO 4 , and Halt Protease Inhibitor (Thermo Fisher).Lysates were incubated on ice for 15 min and then centrifuged at 13,000× g for ten minutes.Following protein quantification, lysates were then diluted to an equal concentration using RPPA buffer before being boiled and denatured using 1% SDS for ten minutes.Lysates were then snap-frozen and shipped on dry ice to MD Anderson Cancer Center for RPPA analysis.

Analysis of Glycolysis
The extracellular acidification rate (ECAR) was measured using an XFp Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA).CaOv3 cells were exposed to taurine for 48 h and then reseeded (8000 cells/well) in taurine-containing media into the wells of an XFp Cell miniplate.Then, 24 h later, the cells were analyzed using an XFp Glycolysis Stress Test Kit (Seahorse Bioscience, North Billerica, MA, USA) following the manufacturer's instructions.
Cells were washed 3 times with 100 µL of XF Base Media (Seahorse Bioscience, North Billerica, MA, USA) containing 2 mM L-Glutamine.Then, the cells were incubated in 180 µL of the same media for 20 min.After three baseline ECAR measurements, cells were subsequently injected with 10 mM glucose, 1 µM oligomycin, and 50 mM 2-deoxyglucose (glycolysis inhibitor).
After both assays, the data obtained were normalized to protein concentrations determined by the bicinchoninic acid (BCA) assay (Pierce, Thermo Fisher Scientific, Waltham, MA, USA).Wave (Agilent Technology, Santa Clara, CA, USA) and GraphPad Prism 8 software were used to analyze and plot the data.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).Statistical significance was determined using an unpaired, twotailed, parametric t-test; an ordinary one-way ANOVA with Tukey's post hoc multiple comparisons test; or a two-way ANOVA with Sidak's post hoc multiple comparisons test, with p ≤ 0.05 considered statistically significant.

Determination of Intracellular Taurine Levels in Patient-Derived and Tissue Culture Cells
To begin investigations of taurine's cell-protecting mechanisms, we first wanted to determine the taurine levels in cells derived from the ascites of OC patients.To do so, we utilized quantitative mass spectrometry to profile the intracellular levels of 36 free amino acids within OC ascites-derived cells obtained from six chemotherapy-naïve patients (Figure 1A).Our analysis established that taurine was one of the most abundant free amino acids among the selected intracellular pool of amino acids (Figure 1B).We found that intracellular taurine concentrations varied (45-900 µm) and averaged 430 µm.
Since our tissue culture conditions do not include taurine supplementations, and taurine is not a component of commonly used OC cell culture media, we wanted to know the basal intracellular taurine levels in tissue culture cells.We began our experiments using fallopian tube non-ciliated epithelial (FNE) cells expressing OC-driving mutant p53 R175H (m-p53) [30].We chose this cell model for several reasons.First, FNE cells are considered to represent precursor cells for most OCs [31].Second, mutations in the TP53 gene are nearly ubiquitous in OC disease [16].Third, the taurine transporter SLC6A6 is negatively regulated by wild-type p53 [32].Fourth, as opposed to a majority of OC cell lines, FNE-m-p53 cells represent a chemotherapy-naïve cell model, ruling out pre-existence of mechanisms of resistance to DNA-damaging agents.Using FNE-m-p53 as a cell culture model, we found that these cells contained, on average, 100 times less taurine compared to ascites-derived cells (Figure 1C).Supplementation of FNE-m-p53 culture media with 160 mM taurine was sufficient to increase intracellular taurine content to within in the range of ascites cells (Figure 1C) without causing cell death (Figure 1D,E).Our data suggest that taurine is one of the most abundant intracellular amino acids in ascites cells isolated from OC patients and that mimicking physiologic intracellular taurine concentrations in cell culture is not toxic and can be accomplished by addition of taurine to media.Since our tissue culture conditions do not include taurine supplementations, rine is not a component of commonly used OC cell culture media, we wanted to k basal intracellular taurine levels in tissue culture cells.We began our experimen fallopian tube non-ciliated epithelial (FNE) cells expressing OC-driving mutant (m-p53) [30].We chose this cell model for several reasons.First, FNE cells are con to represent precursor cells for most OCs [31].Second, mutations in the TP53 g nearly ubiquitous in OC disease [16].Third, the taurine transporter SLC6A6 is ne regulated by wild-type p53 [32].Fourth, as opposed to a majority of OC cell lin m-p53 cells represent a chemotherapy-naïve cell model, ruling out pre-existence o anisms of resistance to DNA-damaging agents.Using FNE-m-p53 as a cell cultur we found that these cells contained, on average, 100 times less taurine compared to

