Golgi-Localized PAQR4 Mediates Antiapoptotic Ceramidase Activity in Breast Cancer Line Pedersen1, Pouda Panahandeh1, Muntequa I

Published OnlineFirst April 14, 2020; DOI: 10.1158/0008-5472.CAN-19-3177

CANCER RESEARCH | METABOLISM AND CHEMICAL BIOLOGY

Golgi-Localized PAQR4 Mediates Antiapoptotic Ceramidase Activity in Breast Cancer Line Pedersen1, Pouda Panahandeh1, Muntequa I. Siraji1, Stian Knappskog2,3, Per Eystein Lønning2,3, Ruth Gordillo4, Philipp E. Scherer4, Anders Molven5,6, Knut Teigen1, and Nils Halberg1

ABSTRACT ◥ The metabolic network of sphingolipids plays important roles compared with matched control tissue and its overexpression in cancer biology. Prominent sphingolipids include ceramides correlated with disease-specific survival rates in breast cancer. and sphingosine-1-phosphate that regulate multiple aspects of Induction of PAQR4 in breast tumors was found to be subtype- growth, apoptosis, and cellular signaling. Although a significant independent and correlated with increased ceramidase activity. number of enzymatic regulators of the sphingolipid pathway These findings establish PAQR4 as Golgi-localized ceramidase have been described in detail, many remained poorly character- required for cellular growth in breast cancer. ized. Here we applied a patient-derived systemic approach to identify and molecularly define progestin and adipoQ receptor Significance: Induction of and cellular dependency on de novo family member IV (PAQR4) as a Golgi-localized ceramidase. sphingolipid synthesis via PAQR4 highlights a central vulnera- PAQR4 was approximately 5-fold upregulated in breast cancer bility in breast cancer that may serve as a viable therapeutic target.

Introduction endows cancer cells with a dual selective advantage through the combined effects of lowering cytotoxic ceramides and generating S1P. Sphingolipids are key regulatory bioactive molecules that play important roles in cancer biology (1). Altered sphingolipid metabolism are linked to several cancer types including liver, colon (2), endome- Materials and Methods trial (3), and breast cancer (4, 5). The levels of sphingolipid pathway Cell lines metabolites, including ceramides and sphingosine-1-phosphate (S1P), MDA-MB-231, HCC1806, MCF7, and T-47D cells were purchased are significantly deregulated in breast tumor tissue compared from ATCC and the murine cell line EO771 from Ch3Biosystems. with normal tissue (4, 5), and elevated tumor ceramide levels have Ishikawa cells were kindly provided by Dr. C. Krakstad. H293T, been shown to be associated with higher tumor grades (4, 6). The SUM149, and MCF10A cells were a kind gift from Dr. J. Lorens. Cells conversion of ceramides to sphingosine and subsequent phosphory- were maintained using standard tissue culture procedures, according lation generates S1P. The balance between the intracellular levels of to the manufacturer's instructions, and grown at 37 C with 5% CO proapoptotic ceramide and prosurvival S1P determines the fate of 2 and atmospheric oxygen. cancer cells (7). By mediating apoptosis, growth arrest, and senescence, Genetic fingerprinting and short tandem repeat profiling cell line ceramides function as tumor-suppressor lipids, whereas S1P is a key authentication of MDA231, MCF7, and MCF10A was performed by tumor-promoting lipid that enhances cell proliferation, migration, and Eurofins Genomics laboratory. angiogenesis (8–10). In this study, we identify PAQR4 as a Golgi-localized ceramidase Patient tumor samples that is highly expressed in breast cancer tissues and whose expression is required for tumor growth. We find that enhanced PAQR4 expression Survival analysis Survival analysis was performed on a quartile normalized cDNA microarray data from 203 breast patient's tumor samples based 1Department of Biomedicine, University of Bergen, Bergen, Norway. 2Depart- on Illumina HumanHT-12v4 Expression platform. The samples ment of Clinical Science, Faculty of Medicine, University of Bergen, Bergen, were treatment na€ve (11). The study was approved by the regional 3 Norway. Department of Oncology, Haukeland University Hospital, Bergen, committees for medical and health research of Western Norway 4 Norway. Touchstone Diabetes Center, Departments of Internal Medicine and (REK-Vest; approval number 273/96-82.96). Simmons Cancer Center, The University of Texas Southwestern Medical Center, Dallas, Texas. 5Gade Laboratory for Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway. 6Department of Pathology, Haukeland Histology University Hospital, Bergen, Norway. Breast cancer tissue sections used in Fig. 1C were from anon- Note: Supplementary data for this article are available at Cancer Research ymized patients included in the same study by Chrisanthar and Online (http://cancerres.aacrjournals.org/). colleagues (11). L. Pederson and P. Panahandeh contributed equally to this article. Ceramidase assay Corresponding Author: Nils Halberg, University of Bergen, Bergen 5020, Female breast cancer patient specimens, used for ceramidase assays, Norway. Phone: 474-147-2368; Fax: 47-55-58-63-60; E-mail: [email protected] were obtained at the Department of Surgery (Haukeland University Hospital, Bergen, Norway) under written informed consent, and Cancer Res 2020;80:2163–74 anonymized. The tumor and normal tissue samples were matched doi: 10.1158/0008-5472.CAN-19-3177 (N ¼ 7 pairs), meaning that the tumor and normal sample were 2020 American Association for Cancer Research. collected from the same patient during mastectomy (12). The matched

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ABTCGA Breast dataset C BIOCARTA_Ceramide_pathway (N = 111 pairs) 0.6 − NES = 1.67 50 log2 FC = 1.5 log2 FC = 1.5 Patient 1 Patient 2 0.4 P = 0.005 B3GALT1 S 0.2 FDR = 0.075 40 T 0.0 PAQR4 T S ) 100 µm adj 30 KIT

( P S1PR1 KEGG_Sphingolipid_metabolism Patient 3 Patient 4 10 20 IGF1 NES = 1.62 MMD DEGS2 T 0.4 FAM57B − log Enrichment score P = 0.021 GAL3ST1 T 10 ST6GALNAC1 FUT7 S S 0.2 FDR = 0.053 P UGT8 adj = 0.01 0.0 0 −4 −2 0 2 4 Tumor Normal log2 (Tumor/Normal) gene expression D E 100 Low PAQR4 100 Low PAQR4 High PAQR4 High PAQR4 75 75

50 50 N = 203 N = 203 25 Hazard ratio = 2.116 25 Hazard ratio = 1.743 95% CI = 1.329−3.368 95% CI = 1.124−2.704 Log-rank P value = 0.0082 Log-rank P value = 0.0301 0 0

% Relapse-free survival 0 0 0 0 0 0 0 0 0 0 5 0 5 0 5 0 5 0 1 1 2 % Disease-specific survival 1 1 2 Time (months) Time (months)