shRNA-Mediated Attenuation of SLC6A6 Expression Decreases Intracellular Taurine Levels in FNE-m-p53 Cells
In most cells, taurine can be transported from outside via a sodium/chloride-dependent symporter, SLC6A6 [33].So, we examined the functional consequences of SLC6A6 expression in the taurine transport of tissue culture cells.We first measured SLC6A6 mRNA expression using RT-qPCR in FNE-m-p53 cells expressing a control plasmid (pLKO vector) or plasmids containing SLC6A6 shRNA sequences (G5 and G8).We found that both shRNA plasmids depleted expression of SLC6A6 mRNA, with G8 being more efficient (Figure 2A).We confirmed SLC6A6 shRNA G8-mediated knock-down using a combination of cell membrane protein extraction and Western blot analysis of FNE-m-p53 cell lysates (Figure 2B).To examine the functionality of SLC6A6 knock-down, we used a commercially available taurine assay kit from Cell Biolabs (MET-5071) that measures intracellular taurine levels.We found that shRNA-mediated attenuation of SLC6A6 using the G8 shRNA induced a three-fold decrease in taurine intracellular levels (Figure 2C).We verified these results using mass spectrometry and found that in FNE-m-p53 cells cultured in the presence of taurine, shRNA-mediated attenuation of SLC6A6 decreased intracellular taurine content from 130 µm to 48 µm (Figure 2D).Our data support the idea that in cultured cells, intracellular taurine accumulation is partially mediated by SLC6A6.
shRNA plasmids depleted expression of SLC6A6 mRNA, with G8 being more efficient (Figure 2A).We confirmed SLC6A6 shRNA G8-mediated knock-down using a combination of cell membrane protein extraction and Western blot analysis of FNE-m-p53 cell lysates (Figure 2B).To examine the functionality of SLC6A6 knock-down, we used a commercially available taurine assay kit from Cell Biolabs (MET-5071) that measures intracellular taurine levels.We found that shRNA-mediated attenuation of SLC6A6 using the G8 shRNA induced a three-fold decrease in taurine intracellular levels (Figure 2C).We verified these results using mass spectrometry and found that in FNE-m-p53 cells cultured in the presence of taurine, shRNA-mediated attenuation of SLC6A6 decreased intracellular taurine content from 130 µM to 48 µM (Figure 2D).Our data support the idea that in cultured cells, intracellular taurine accumulation is partially mediated by SLC6A6.

Taurine Supplementation Inhibits OC Cell Proliferation in Tissue Culture
Our data suggest that the addition of exogenous taurine to culture media can elevate intracellular taurine in cultured cells to levels similar to cells in ascites without causing cell death (Figure 1D,E).Based on these data, we decided to propagate FNE-m-p53 and various OC cells in media containing 160 mM taurine, which can increase taurine intracellular levels to those observed in ascites-derived cells (Figure 2D).Live-cell phase-contrast micrography of taurine-supplemented FNE-m-p53 cell monolayer cultures showed fewer cells undergoing cell division and an increase in the cell adhesion area (Figure S1A-C, MOVIE S1).Because taurine is an osmolyte and intracellular accumulation of osmolytes increases cell size and promotes G1 arrest [34], we hypothesized that elevation of intracellular taurine levels suppresses the proliferation of cultured cells.To test this hypothesis, we assessed the proliferation of multiple cell lines and cell culture models supplemented with taurine.We found that taurine supplementation suppressed cell proliferation in all tested cell lines, which were cultured as monolayers or in some cases under conditions of three-dimensional reconstituted basement membranes (Figure 3A-D).We also observed

Taurine Supplementation Inhibits OC Cell Proliferation in Tissue Culture
Our data suggest that the addition of exogenous taurine to culture media can elevate intracellular taurine in cultured cells to levels similar to cells in ascites without causing cell death (Figure 1D,E).Based on these data, we decided to propagate FNE-m-p53 and various OC cells in media containing 160 mM taurine, which can increase taurine intracellular levels to those observed in ascites-derived cells (Figure 2D).Live-cell phase-contrast micrography of taurine-supplemented FNE-m-p53 cell monolayer cultures showed fewer cells undergoing cell division and an increase in the cell adhesion area (Figure S1A-C, Movie S1).Because taurine is an osmolyte and intracellular accumulation of osmolytes increases cell size and promotes G1 arrest [34], we hypothesized that elevation of intracellular taurine levels suppresses the proliferation of cultured cells.To test this hypothesis, we assessed the proliferation of multiple cell lines and cell culture models supplemented with taurine.We found that taurine supplementation suppressed cell proliferation in all tested cell lines, which were cultured as monolayers or in some cases under conditions of three-dimensional reconstituted basement membranes (Figure 3A-D).We also observed that growth suppression by taurine was associated with cell cycle arrest (Figure S1D,E), suggesting the activation of cell cycle inhibitory programs.Interestingly, taurine washout demonstrated phenotype reversal (Figure S1F), supporting the idea that maintaining taurine pools is required for the suppression of proliferation.Nevertheless, we were motivated by these experiments and wanted to determine whether taurine supplementation suppresses the growth of patient-derived organoids (PDOs).We provide evidence that taurine significantly decreased the growth of various PDOs (Figure 4A,B).Taurine's PDO growth suppression was associated with a decrease in ATP production as measured by CellTiter-Glo ® assays (Figure 4C).Our data support the idea that taurine intracellular accumulation is associ-ated with suppression of cell proliferation and the activation of cell cycle and metabolic regulatory mechanisms.
rine pools is required for the suppression of proliferation.Nevertheless, we were motivated by these experiments and wanted to determine whether taurine supplementation suppresses the growth of patient-derived organoids (PDOs).We provide evidence that taurine significantly decreased the growth of various PDOs (Figure 4A,B).Taurine's PDO growth suppression was associated with a decrease in ATP production as measured by CellTiter-Glo ® assays (Figure 4C).Our data support the idea that taurine intracellular accumulation is associated with suppression of cell proliferation and the activation of cell cycle and metabolic regulatory mechanisms.

Taurine Promotes Mutant and WT-p53 Binding to DNA for the Activation of p53-Mediated Cyclin-Dependent Kinase 1A (p21) Expression
To gain mechanistic insight into taurine's suppression of growth and metabolism, we performed transcriptomic analysis of FNE-m-p53 cell monolayer cultures supplemented with taurine and found activation of the p53 pathway (Figure 5A).These results encouraged us to test whether taurine induces mutant p53 binding to DNA.We used a commercially available p53-DNA binding assay kit (Cayman, #600020) that measures the extent of p53 protein interaction with DNA p53 response elements (Figure 5B).As a negative control, we used a nuclear extract of CaOv3 cells, which do not express p53, and a positive control was represented by treatment of MCF7 cells (expressing WT p53) with Nutlin (a p53 protein stabilizer).We found that taurine increased the interaction of various mutant p53 proteins with DNA (Figure 5B).

Taurine Promotes Mutant and WT-p53 Binding to DNA for the Activation of p53-Mediated Cyclin-Dependent Kinase 1A (p21) Expression
To gain mechanistic insight into taurine's suppression of growth and metabolism, we performed transcriptomic analysis of FNE-m-p53 cell monolayer cultures supplemented with taurine and found activation of the p53 pathway (Figure 5A).These results encouraged us to test whether taurine induces mutant p53 binding to DNA.We used a commercially available p53-DNA binding assay kit (Cayman, #600020) that measures the extent of p53 protein interaction with DNA p53 response elements (Figure 5B).As a negative control, we used a nuclear extract of CaOv3 cells, which do not express p53, and a positive control was represented by treatment of MCF7 cells (expressing WT p53) with Nutlin (a p53 protein stabilizer).We found that taurine increased the interaction of various mutant p53 proteins with DNA (Figure 5B).To extend these studies to living cells, we engineered, based on el-Deiry's p21 reporter [35], a live p21-mCherry promoter nuclear biosensor that produces fluorescence in living cells through the engagement of a p53 response element.As a control, we used a CSII-mCherry plasmid, which expresses nuclear mCherry through activity of the EF1-α promoter (Addgene #125262).We found, using flow cytometry, that taurine supplementation to OC cells expressing WT p53 (Hey-A8) or mutant p53 (OVCAR4) increased p21 promoter activity, but not EF1-α promoter activity (Figure 5C).Consistent with these data, Western blot analysis demonstrated taurine-induced p21 protein regulation in a panel of cell lines expressing various mutant p53 proteins or WT p53 protein (Figure 5D).Our findings prompted us to evaluate whether taurine-induced growth suppression involved activation of other cyclin-dependent kinase inhibitors implicated in the negative regulation of the cell cycle.We found that while taurine supplementation of FNE-m-p53 cell cultures induced p21 transcription, it did not induce C2KN2A (p14/p16) or CDKN1B (p27) transcription (Figure 5E).Furthermore, supplementation of FNE-m-p53 monolayers with an equimolar concentration of valine did not induce p21 activation, supporting taurine specificity in activation of the p53 pathway (Figure 5F).Importantly, shRNA-mediated depletion of mutant or WT-p53 expression attenuated taurine-induced p21 activation (Figure 5G).Furthermore, taurine failed to activate p21 in CaOv3 cells lacking p53 protein expression (Figure S2A).These results support the possibility that p53 (mutant or WT) is partially required for taurine-mediated activation of p21.
In addition to suppression of cell proliferation, we observed that taurine negatively regulated expression of genes supporting glycolysis (Figure 6A).Western blot analysis revealed activation of the TP53-inducible glycolysis and apoptosis regulator (TIGAR) (Figure 6B), a phosphatase that directly inhibits glycolysis [36].These results suggested a possible inhibition of glycolysis by taurine.To address this possibility, we examined whether taurine inhibits the extracellular acidification rate (ECAR) as a proxy of glycolytic activity.ECAR analysis of FNE-m-p53 cell monolayers reveled that taurine suppressed glycolysis and glycolytic capacity, the maximum rate of conversion of glucose to pyruvate/lactate (Figure 6C,D).Furthermore, we obtained similar results in TYK-nu cell monolayers supplemented with taurine (Figure S2B,C).Taken together, our results provide evidence that taurine promotes mutant or WT-p53 interaction with DNA, which is associated with the activation of suppressive mechanisms for growth and metabolism in cultured FNE and OC cells.