Figure 1. Expression of the sphingolipid metabolism–related gene PAQR4 is induced in tumors and negatively correlates with breast cancer patient survival. A, Functional analysis of DEGs comparing the transcriptomics of paired normal and tumor tissues from patients with breast cancer (n ¼ 111) obtained from TCGA revealed an enrichment of genes in the ceramide signaling and sphingolipid pathway in tumor tissue. NES, normalized enrichment score; FDR, false discovery rate. B, Volcano plot showing deregulated sphingolipid-related genes when comparing transcriptomic proﬁles of paired normal and tumor tissue derived from patients with breast

cancer (n ¼ 111). The signiﬁcant (Padjusted < 0.01) overexpressed genes (red circles) are represented as log2 (tumor/normal) gene expression >1.5 and downregulated (blue circles) as log2 (tumor/normal) gene expression <1.5-fold changes. C, Representative PAQR4 staining in patient with breast cancer tissue. S, stroma; T, tumor tissue. D and E, PAQR4 protein expression negatively correlates with patient survival (n ¼ 203) as determined by Kaplan–Meier curves of RFS and DSS based on high (equal or more than 25% quartile, red curve) or low (25% quartile, blue curve) PAQR4 expression using log-rank test.

normal tissue was collected from a region of the breast with a clear viable MDA-MB-231 cells in PBS were mixed 1:1 by volume with physical separation from the malignant lesion. Matrigel (Corning, 356231) and injected in a total volume of 50 mL. At endpoint, the mice were humanely euthanized, and tumors Animal models harvested for weight measurements and histology. Animal experiments were approved by the Norwegian Animal Research Authority and conducted according to the European Conven- Transcriptomics analysis tion for the Protection of Vertebrates Used for Scientific Purposes, Total transcriptomics profiles of paired normal and tumor tissues Norway. The Animal Care and Use Programs at University of Bergen breast invasive patients with carcinoma were obtained from The are accredited by the Association for Assessment and Accreditation of Cancer Genome Atlas (v1.5.2 TCGA, n ¼ 111) and Gene Expression Laboratory Animal Care International. The laboratory animal facility at Omnibus (GSE70947, n ¼ 147). To identify the differentially expressed P University of Bergen was used for the housing and care of all mice. genes (DEG, with cut-off adjusted value 0.01 and log2 tumor/normal Female NOD-SCID gamma (005557, RRID:IMSR_JAX:005557) and fold-change expression 1.5), a supervised paired sample t test was C57BL/6J (000664, RRID: IMSR_JAX:000664) mice were purchased implemented using limma Bioconductor package in R (13). To from Jackson laboratories. Mice were kept in IVC-II cages (Sealsafe investigate the DEGs involved in sphingolipid metabolism, we com- IVC Blue Line 1284L, Tecniplast). For both strains, 5–6micewere piled a gene set containing 203 genes involved in the sphingolipid and housed together and maintained under standard housing conditions at ceramide biosynthesis, catabolism, and signaling pathways based on 21C 0.5C, 55% 5% humidity, and 12-hour artificial light-dark the Molecular Signatures Database (v6.1 MsigDB; ref. 14). As adipo- cycle. Mice were provided with standard rodent chow (Special Diet nectin receptor 1 and 2 (AdipoR1 and AdipoR2) are known as Services RM1, 801151, Scanbur BK) and water ab libitium. ceramidases, all 11 genes in the progestin and adipoQ receptor (PAQR) family were included in the gene set. Syngeneic and xenograft model PAQR4-depleted cells were orthotopically implanted in the Gene set enrichment analysis inguinal mammary fat pad of 8 (C57BL/6J) or 6 (NOD-SCID) Functional analysis of the DEGs comparing the transcriptomics of weeks old female mice. A total of 5 104 viable EO771 or 5 105 the paired normal and tumor tissues from TCGA was performed using