Taurine Supports Cell Survival under Genotoxic Stress
So far, our data are consistent with the idea that taurine supplementation reprograms cells into low-proliferation and reduced-energy metabolic states.Proliferating cells are usually metabolically active and more sensitive to genotoxic stress [37], suggesting a possibility that taurine supplementation, which we demonstrated inhibits proliferation and glycolysis, could protect cells from genotoxic stress.To test this possibility, we used a combination of live cell imaging, flow cytometry, and propidium-iodide (PI)-based measurements of cell death, taurine supplementation, and DNA damage agent (cisplatin) treatment.We found that 160 mM taurine supplementation protected FNE-m-p53, FNE cells (WT p53), and additional OC cell lines from genotoxic stress associated with cisplatin treatment (Figures 7A,B and S3A,B, (MOVIE S2).In accordance with these results, assessment of γH2AX expression (flow cytometry) and DNAPK phosphorylation (Western blot) in FNE-m-p53 cells revealed that taurine decreased cisplatin-induced DNA damage (Figure 7C-E).Furthermore, RNA-seq data analysis of taurine-supplemented FNE-m-p53 cells showed enrichment of genes associated with cell protection from cisplatin, as defined by multiple studies [38][39][40][41](Figure 7F).Our data support the idea that taurine activates p53 and decreases DNA damage to protect cells from genotoxic stress.

Taurine Supports Cell Survival under Genotoxic Stress
So far, our data are consistent with the idea that taurine supplementation reprograms cells into low-proliferation and reduced-energy metabolic states.Proliferating cells are usually metabolically active and more sensitive to genotoxic stress [37], suggesting a possibility that taurine supplementation, which we demonstrated inhibits proliferation and glycolysis, could protect cells from genotoxic stress.To test this possibility, we used a combination of live cell imaging, flow cytometry, and propidium-iodide (PI)-based measurements of cell death, taurine supplementation, and DNA damage agent (cisplatin) treatment.We found that 160 mM taurine supplementation protected FNE-m-p53, FNE cells (WT p53), and additional OC cell lines from genotoxic stress associated with cisplatin treatment (Figures 7A,B and S3A,B, (Movie S2)).In accordance with these results, assessment of γH2AX expression (flow cytometry) and DNAPK phosphorylation (Western blot) in FNEm-p53 cells revealed that taurine decreased cisplatin-induced DNA damage (Figure 7C-E).Furthermore, RNA-seq data analysis of taurine-supplemented FNE-m-p53 cells showed enrichment of genes associated with cell protection from cisplatin, as defined by multiple studies [38][39][40][41] (Figure 7F).Our data support the idea that taurine activates p53 and decreases DNA damage to protect cells from genotoxic stress.

Attenuation of p53 Does Not Affect Cell Protection by Taurine
To explore the possibility of p53 involvement in cell protection downstream of taurine, we used p53 shRNA to attenuate the expression levels of the p53 protein.Our experiment revealed that shRNA-mediated attenuation of mutant or WT-p53 in FNE-m-p53 or FNE cells decreased activation of p21 in response to taurine (Figure 5G).Next, we wondered whether loss of p53 uncouples taurine from cell protection.Our experiments demonstrated that attenuation of mutant or WT-p53 expression did not affect taurine's ability to protect cells from cisplatin (Figure 8A-D).These results suggest that taurine's protection of cells from cisplatin is independent of p53 activation.

Attenuation of p53 Does Not Affect Cell Protection by Taurine
To explore the possibility of p53 involvement in cell protection downstream of taurine, we used p53 shRNA to attenuate the expression levels of the p53 protein.Our experiment revealed that shRNA-mediated attenuation of mutant or WT-p53 in FNE-m-p53 or FNE cells decreased activation of p21 in response to taurine (Figure 5G).Next, we dered whether loss of p53 uncouples taurine from cell protection.Our experi demonstrated that attenuation of mutant or WT-p53 expression did not affect tau ability to protect cells from cisplatin (Figure 8A-D).These results suggest that tau protection of cells from cisplatin is independent of p53 activation.