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Gene Set Enrichment Analysis (GSEA) software (14). The significant Mutagenesis enriched gene sets were selected as FDR < 0.25. Site-directed mutagenesis was performed using the PCR-based QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Survival analysis Technologies, 210515) according to the manufacturer's instructions. Disease-specific survival (DSS) and relapse-free survival (RFS) were Primers are listed in the Supplementary Table S1. Sanger sequencing calculated with GraphPad Prism software using log-rank test. Patients analysis was performed to confirm mutated PAQR4 products. with 25% quartile of PAQR4 expression were considered as low PAQR4 group and patients with ≥25% quartile were classified as patients with qRT-PCR highly expressed PAQR4 tumors. In addition, Gene Expression–Based Total RNA extraction and cDNA synthesis were performed using Outcome for Breast Cancer Online (GOBO, v.1.0.3; ref. 15) was used to total RNA purification Kit (Norgen Biotek, 37500) and Superscript III graph the distant metastasis-free survival based on PAQR4 expression reverse transcriptase (Invitrogen, 18080051). qRT-PCR was per- in 1,176 available breast tumor. formed using designed primers (Supplementary Table S1) and SYBR Green (Roche, 4707516001) on the LC480 instrument (Roche). The DDC Structural prediction and molecular dynamic simulation relative amount of cDNA was calculated by the t method using PAQR2 and PAQR4 amino acid sequence alignment was per- human Hypoxanthine-guanine phosphoribosyltransferase (hHPRT) formed using Clustal Omega. Swiss-Model (16) was used to prepare and mouse actin (mActin) RNA expression as controls for human and a homology-model of PAQR4, based on the structure of AdipoR2 mouse cell lines, respectively. (PDBID:5LXA).ThePAQR4modelwasembeddedinalipid bilayer using the Charmm membrane builder (17) and subjected Proliferation assay tomoleculardynamicssimulations with the Amber molecular Proliferation of cells was determined in triplicates by high content modeling package as described below. imaging using the IncuCyte Zoom (Essen Bioscience) according to the Simulations of PAQR4 in complex with ceramide were based on manufacturer's instructions. In all experiments, four fields were docking of ceramide to the homology model of PAQR4. The PAQR4- imaged per well under 10 magnification every 2 hours for 3–5 days. ceramide complex was embedded in an equilibrated bilayer of POPC The IncuCyte Zoom (v2018A) software was used to calculate con- (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The system was fluency values. solvated in a water box encompassing the protein with extensions in the XY-dimensions identical to the lipid bilayer. Starting coordinates Tissue IHC for PAQR4 and immunofluorescence for cleaved for simulations of PAQR4 with the three zinc-coordinating histidine caspase-3 residues mutated to alanine were prepared by removing zinc and About 5-mm tissue sections were deparaffinized and antigen retriev- modifying histidine to alanine in the coordinate file. Simulations were al performed using a high pH buffer (Vector Laboratories, H-3301). performed using the GPU-accelerated Particle Mesh Ewald Molecular Sections were blocked (Vector Laboratories, PK-4001) and incubated Dynamics (PMEMD) module (18) implemented in the AMBER18 overnight at 4C with PAQR4 primary antibody (Supplementary molecular dynamics software package (19), applying the ff14SB (20) Table S1) diluted in 1% BSA in PBS. Following signal amplification and lipid14 force fields (21). Periodic boundary conditions with by the Vectastain ABC reagent, the HRP signal was developed particle mesh Ewald summation (22) of electrostatic interactions were by incubating the sections with HRP substrate (Vector Laboratories, applied and van der Waals interactions were truncated with a 10 Å SK-4105). Counterstaining was performed by haematoxylin. The cutoff. SHAKE (23) was used to constrain bonds involving hydrogen images were acquired using Hamamatsu slide scanner and Aperio atoms. After 10,000 steps of minimization, the systems were subjected ImageScope software. to 5 ps of gradual constant volume heating (NVT) from 0 to 100 K For immunofluorescence of cleaved caspase-3, mouse tumor tissues followed by 100 ps gradual constant pressure heating (NPT) applying were fixed in 4% paraformaldehyde (PFA) overnight and embedded anisotropic Berendsen coupling (24) with reference pressure set to 1 in paraffin for sectioning. After deparaffinization and rehydration, bar. Temperature was regulated with the Langewin thermostat (25) antigen unmasking was performed in pH 6.0 (Vector Laboratories, gradually over 100 ps from 100 to 303 K. Weak restraints maintained H-3300). The sections were blocked in 4% goat serum in PBS with by a force constant of 10 kcal mol 1 Å 2 were applied to the protein 1% BSA and stained with cleaved caspase-3 antibody. Images were and lipids throughout both heating steps. The systems were finally acquired using the Leica SP5 confocal microscope. Cleaved caspase-3 simulated at constant pressure conditions (NPT) without restraints for staining was quantified as stained area per field (0.15 mm2)infive 1,500 ns at 303 K and the resulting coordinates were saved every 100 ps different fields per tumor (n ¼ 5 per group). for analysis. Cell immunofluorescence and confocal microscopy Generation of knockdown and overexpression cell lines Cell staining for immunofluorescence was performed as described Short hairpin RNA (shRNA) constructs targeting PAQR4 and previously (27). The type, source, and dilution of antibodies are scramble (shCtrl) were purchased from Sigma (Supplementary described in the Supplementary Table. Immunostained samples were Table S1). Full-length PAQR4 (Isoform 1) was PCR amplified from imaged using Leica SP5 confocal microscope. Images for PAQR4 cDNA of Ishikawa cells and cloned into the retroviral pBabe-puro localization were acquired using Leica SP8 confocal microscope. Image vector (Addgene, catalog no. 1764) by conventional restriction analysis was performed using ImageJ Fiji. The surface-rendering tool enzyme-based cloning. Sanger DNA sequencing analysis confirmed in the Imaris 9.1.2 Bitplane software was used to generate Fig. 5A. successful cloning. Lentivirus (knockdown) and retrovirus (overexpression) production were performed as described previously (26). Flow cytometry After selection, cells were allowed to recover for at least 48 hours before Apoptosis and cell-cycle assays were determined by the Annexin V tested for overexpression or knockdown of PAQR4. Knockdown cells assay (Thermo Fisher Scientific, A13201) and incubation of the cells were never passaged for more than three passages after infection. with PI and RNase (BD Biosciences, 550825), respectively, according

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to the manufacturer's protocol. Flow cytometry was performed using concentration of each metabolite was determined using the peak-area the Accuri C6 (BD Biosciences) and data were analyzed with FlowJo ratio of analyte versus corresponding internal standard. Data are software (Tree Star, Inc). reported as analyte peak area/internal standard peak area and normalized according to the protein content. Cellular sphingolipidomics MDA-MB-231 and MCF7 cells were seeded in triplicates and C6-NBD ceramidase assay allowed to adhere overnight. The following day the media were C6 ceramide-NBD (Thermo Fisher Scientific, N-22561) was pre- renewed with fresh culture media. For cells subjected to S1P measure- pared according to manufacturer's instruction. Cells grown on cover- ments, the media were replaced with DMEM phenol-free media slips were washed twice in Hanks' Balanced Salt Solution (Sigma, (Sigma, D1145) containing 0.02% FBS. Two days after plating, cells H8264) supplemented with 10 mmol/L HEPES (HBSS/HEPES), before were washed once in PBS and harvested by scraping. Snap-frozen adding 5 mmol/L C6-NBD ceramide dissolved in HBSS/HEPES. The cell pellets were quantified by LC/MS-MS as described (28) by the cells were then incubated at 4C for 30 minutes and washed five times VCU Lipidomics/Metabolomics Core. The measured sphingolipid in ice cold HBSS/HEPES before incubated at 37C for 30 minutes. levels were normalized to protein concentration as determined using Finally, cells were washed four times before fixation with 4% PFA for Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fischer 15 minutes. For immunofluorescence staining of the Golgi, cells were Scientific, 23225). permeabilized with 0.03 mg/mL digitonin for 10 minutes at 4C and stained with RCAS1 as described above. Images were acquired using Ceramidase activity assay and sphingolipid measurement the Leica SP5 confocal microscope. ImageJ Fiji was used to measure the Tumor biopsies and frozen cell pellets were homogenized in 500 mL C6 ceramide-NBD signal in RCAS1-positive areas. cold Dubelco PBS solution containing calcium and magnesium and EDTA-free protease inhibitor cocktail (Roche) with a mechanical C6-ceramide treatment of cells tissue homogenizer (tissue biopsies) and tissue dismembrator probe C6-ceramide/cholesteryl phosphocholine formulations were pre- (cell pellets, Thermo Fisher Scientific Model FB50, 8 short 1-second pared following the manufacturer's instructions (Avanti Polar Lipids, pulses at 15% of amplitude). After incubation of the samples on ice, 640001). For assessing CDK4 expression and cytochrome C release 10 mL of an ethanolic mixture of deuterated ceramides-d7 was upon C6-ceramide treatment, cells were treated with 20 mmol/L added to each sample [Ceramide d18:1-d7/16:0 (Cayman Chemicals, (48 hours) and 5 mmol/L (overnight) C6-ceramide before collection/ 22787, 4.9 mmol/L), Ceramide d18:1-d7/18:0 (Cayman Chemicals, fixation of cells, respectively. 22788, 4.6 mmol/L), Ceramide d18:1-d7/24:1 (Avanti Polar Lipids, 10 mmol/L), Ceramide d18:1-d7/24:0 (Avanti Polar Lipids, 860679, Western blotting 10 mmol/L)]. The homogenates were incubated at 37C with contin- Cells were lysed in RIPA buffer containing EDTA-free protease uous shaking for 3 hours and the reaction was quenched by adding inhibitor cocktail (Pierce RIPA buffer, Thermo Fisher Scientific, 2 mL of organic extraction solvent (Isopropanol/Ethyl Acetate 89901; Sigma, 5892791001). Protein samples were immunoblotted 1:2, vol/vol). Immediately afterward, 20 mL of organic internal stan- using standard methods. Flag-tagged PAQR4 expression was detected dard solution was added (Ceramide/Sphingoid Internal Standard using an anti-Flag antibody conjugated to HRP (Supplementary Mixture II diluted 1:10 in ethanol, Avanti Polar Lipids). After Table S1) for 1 hour at room temperature, whereas CDK4 expression short-vortexing, a two-phase liquid–liquid extraction was performed. was detected by incubating membranes overnight at 4C with The organic phases were combined and dried under nitrogen stream at anti-CDK4 antibody. b-actin served as a loading control. 40C. In the case of tissue samples, the dried residue was reconstituted in 200 mL of methanol. The aqueous phase was dried in a SpeedVap Quantification and statistical analysis solvent evaporation system and the dried residue was reconstituted in Statistical computation on the transcriptomics data and principle 500 mL RIPA buffer containing 5% TritonTM X-100 and total soluble component analysis on clinical ceramidase assay data were per- protein content was determined by the BCA assay. formed in RStudio. Several R packages were used to prepare the data About 5 mL samples were injected into an LC/MS-MS system for the and analyze the RNA-seq. The in-house script is available upon analysis of ceramides and sphingoid bases, 1 mL injection was required request. for the analysis of sphingomyelins. The system consisted of a Shimadzu For the ceramidase assays (Fig. 4C and D) on the clinical patient LCMS-8050 triple quadrupole mass spectrometer with the dual ion samples, the results were analyzed by Wilcoxon matched-pair source operating in electrospray positive ionization mode in the case of signed rank test using GraphPad Prism 7 software. For all the sphingoid bases and ceramides and in negative mode in the case of comparisons, Student two-tailed t test was used in GraphPad sphingomyelins. The mass spectrometer was coupled to a Shimadzu Prism 7 software. The significant symbols used in the figures are Nexera X2 UHPLC system equipped with three solvent delivery as , P < 0.05; , P < 0.01; , P < 0.001. modules LC-30AD, three degassing units DGU-20A5R, an autosam- pler SIL-30ACMP and a column oven CTO-20AC operating at 40C (Shimadzu Scientific Instruments). Analysis of sphingolipid species Results was achieved using selective reaction monitoring scan mode. The sphingolipid metabolism–related gene PAQR4 is required Sphingoid lipid separation was achieved by reverse phase LC on a for breast cancer cellular growth and negatively correlated 2.1 (i.d.) 150 mm Ascentis Express C8, 2.7 mm (Supelco) column with breast cancer patient survival under gradient elution, using three different mobile phases: eluent A GSEA of DEGs between 111 paired tumor and nontumor tissue consisting of methanol/water/formic acid, 600/400/0.8, vol/vol/vol revealed an enrichment of genes in the ceramide signaling pathway with 5 mmol/L ammonium formate, eluent B consisting of metha- (NES ¼ 1.67; FDR ¼ 0.075) and sphingolipid metabolism (NES ¼ 1.62; nol/formic acid, 1,000/0.8, vol/vol with 5 mmol/L ammonium formate, FDR ¼ 0.053) in tumor tissues (Fig. 1A; Supplementary Fig. S1A). To and eluent C consisting of CH3OH/CH2Cl2 350/650. The relative uncover key regulatory members of the sphingolipid pathway, we

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performed a supervised differential expression analysis of sphingoli- (Fig. 2E; Supplementary Fig. S2C). Collectively, these data establish pid-related genes (203 genes) between matched tumor and nontumor that the sphingolipid metabolism–related gene PAQR4 is required for tissue samples. We identified PAQR4 as the most upregulated gene breast cancer cell growth. P < PAQR4 transcript (fold change 5, adj 0.01; Fig. 1B). deregulation was breast cancer subtype-independent, as enhanced PAQR4 expres- Homology modeling suggests that PAQR4 functions as a sion was detected in tumors irrespective of estrogen, progesterone ceramidase receptor, and HER2 receptor profile in both the TCGA and a validation To investigate the mechanism by which PAQR4 regulates tumor dataset (Supplementary Fig. S1B and S1C). Consistent with the growth, we next sought to determine the structural basis of its function. transcriptomic analysis, detection of PAQR4 by IHC in breast cancer PAQR4 belongs to the PAQR protein family consisting of 11 members tissue sections showed enhanced staining intensity in the characterized by a 7-transmembrane domain. The close family mem- tumor compartment compared with the stromal/normal tissue areas ber PAQR2 (also known as AdipoR2) is known to possess ceramidase (Fig. 1C). PAQR4 expression was further negatively correlated with activity and has been cocrystalized with oleic acid, that is, the byprod- patient RFS (log-rank P value ¼ 0.0082; Fig. 1D) and DSS (log-rank uct of its enzymatic activity (30, 31). Within its catalytic site, a zinc ion P value ¼ 0.0301; Fig. 1E) in an in-house dataset (11), as well as coordinated by three histidine residues (His202, His348, and His352), metastasis-free survival in an independent validation set (Supplemen- is essential for catalysis. In concert with the zinc ion, an aspartic acid tary Fig. S1D). These findings establish that PAQR4 expression is residue (Asp219) facilitates the cleavage of ceramide into sphingosine induced in tumor tissues and negatively correlates with patient and a fatty acyl chain. Interestingly, these histidine residues and the survival. aspartic acid are conserved in PAQR4 (His100, His240, His244, and To investigate the mechanism by which PAQR4 affect tumor cells, Asp119; Supplementary Fig. S3A and S3B). we next sought to determine the cellular phenotypes it governs. We therefore constructed a homology model of PAQR4 based on Depletion of endogenous PAQR4 in triple-negative (MDA-MB-231 the resolved crystal structure of AdipoR2. We then did a structural and HCC1806) and estrogen receptor–positive (MCF7 and T47D) superimposition of the AdipoR2 structure to our PAQR4 model to human breast cancer cell lines as well as the murine breast cancer cell position oleic acid inside the channel of PAQR4 and performed a line EO771, using lentiviral delivered shRNAs (Supplementary 100 ns molecular dynamics simulation of the complex embedded in a Fig. S2A), significantly reduced cellular growth as determined by high lipid bilayer to relax the structure. This model revealed conservation of content imaging (Fig. 2A–C). Such conserved effect across tumor cell a 7-transmembrane structure that forms a barrel domain with an genotypes is consistent with the observed PAQR4 induction between amphipathic pore (Fig. 3A). The interactions of the fatty acid with breast cancer subtypes. In contrast to the findings of Zhang and PAQR4 were essentially the same as observed for AdipoR2. The colleagues (29), the observed reduction of cellular growth in carboxylic group of the fatty acyl locates at the proximity of the zinc PAQR4-depleted cells was independent of changes in cell-cycle status coordination in PAQR4 (Fig. 3A and B). We then repeated the (Supplementary Fig. S2B). Consistent with the observed growth simulation by docking a C18:1-ceramide molecule in the PAQR4 inhibition in vitro, PAQR4 depletion in human MDA-MB-231 and model using the oleic acid as an anchor. Independent simulations mouse EO771 triple-negative breast cancer cells abrogated orthotopic over 1.5 ms showed that PAQR4, as was shown for PAQR2 (31), readily tumor growth in immune-compromised NOD-SCID and immuno- accommodated the fatty acyl part of the ceramide molecule inside the competent C57BL/6J mice, respectively (Fig. 2D). Finally, ectopic pore, with the cleavage site (amide bond) in close proximity to the expression of PAQR4 in SUM149 cells increased the cellular growth active site zinc center (Fig. 3C; Movie S1). Interestingly, in both