Taurine Mediates Cell Protection through mTOR/DNAPK/ATM/ATR Signal Transdu
To identify possible cell protective mechanisms downstream of taurine, w formed reverse-phase protein array (RPPA) analysis to probe activation (express phosphorylation) of hundreds of proteins in response to taurine in FNE-m-p53 cell RPPA experiments revealed activation of several major signal transduction pathwa cluding S6K and ERK (Figure 9A).We confirmed these results using Western blot an (Figure 9B).Furthermore, our RNA-seq analysis indicated that intracellular accumu of taurine in FNE-m-p53 cells was associated with increased mRNA expression of s DNA damage repair genes, including the Phosphatidylinositol 3-kinase-related (PIKK) family members DNAPK, ATM and ATR (Figure 9C).Reconciling these r with the activation of S6K, a well-accepted effector of mTOR that also belongs to family, we hypothesized that inhibition of PIKK signaling sensitizes taurine-s mented cell cultures to cisplatin.To test this, we used Torin2, a clinically relevan competitive inhibitor of mTOR, DNAPK, and ATM/ATR activity [42].Treatment of

Taurine Mediates Cell Protection through mTOR/DNAPK/ATM/ATR Signal Transduction
To identify possible cell protective mechanisms downstream of taurine, we performed reverse-phase protein array (RPPA) analysis to probe activation (expression or phosphorylation) of hundreds of proteins in response to taurine in FNE-m-p53 cells.Our RPPA experiments revealed activation of several major signal transduction pathways, including S6K and ERK (Figure 9A).We confirmed these results using Western blot analysis (Figure 9B).Furthermore, our RNA-seq analysis indicated that intracellular accumulation of taurine in FNE-m-p53 cells was associated with increased mRNA expression of several DNA damage repair genes, including the Phosphatidylinositol 3-kinase-related kinase (PIKK) family members DNAPK, ATM and ATR (Figure 9C).Reconciling these results with the activation of S6K, a well-accepted effector of mTOR that also belongs to PIKK family, we hypothesized that inhibition of PIKK signaling sensitizes taurine-supplemented cell cultures to cisplatin.To test this, we used Torin2, a clinically relevant ATP-competitive inhibitor of mTOR, DNAPK, and ATM/ATR activity [42].Treatment of FNE-m-p53 monolayers with Torin2 abrogated phosphorylation of S6 ribosomal protein (Figure S3C), and sensitized taurine supplemented cultures to cisplatin (Figure 10A,B).However, inhibition of mTOR alone with rapamycin did not break taurine protection, and nor did inhibition of its downstream effector S6K1 with PF-4708671.Inhibition of ERK with the MEK inhibitor PD03255901 also failed to break taurine's protection from cisplatin (Figure S3D-H).Our data suggest that taurine protects cells from cisplatin through activation of DNA-damagesensing mechanisms, including DNAPK and ATM/ATR (Figure 11).However, inhibition of mTOR alone with rapamycin did not break taurine protection, and nor did inhibition of its downstream effector S6K1 with PF-4708671.Inhibition of ERK with the MEK inhibitor PD03255901 also failed to break taurine's protection from cisplatin (Figure S3D-H).Our data suggest that taurine protects cells from cisplatin through activation of DNA-damage-sensing mechanisms, including DNAPK and ATM/ATR (Figure 11).

Discussion
These studies will provide potentially new insights into the mechanisms growth suppression and cell protection by taurine, a non-proteogenic sulfonic amin present in the cytoplasm of ascites cells derived from OC patients.Mass-spectro based quantitative examination of intracellular taurine pools revealed taurine con tions ranging from 45 µM to 900 µM among ascites cells.These taurine levels w average 100-fold higher than taurine intracellular pools found in cultured cells.M ing ascites cell-associated intracellular taurine concentrations in cell culture supp cell proliferation and glycolysis and induced cell protection from genotoxic stress.T decreased cisplatin-mediated DNA damage, indicating that intracellular accumula of pro-survival mechanisms.Here, we provide evidence that taurine suppresses OC cell proliferation and activates a p53 effector, p21, a molecule directly involved in cell cycle arrest.However, p53 activation was not required for taurine-induced protection of cells from cisplatin, indicating that p53 is dispensable in taurine cell protection.
Taurine is an osmolyte [47], and recent results support the idea that accumulation of intracellular osmolytes promotes an increase in cell size and cell cycle arrest [34].Our cell culture data demonstrate that elevation of intracellular taurine to the levels found in cells isolated from OC ascites significantly increases OC cell size, and this phenotype is associated with suppression of proliferation.Based on these results, it is possible that one of the mechanisms supporting evolution of growth-suppressed tumor cell clones involves an influx of free amino acid osmolytes and cell size changes.
Recent in silico and cell-free biochemical studies [48] have demonstrated that taurine can directly bind cyclin-dependent kinase 6 (CDK6).This interaction appears to inhibit CDK6 activity in solution, supporting the possibility that intracellular taurine accumulation could directly suppress CDK6/cyclin D activity, which could lead to activation of p53 [49,50] and inhibition of proliferation.Our experiments also provide evidence that taurine increases mutant or wild-type p53 binding to DNA.There is existing biochemical evidence [51] that taurine interacts with casein proteins to change their conformation.Thus, it is possible that taurine could directly interact with p53 and change its conformation to support DNA binding and subsequent activation of effector molecules including p21 and TIGAR.However, taurine's ability to protect cells from cisplatin did not depend on p53 but required mTOR/DNAPK/ATM/ATR activity.These results suggested the idea that emergence of therapy-resistant cell clones is driven by the mechanisms of cell cycle suppression and the activation of major cell survival and DNA repair signal transduction pathways including mTOR/DNAPK/ATM/ATR.How taurine activates mTOR/DNAPK/ATM/ATR is not known, and our data do not support that taurine itself induces DNA damage (as measured by γH2AX levels), thus implying the existence of taurine-induced DNA-damageindependent mechanisms of transcriptional regulation of DNA-damage-sensing kinases.