Figure 2. A B PAQR4 MDA-MB-231 HCC1806 MCF7 T-47D is required for cellular growth shCtrl of breast cancer cells. A–C, Growth shCtrl 3 sh1PAQR4

40 ** curve of triple-negative (MDA-MB- 20 sh PAQR4 sh2PAQR4 15 ***

1 * ** * **** ****

231 and HCC1806), hormone recep- t = 0 sh PAQR4 30 * 15 2 t = 0 2 tor–positive (MCF7 and T-47D) and 10 * 10 20 murine EO771 cells transduced with 1 5 lentiviral-delivered shRNAs targeting 5 10 PAQR4. Cell growth was determined 0 0 relative to 0 0 relative to 0 0 0 6 2 by high content imaging and is repre- 0 0 0 0 6 2 1 0 6 2 6 2 3 7 3 7 Growth (% confluence) sented as percentage of conﬂuence 1 Growth (% confluence) Time (hr) Time (hr) normalized to t ¼ 0, n ≥ 6. D, Tumor Time (hr) Time (hr) weight of orthotopically implanted MDA-MB-231 and EO771 cells with the C D E PAQR4 SUM149 indicated shRNAs targeting . EO771 MDA-MB-231 EO771 One representative of two experi- shCtrl Ctrl OE ments is shown, n ¼ 4 mice (MDA- shmPAQR4 0.6 *** 60 1.5 ** 60 PAQR4 WT OE n ¼ – * MB-231) or 4 5 mice (EO771). ** * t = 0

t = 0 0.4 E, Growth curve of SUM149 cells over- 40 1.0 40 expressing PAQR4 in starved (1% FBS) 20 medium. Cell growth was deter- 20 0.2 0.5 mined by high content imaging and 0 relative to 0 Tumor weight (g) 0.0 0.0 relative to 0 6 2 is represented as percentage of con- 0 6 2 l 3 7 3 7 r ﬂuence normalized to t ¼ 0, n ¼ 6. All R4 R4 Ct Ctrl Growth (% confluence) Time (hr) Growth (% confluence) Q h Q Time (hr) sh A s A plots in this ﬁgure are presented as P mean SEM. , P < 0.05; , P < 0.01; h 2 mP s sh , P < 0.001; , P < 0.0001.

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Wild-type PAQR4 Mutant PAQR4 A C-terminal C E

C18:1-Cer (Fatty acyl moiety) C18:1-Cer

2+ Oleic acid Zn (C18:1) Transmembrane

C18:1-Cer (Sphmoiety) N-terminal

0 1.5 µs 0 1.5 µs

B Top view D F 3.5 3.5 H244 H100 3.0 3.0 H240 zinc 2.5 2.5 D119 2.0 2.0 FA (C18:1) 1.5 1.5 RMSD (Å) RMSD (Å) 1.0 1.0 o 180 0.5 0.5 0.0 0.0 0 0 Bottom view 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Time (µs) Time (µs) FA D119 (C18:1) zinc

H244 H100 H240

Figure 3. Homology modeling suggests that PAQR4 function as a ceramidase. A and B, Homology model of PAQR4 resolved within the membrane plane using PAQR2 as template structure. Top and bottom view of the hydrophobic binding pocket of PAQR4 is shown in B. Oleic acid (C18:1) in the amphipathic pore of PAQR4 is shown in ball and stick representation (carbons, gray; oxygen, red). The three Zn-coordinated His residues together with Asp 219 are shown in stick representation. The Zn ion is shown as a light blue sphere. C, Schematic molecular dynamic simulation of wild-type PAQR4 in complex with C18:1-ceramide, embedded in a lipid bilayer. Frames of the receptor-ceramide complex, showing one frame every mirosecond of the 1.5-ms simulation, color coded blue to red through the trajectory. Lipid molecules and water are omitted from the presentation for clarity. D, Root-mean-square deviation (RMSD) of the wild-type PAQR4 protein atomic positions relative to ﬁrst frame in the trajectory, as a function of simulation time. E, Schematic of molecular dynamic simulation of mutant PAQR4 in complex with C18:1-ceramide, where the three Zn-coordinated histidines are mutated to alanine, embedded in a lipid bilayer. Frames of the mutated receptor-ceramide complex, showing one frame every micosecond of the 1.5-ms simulation, color coded blue to red through the trajectory. Lipid molecules and water are omitted from the presentation for clarity. F, RMSD of the mutant PAQR4 protein atomic positions relative to ﬁrst frame in the trajectory, as a function of simulation time.

structures, the sphingosine moiety of the ceramide molecule is pre- Interestingly, this simulation showed that both the fatty acid and the dicted to localize outside of the pore. sphingosine arm of ceramide can be accommodated inside the We further found that the homology model was remarkably amphipathic pore (Fig. 3E). Although the overall structure of stable over time (Fig. 3D). To better understand the role of the zinc mutated PAQR4 was stable (Fig. 3F), the more relaxed structure molecule, we performed another simulation of C18:1-ceramide revealed that ceramide can interact in two different conformations. docked in PAQR4 wherein the three zinc-binding histidine residues In sum, these modeling experiments suggest that PAQR4 possess were mutated to alanine, thereby excluding zinc from the structure. ceramidase activity.

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PAQR4 is a Golgi-associated ceramidase with long (C16:0 and C18:0) and very long fatty acyl chains (C24:1 and To experimentally test whether PAQR4 harbors intrinsic cerami- C24:0) were increased in knockdown lysates (Fig. 4A). dase activity, we incubated deuterium-labeled ceramides with cellular Given that we found PAQR4 to be induced in breast cancer, we lysates from control and PAQR4 knockdown cells. Following the wondered whether this induction was paralleled by higher ceramidase incubation period, ceramidase activity was assessed by measuring the activity in tumor compared with nontumor tissue. We therefore abundance of exogenous deuterated ceramide species by LC/MS-MS. performed the ceramidase assay in matched normal and breast cancer Consistently, we found that lysates from PAQR4-depleted cells dis- tissue samples. Interestingly, a principle component analysis was able played reduced ceramidase activity when compared with control cells to differentiate tumor tissues from the corresponding normal tissues (Fig. 4A). We did not detect any PAQR4-related biases for speciﬁc (Fig. 4B). We further found that tumor tissue lysates exhibited ceramide fatty acyl chain length, as the levels of both ceramide species increased ceramidase activity for C16:0, C18:0, and C24:1 ceramide

A B (N = 7 pairs) Tumor Normal 0.015 *** *** *** *** 0.010 P1 5 8 600 300 P4 P5 standard 4 0.005 6 400 P3 P7 P7 200 P2 3 0.000 P5 P2 4 P1 2 P6 P4 200 100 −0.005 P3 /internal 1 2 -4 C16:0-Cer C18:0-Cer C24:0-Cer C24:1-Cer PC2 (17.33%) − 0 0 0 0.010 P6 0 l l 4 r 4 4 ×10 r 4 trl 4 R4 R4 R Ct R R −0.015 Ctrl R4 R Ct R C Q Q Q Q Q Q Q Q sh A A sh A A sh A A sh A A P P P P 2 0 0 1 P P P P 2 1 2 .0 1 .0 .02 peak area/mg protein) 1 0 1 2 1 2 h h sh −0 . 0 0 sh sh sh sh sh s s −0 Exogenous deuterated Cer PC1 (82.41%) (peak area

C D * * * NS 15 25 800 * 2.5 600 20 600 2.0 10 15 400 1.5 400 10 1.0 5 200

/internal standard 200 5 -3 0.5 -4 C16:0-Cer C18:0-Cer C24:0-Cer C24:1-Cer 0 10 0 0 area/mg protein) 0.0 0 ×

×10 l l l r r l r l r a r ma mo ma mo ma mo ma mo m mo r u r u r r u r No T No T Tu T peak No Tu

peak area/mg protein) No No Exogenous deuterated Cer (peak area /internal standard (peak area Exogenous deuterated sphingosine

-0.7 -5.6 -12.8 -34.7 -7.3 -7.6 -0.8 -2.6 -0.9 -27.5 -1.4 -1.4 -0.5 -0.4 -3.0 -0.3 -16.4 3

2 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 10 adj adj adj adj adj adj adj adj adj adj adj adj 1 P P P P adj P adj P adj P adj P P P P P P P P P adj P 0 −

FC Expression 1 2 −2 (tumor/normal) log −3 1 B 4 8 D H H2 R1 R2 R3 R1 R2 R D2 M A A E E E O O QR3QR QR5QR6QR7Q QR9 M S S AH2C C C IP IP A A A A A A A MM A A S A A A D D P P P P P P P A A A

Figure 4. PAQR4 possesses intrinsic ceramidase activity. A, PAQR4 ceramidase activity determined by incubating cell lysates from control and PAQR4-depleted cells with isotope (deuterium)-labeled ceramides. Ceramide content was determined by LC/MS-MS and normalized to internal standard peak area and mg protein. One of two independent experiments is shown. Dot plot shows mean SEM (n ¼ 3/group). B, Principal component analysis (PCA) of the ceramidase assay (as described in A)on normal and breast cancer tissues. The principal component analysis on the deuterated-labeled ceramides (C16, C18, C24:0, and C24:1) and sphingosine display the clustering of tumor (red) from normal (blue) tissues. C, Ceramidase activity in paired breast cancer and normal tissue (n ¼ 7 patients) determined as described in A. Black lines represent patients with increased ceramidase activity in the tumor compared with normal tissue; gray lines represent patients with reduced activity in the tumor tissue. D, In addition to the levels of deuterium-labeled ceramides, the cleavage product, deuterium-labeled sphingosine, was determined by LC/MS-MS in the same lysates as in C. E, Relative gene expression of known ceramidases in the paired breast tumor and normal tissues. The transcriptomics data were obtained from TCGA database. The adjusted P value of the fold change in each gene expression is presented on top of each bar in the graph. , P < 0.05; , P < 0.001; NS, nonsigniﬁcant.

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compared with their paired normal tissues lysates (Fig. 4C). However, reduced the NBD-labeled ceramide in the Golgi as determined by C24:0 ceramide abundance was unchanged. In further support of cosignal between NBD and RCAS1 (Fig. 5B). Furthermore, this effect increased ceramidase activity in tumors, we found significant increase was significantly reduced when the three histidine residues coordi- of labeled sphingosine—the downstream product of the ceramidase nated with the zinc ion in the catalytic domain were mutated to alanine reaction—in the tumor compared with the matched control tissues (PAQR4HtriAOE; Fig. 5B; Supplementary Fig. S4C). At the cellular (Fig. 4D). Combined, our studies strongly suggest that PAQR4 level, mutation of the three histidine residues, ablated the ability of possesses ceramidase activity. In addition to the aforementioned PAQR4 to promote cancer growth (Fig. 5C). PAQR2, cancer cells contain a number of other ceramidases. A Ceramides accumulation in the Golgi has been shown to induce transcriptional analysis of these ceramidases revealed that PAQR4 is local Golgi fragmentation (35). Consistent with PAQR4 having cer- induced to a larger extent compared with other ceramidases (Fig. 4E). amidase activity, we observed a significant Golgi fragmentation in the Sphingolipid metabolism is highly compartmentalized within the PAQR4-depleted cells compared with the control cells by confocal cell (32); thus, we next used three-dimensional (3D) confocal imaging immunofluorescence (Fig. 5D). Collectively, our findings suggest that to determine the subcellular localization of PAQR4. Detection of PAQR4 acts as a ceramidase in the Golgi compartment. expressed flag-tagged PAQR4 revealed a perinuclear staining that colocalized with the Golgi marker RCAS1 both in MCF10A cells and PAQR4 depletion causes accumulation of de novo sphingolipid in the breast cancer cell line SUM149 (Fig. 5A; Supplementary Fig. S4A intermediates and ceramide-induced apoptosis and S4B). To further determine whether PAQR4 possesses ceramidase Having identified PAQR4 as a ceramidase, we next sought to activity in the Golgi compartment, we took advantage of the fact that determine how PAQR4 depletion alters cellular sphingolipid homeo- NBD fluorescent-labeled C6 ceramide readily accumulates in the Golgi stasis. To this end, we performed an unbiased sphingolipidomics when added exogenously to cells (33, 34). We therefore added NBD- analysis of both MDA-MB-231 and MCF7 cells with depleted levels labeled ceramides to control cells and PAQR4 overexpressing cells and of PAQR4. Consistent with a ceramidase activity, we found overall measured Golgi-localized fluorescence intensities by confocal imaging. elevated levels of ceramides in the PAQR4 knockdown cells in both cell Interestingly, we found that overexpression of PAQR4 significantly lines (Fig. 6A). Similar to what we observed for ceramidase activity