Conclusions
In conclusion, our studies provide a possible mechanism whereby intracellular accumulation of taurine can activate p53 and mTOR/DNAPK/ATM/ATR signaling pathways to suppress cell growth and support the emergence of slowly proliferating cells resistant to DNA-damage-based therapies (Figure 11).We identified that taurine-mediated cisplatin protection was abolished by treatment with Torin2, a clinically relevant small-molecule inhibitor of mTOR/DNAPK/ATM/ATR.

024, 16 ,Figure 1 .
Figure 1.Modeling of intracellular taurine levels associated with OC in vitro.(A,B) Qu mass spectrometry workflow for the determination of free intracellular amino acid concen cells isolated from patient ascites; n = 6 patient samples.Data are reported as mean ± SD. spectrometry quantification of intracellular taurine concentration in FNE-m-p53 cells cu control media or media supplemented with 160 mM taurine.Each dot is one replicate.(D sentative histograms and (E) quantification of cell death for FNE-m-p53 cells treated with taurine or 10 µM cisplatin for 72 h.Viability was determined by propidium iodide (PI) stai quantified using flow cytometry.n = 3 replicates.Data are reported as mean ± SD and were using a one-way ANOVA.

Figure 1 .
Figure 1.Modeling of intracellular taurine levels associated with OC in vitro.(A,B) Quantitative mass spectrometry workflow for the determination of free intracellular amino acid concentration in cells isolated from patient ascites; n = 6 patient samples.Data are reported as mean ± SD. (C) Mass spectrometry quantification of intracellular taurine concentration in FNE-m-p53 cells cultured in control media or media supplemented with 160 mM taurine.Each dot is one replicate.(D) Representative histograms and (E) quantification of cell death for FNE-m-p53 cells treated with 160 mM taurine or 10 µm cisplatin for 72 h.Viability was determined by propidium iodide (PI) staining and quantified using flow cytometry.n = 3 replicates.Data are reported as mean ± SD and were analyzed using a one-way ANOVA.

Figure 2 .
Figure 2. Intracellular taurine accumulation is mediated by the SLC6A6 transporter.(A) RT-q-PCR analysis of SLC6A6 mRNA expression in FNE-m-p53 cells transduced with scramble shRNA (pLKO) or SLC6A6 shRNA (G5 and G8).Each dot is one replicate.Statistical analysis was performed using a one-way ANOVA.(B) Western blot of SLC6A6 expression in FNE-m-p53 cells transduced with scramble shRNA or SLC6A6 shRNA G8. (C) Fold change in intracellular taurine content following treatment with control media or media containing 160 mM taurine for 72 h.Taurine concentration was determined using a Cell Biolabs taurine assay kit.Each dot is one replicate.Statistical analysis was performed using a one-way ANOVA comparing no taurine vs. taurine groups.The line indicates the median.(D) Mass spectrometry quantification of intracellular taurine content in cells treated with 160 mM taurine for 72 h.Each dot is one replicate.

Figure 2 .
Figure 2. Intracellular taurine accumulation is mediated by the SLC6A6 transporter.(A) RT-q-PCR analysis of SLC6A6 mRNA expression in FNE-m-p53 cells transduced with scramble shRNA (pLKO) or SLC6A6 shRNA (G5 and G8).Each dot is one replicate.Statistical analysis was performed using a one-way ANOVA.(B) Western blot of SLC6A6 expression in FNE-m-p53 cells transduced with scramble shRNA or SLC6A6 shRNA G8. (C) Fold change in intracellular taurine content following treatment with control media or media containing 160 mM taurine for 72 h.Taurine concentration was determined using a Cell Biolabs taurine assay kit.Each dot is one replicate.Statistical analysis was performed using a one-way ANOVA comparing no taurine vs. taurine groups.The line indicates the median.(D) Mass spectrometry quantification of intracellular taurine content in cells treated with 160 mM taurine for 72 h.Each dot is one replicate.

Figure 3 .
Figure 3. Taurine supplementation suppresses the growth of OC cell lines.(A) Proliferation of OC cell lines was quantified by cell counting.OC cells were treated with 160 mM taurine for 72 h.Data were normalized to the mean cell count for each respective control group and are reported as mean ± SD.Each dot represents one replicate count.Statistical analysis was performed using Student's ttest.(B-D) Bright-field microscopy images of reconstituted basement membrane organotypic structures.The size of each structure was quantified based on area.Each dot represents one structure, and the bar is the mean.Statistical analysis was determined using Student's t-test.Scale bars are 50 µm.