A B DAPI/RCAS1/ C PAQR4-FLAG Zoom NBD-C6 Cer Ctrl OE PAQR4 WT OE HtriA

= 0) PAQR4 OE 3 t 60

Ctrl OE ****** ) DAPI / FLAG

5 µm 20 µm 3 2 40 *** OE T 1

W 20 Growth (A.U × 10 0 0 DAPI / RCAS1 PAQR4 E E E 0244872 A O lOT O ri tr W t C H Time (hr) Mean C6 Cer-NBD in Golgi OE QR4 A QR4 (% confluenceto relative HtriA A P P DAPI / FLAG RCAS1 PAQR4

*** D 100 shCtrl sh1PAQR4 sh2PAQR4 80 60 40 20 DAPI / RCAS1 20 µm 0 covered nuclei) R4 R4 CtrlQ Q

Golgi extent (% RCAS1 sh A A P P 1 2 sh sh

Figure 5. PAQR4 is a Golgi-associated ceramidase. A, 3D confocal immunofluorescent images of MCF10A cells expressing flag-tagged PAQR4. The tagged protein colocalizes with the Golgi marker RCAS1. Presented images were made by using the surface-rendering tool in the Imaris 9.1.2 Bitplane software. B, Ceramidase activity of PAQR4 in the Golgi determined by quantifying NBD-C6 ceramide fluorescence intensity in the Golgi (RCAS1-positive area) in SUM149 cells expressing wild-type (WT) or mutated PAQR4. The graph shows mean SEM from one of three independent experiments (n ≥ 30 cells/condition). C, Growth of SUM149 cells overexpressing wild-type or mutated PAQR4 incubated in starved (1% FBS) medium for 72 hours. Cell growth was determined by high content imaging and is represented as percentage of confluence normalized to t ¼ 0, n ¼ 4. Data are shown as mean SEM. D, Confocal immunofluorescent images of Golgi structure in MDA-MB-231 cells depleted for PAQR4. Golgi extent was measured as percentage of RCAS1-covered perinuclei. Data are presented as mean SEM from one of three independent experiments (n ≥ 30 cells/condition). , P < 0.001.

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ABC MDA-MB-231 MCF7 MDA-MB-231 MCF7 MDA-MB-231 MCF7 C14:0 18 21 34 8 8 13 C14:0 1 1 2 0 00 C16:0 491 541 ***1167 142 172 234 C16:0 20 25 ***46 6 9 10 * 0.064 3 11 4 1 1 1 1 1 0 000 ** C18:1 C18:1 40 *** C18:0 74 119 149 21 27 32 C18:0 4 13 7 1 2 2 10 30 C20:0 33 67 63 14 19 22 C20:0 2 6 5 1 1 1 C22:0 178 445 413 55 105 146 C22:0 4 10 17 3 6 *7 20 5 C24:1 325 603 538 202 249 238 C24:1 3 10 10 2 **8 4 (pmol/mg protein) C24:0 591 ***1270 1005* 121 161 225 C24:0 2 9 15* 1 3 2 10 C26:1 16 35 25 14 9 8 C26:1 000 000 0 0 C26:0 17 50 36 3 3 3 C26:0 0 2 1 000 4 Ctrl R4 R4 Ctrl R4 R Total 1746 ***3161 ***3434 580 754** ***922 Total 37 ***77 ***103 15 28 ****** 26 Q Q Q Q sh A A sh A A P P P P Ceramides (pmol/mg protein) 4 4 1 2 1 2 trl Sphinganine h C Ctrl Ctrl sh sh s sh Ctrl QR4 QR4 QR4 QR h QR QR4 QR4 QR4 sh A A sh A A s A A sh A A P P P P P P P P h 1 2 1 2 Dihydroceramide (pmol/mg protein) 1 2 1 h 2 s sh sh sh sh sh sh s

D E MDA-MB-231 MCF7 MDA-MB-231 MCF7 C14:0 7 3 26 7 3 6 C14:0 759 507 478 256 222 317 C16:0 446 159 ***1315 507 288 431 C16:0 6029 ***3906 ****3125 3053 2373 2886 C18:1 5 3 6 4 3 3 C18:1 150 133 103 83 64 79 C18:0 50 38 102 44 42 50 C18:0 528 504 282 352 365 344 C20:0 15 17 31 30 25 34 C20:0 2661 2230 1576 1926 2020 2074 C22:0 250 253 498 329 307 485 C22:0 841 1284 502 573 725 904 C24:1 309 223 462 526 279 418 C24:1 2148 2374 1025 1810 1603 1536 C24:0 1194 1408 ****3169 639 587 985** C24:0 1913 3003 1258 667 992 1120 (pmol/mg protein) C26:1 21 24 67 51 25 30 C26:1 76 102 47 318 468 477 Monohexosylceramide C26:0 32 50 159 15 13 16 C26:0 47 86 38 89 241 283 Total 2328 2177 ****5836 2154 1571 2457 Total 15153 14127 ****8435 9125 9074 10020 l l l tr r 4 4 tr Ctrl R4 C Ct R C QR4 Q QR4 QR4 Sphingomyelins (pmol/mg protein) Q QR QR4 QR4 sh A A sh A A sh A A sh A A P P P P P P P P h 1 2 1 2 1 2 1 h 2 s sh sh sh sh sh sh s

Figure 6. PAQR4 depletion causes accumulation of de novo sphingolipid intermediates. Ceramides (A), dihydroceramides (B), sphinganine (C), monohexosylceramides (D), and sphingomyelins (E) levels in lysates from MDA-MB-231 and MCF7 cells depleted for PAQR4 determined by LC/MS-MS. Sphingolipid levels were normalized to the protein concentration and are presented as mean in the heatmap and mean SEM in dot plots, n ¼ 3 per group from one of two independent experiments. , P < 0.05; , P < 0.01; , P < 0.001.