Figure 3 .
Figure 3. Taurine supplementation suppresses the growth of OC cell lines.(A) Proliferation of OC cell lines was quantified by cell counting.OC cells were treated with 160 mM taurine for 72 h.Data were normalized to the mean cell count for each respective control group and are reported as mean ± SD.Each dot represents one replicate count.Statistical analysis was performed using Student's t-test.(B-D) Bright-field microscopy images of reconstituted basement membrane organotypic structures.The size of each structure was quantified based on area.Each dot represents one structure, and the bar is the mean.Statistical analysis was determined using Student's t-test.Scale bars are 50 µm.

Figure 4 .
Figure 4. Taurine supplementation suppresses the growth of patient-derived organoids.(A) Lowand high-(inset) magnification bright-field images of entire wells representing three PDO cultures at day 0 and day 10.Inset scale bar is 200 µm.(B) Quantification of single organoid area at day 0 and day 10.Some 100-700 organoids were quantified per group.Each dot represents one organoid.(C) End-point measurement of ATP levels using a CellTiter-Glo ® assay.Each dot represents a luminescence value recorded from one well.Unpaired, non-parametric two-tailed t-tests with Welch's correction were used to determine the difference between groups.

Figure 4 .
Figure 4. Taurine supplementation suppresses the growth of patient-derived organoids.(A) Low-and high-(inset) magnification bright-field images of entire wells representing three PDO cultures at day 0 and day 10.Inset scale bar is 200 µm.(B) Quantification of single organoid area at day 0 and day 10.Some 100-700 organoids were quantified per group.Each dot represents one organoid.(C) End-point measurement of ATP levels using a CellTiter-Glo ® assay.Each dot represents a luminescence value recorded from one well.Unpaired, non-parametric two-tailed t-tests with Welch's correction were used to determine the difference between groups.

Figure 5 .
Figure 5. Taurine promotes mutant and wild-type p53 binding to DNA and p53-mediated p21 activation.(A) Differential transcriptomic analysis of the TP53 gene pathway in FNE-m-p53 cell monolayers grown under normal conditions or in media supplemented with taurine.(B) Quantification of p53 binding to its response element using a p53 Transcription Factor Assay Kit (Cayman).For each cell line, data were normalized to the mean of the respective control group and are reported as a fold change.(C) Histogram representation of flow cytometry-based analysis of taurine-induced

Figure 5 .
Figure 5. Taurine promotes mutant and wild-type p53 binding to DNA and p53-mediated p21 activation.(A) Differential transcriptomic analysis of the TP53 gene pathway in FNE-m-p53 cell monolayers grown under normal conditions or in media supplemented with taurine.(B) Quantification of p53 binding to its response element using a p53 Transcription Factor Assay Kit (Cayman).For each cell

Figure 6 .
Figure 6.Taurine activates genes involved in the suppression of glycolysis and attenuates glycolytic capacity.(A) Enrichment analysis of transcripts corresponding to genes implicated in the regulation of glycolysis in FNE-m-p53 cells cultured in taurine.(B) Western blot of TIGAR and p21 expression in FNE-m-p53 cells cultured in the absence or presence of taurine.(C) Analysis of ECAR over time showing glycolysis and glycolytic capacity.(D) Bar graph shows average glycolytic capacity with standard deviations (maximal whiskers) across three experiments.An unpaired, two-tailed t-test was used to determine statistical differences between the means.2-DG: 2-deoxy-D-glucose.

Figure 6 .
Figure 6.Taurine activates genes involved in the suppression of glycolysis and attenuates glycolytic capacity.(A) Enrichment analysis of transcripts corresponding to genes implicated in the regulation of glycolysis in FNE-m-p53 cells cultured in taurine.(B) Western blot of TIGAR and p21 expression in FNE-m-p53 cells cultured in the absence or presence of taurine.(C) Analysis of ECAR over time showing glycolysis and glycolytic capacity.(D) Bar graph shows average glycolytic capacity with standard deviations (maximal whiskers) across three experiments.An unpaired, two-tailed t-test was used to determine statistical differences between the means.2-DG: 2-deoxy-D-glucose.

Figure 7 .
Figure 7. Taurine protects cells both mutant and wild-type p53 expressing cells from cisplatin-induced cell death and DNA damage.(A,B) Representative histograms and corresponding bar graphs of cell viability for FNE-m-p53 and FNE WT cells cultured in taurine, cisplatin, or combination for 72 h.Viability was determined by PI incorporation and quantified using flow cytometry.(C) Representative histograms and (D) quantification of phosphorylated γH2AX staining in FNE-m-p53 cells cultured in taurine, cisplatin, or both for 72 h.Each dot is one replicate.Data are normalized to the mean fluorescence of the control group.The p-value differentiates cisplatin vs. taurine + cisplatin, as determined by a one-way ANOVA.(E) Western blot of phosphorylated and total DNA-PKcs protein levels in FNE-m-p53 treated for 72 h in the indicated conditions.(F) Enrichment analysis of gene sets associated with cisplatin resistance published in the indicated studies [38-41].Taurine treatment of FNE-m-p-53 cells positively correlates with cisplatin resistance.

Figure 7 .
Figure 7. Taurine protects cells both mutant and wild-type p53 expressing cells from cisplatin-induced cell death and DNA damage.(A,B) Representative histograms and corresponding bar graphs of cell viability for FNE-m-p53 and FNE WT cells cultured in taurine, cisplatin, or combination for 72 h.Viability was determined by PI incorporation and quantified using flow cytometry.(C) Representative histograms and (D) quantification of phosphorylated γH2AX staining in FNE-m-p53 cells cultured in taurine, cisplatin, or both for 72 h.Each dot is one replicate.Data are normalized to the mean fluorescence of the control group.The p-value differentiates cisplatin vs. taurine + cisplatin, as determined by a one-way ANOVA.(E) Western blot of phosphorylated and total DNA-PKcs protein levels in FNE-m-p53 treated for 72 h in the indicated conditions.(F) Enrichment analysis of gene sets associated with cisplatin resistance published in the indicated studies [38-41].Taurine treatment of FNE-m-p-53 cells positively correlates with cisplatin resistance.

Figure 8 .
Figure 8. Taurine-induced rescue from cisplatin is not p53-dependent.(A,B) Viability of FNE and FNE (WT p53) cell lines as determined by PI staining and quantified using flow cytometr were transduced with pLKO scramble shRNA or p53 shRNA and treated with taurine, cispl both for 72 h.Each dot represents one replicate.The p-values represent cisplatin vs. taurine + tin for each respective shRNA and were determined by a one-way ANOVA.(C,D) Represe histograms from (A,B).The percentage of PI-positive cells is indicated.

Figure 8 .
Figure 8. Taurine-induced rescue from cisplatin is not p53-dependent.(A,B) Viability of FNE-m-p53 and FNE (WT p53) cell lines as determined by PI staining and quantified using flow cytometry.Cells were transduced with pLKO scramble shRNA or p53 shRNA and treated with taurine, cisplatin, or both for 72 h.Each dot represents one replicate.The p-values represent cisplatin vs. taurine + cisplatin for each respective shRNA and were determined by a one-way ANOVA.(C,D) Representative histograms from (A,B).The percentage of PI-positive cells is indicated.

Figure 9 .
Figure 9. Proteomic and transcriptomic analysis of taurine response.(A) Reverse-phase protein array analysis of FNE-m-p53 cells treated with control culture media or media supplemented with 160 mM taurine.Arrows indicate mTOR activity (phosphorylated S6 ribosomal protein) and phosphorylated MAPK/ERK.(B) Western blot of phosphorylated S6 ribosomal protein and phosphorylated ERK for FNE-m-p53 treated with taurine.(C) RNA-seq heatmap for DNA damage response

Figure 9 .
Figure 9. Proteomic and transcriptomic analysis of taurine response.(A) Reverse-phase protein array analysis of FNE-m-p53 cells treated with control culture media or media supplemented with 160 mM taurine.Arrows indicate mTOR activity (phosphorylated S6 ribosomal protein) and phosphorylated MAPK/ERK.(B) Western blot of phosphorylated S6 ribosomal protein and phosphorylated ERK for

Figure 10 .
Figure 10.Torin2 sensitizes taurine-treated cells to cisplatin.(A) Representative histogra quantification of PI incorporation for FNE-m-p53 cells treated under the indicated condition h.Cells were treated with Torin2 (1 µM) for four hours before exposure to cisplatin (10 µ Representative histograms and quantification of PI incorporation for OVCAR4 cells treated the indicated conditions for 24 h.Data are reported as mean ± SD. p-values represent DMS platin vs. DMSO + taurine + cisplatin and Torin2 + cisplatin vs. Torin2 + taurine + cisplatin.St analysis was performed using a two-way ANOVA.

Figure 10 .
Figure 10.Torin2 sensitizes taurine-treated cells to cisplatin.(A) Representative histograms and quantification of PI incorporation for FNE-m-p53 cells treated under the indicated conditions for 72 h.Cells were treated with Torin2 (1 µm) for four hours before exposure to cisplatin (10 µm).(B) Representative histograms and quantification of PI incorporation for OVCAR4 cells treated under the indicated conditions for 24 h.Data are reported as mean ± SD. p-values represent DMSO + cisplatin vs. DMSO + taurine + cisplatin and Torin2 + cisplatin vs. Torin2 + taurine + cisplatin.Statistical analysis was performed using a two-way ANOVA.