(Figs. 4A and 5B), we did not detect any PAQR4-related biases for levels of cytoplasmic cytochrome C in PAQR4-depleted cells as well as specific ceramide fatty acyl chain length. Interestingly, we also detected in cells treated with exogenous C6-ceramides (Fig. 7D and E). Fur- an accumulation of upstream intermediates (i.e., dihydroceramides thermore, ectopic overexpression of PAQR4 abolished C6-ceramide and sphinganine) in the de novo sphingolipid synthesis pathway induced release of cytochrome C to the cytoplasm (Fig. 7F; Supple- (Fig. 6B and C), but not the downstream metabolites (i.e., glycosyl- mentary Fig. S5). Considering the selective advantage of PAQR4 ceramides and sphingomyelins; Fig. 6D and E). This suggests that the induction in tumors, we postulated that in addition to reducing de novo synthesis pathway is highly active in breast cancer cells. cytotoxic ceramides, induction of PAQR4 could provide intermediates Accumulation of cellular ceramides is tightly linked to apopto- for S1P production. In support of this, we found that overexpression of sis (36, 37). We therefore asked whether the reduced growth rates wild-type PAQR4 increased cellular S1P levels, while overexpression of observed in the PAQR4-depleted cells is caused by increased apoptosis the ceramidase-dead HtriA mutation did not (Fig. 7G). Taken togeth- by assessing externalized phosphatidylserine in MDA-MB-231, er, we find that PAQR4 depletion results in ceramide-induced apo- HCC1806, MCF7, and T-47D PAQR4 knockdown cells by flow ptosis and an accumulation of the de novo sphingolipid synthesis cytometry. Accordingly, we found increased rates of apoptosis across intermediates. all cell lines (Fig. 7A and B). Furthermore, immunostaining of cleaved caspase-3 in tumor sections from control and PAQR4 knockdown tumors grown in vivo, confirmed activation of the apoptotic pathway Discussion in PAQR4-depleted cells (Fig. 7C). Beyond their structural role in membrane homeostasis, sphingoli- Ceramides are linked to apoptosis by forming pores in the outer pids control critical signaling transduction pathways within cancer mitochondrial membrane to facilitate the release of cytochrome C cells to drive growth, proliferation, migration, and invasion. Altered from mitochondria into the cytoplasm (38, 39). Consistent with our levels of ceramide species and other metabolites in the sphingolipid hypothesis and cellular buildup of ceramides, we found increased metabolism network of cancer cells are related to the deregulation of

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A MDA-MB-231 HCC1806 B MCF7 T-47D *** ** 80 *** *** ls 10 l 16 30 e 8 60 c shCtrl

shCtrl 12 c cells 6 20 40 8 4

PI 10 PI 20 4 2 (early and late) (early and late) 0 0 0

0 % Apoptotic % Apoptoti

PAQR4 4 4 PAQR4 4 l trl

r 1 1 R R4 R R4 Ctrl R R4 R4 R4 C Ctrl h Q Q Q Q Q Q Ct Q sh Q s A A sh sh sh A A A A P P sh A A P P P P 1 2 P P 1 h 2 1 2 1 2 sh s sh sh AnnexinV sh sh sh sh AnnexinV

C MDA-MB-231 D MDA-MB-231 DAPI/cCasp3 shCtrl sh1PAQR4 sh2PAQR4 * ** 80 0.10

/ ATBP 60 shCtrl

DAPI 40 0.05 20 0.00 0 (% area per field) (% rl CytC levels (A.U) rl Cleaved caspase-3 t R4 Ct R4 R4 C Q Q Q PAQR4 sh sh A A 2 A / ATBP CytC P Cytoplasmic/mitochondrial P P 2 1 2

sh h 100 µm sh 10 µm s sh DAPI

MDA-MB-231 MDA-MB-231 E F OE Cntrl+ OE Cntrl+ PAQR4 OE+ DAPI/ATBP/CytC Vehicle Cer Cer NS 1.5 *** 8 * 1.0 6 / ATBP Vehicle

4 DAPI 0.5

2 CytC levels (A.U) 0.0 CytC CytC levels (A.U) 0 / Cytoplasmic/mitochondrial ATBP Cytoplasmic/mitochondrial C6-Cer 10 µm VehicleC6-Cer 10 µm OE Cntrl+Cer PAQR4 OE+Cer DAPI / OE Cntrl+Vehicle

G *** 1.5

1.0 S1P 0.5

0.0 (pmol/mg protein) E E E O T O iA O trl W tr C H

QR4QR4 A A P P

Figure 7. PAQR4 regulates ceramide-induced apoptosis and sphingosine-1-phosphate production. A and B, Percentage of apoptotic cells (early and late) upon loss of PAQR4 in MDA-MB-231, HCC1806, MCF7, and T-47D cells determined by Annexin V and propidium iodide (PI) staining measured by flow cytometry. Dot plots show mean SEM, n ¼ 3 from one of more than three individual experiments. C, Confocal immunofluorescent staining of cleaved caspase-3 (cCasp3) as an apoptosis marker in tumor sections of MDA-MB-231 tumors lacking PAQR4 expression. cCasp3 staining was quantified as %cCasp3-positive area per field and data are shown in dot plot as mean SEM, n ¼ 5 per group. D–F, Immunofluorescent staining of cytochrome C as an indicator of ceramide-induced apoptosis in MDA-MB-231 cells with depletion of PAQR4 (D), C6-ceramide (5 mmol/L) treatment (E), and PAQR4 overexpression plus C6-ceramide treatment (5 mmol/L; F). Cytochrome C levels were quantified in the cytoplasm and in the mitochondria (ATPB-positive area). Ratios are presented in the dot plot, which shows mean SEM (n ≥ 30 cells) from one of three individual experiments. G, S1P levels in lysates from MDA-MB-231 cells with PAQR4 overexpression were measured by LC/MS-MS. S1P levels were normalized to protein concentration and are presented as mean, n ¼ 3 per group. One of two independent experimentsisshown., P< 0.05; , P < 0.01; , P < 0.001; NS, nonsignificant.

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expression of the genes encoding the enzymes within this network (4). of mitochondrial cytochrome C to the cytoplasm strongly suggests a Here, we identify and molecularly define the role of the ceramidase mitochondrial buildup of ceramides. Future work will determine PAQR4 in breast tumors. We find that PAQR4 is highly expressed in whether this is a result of direct Golgi-mitochondrial interaction the tumor tissue of the patients with breast cancer compared their sites (46) or vesicular lipid transfer (47). corresponding normal tissue and that its expression negatively corre- In summary, we here describe how PAQR4 may provide cancer cells lates with patient survival. On the basis of our findings, we propose that with an adaptive advantage for growth through modulation of the PAQR4 acts as a Golgi-localized ceramidase that provides a selective balance between ceramides and S1P. This suggests that development of advantage to cancer cells through reducing cytotoxic ceramides as well small-molecule inhibitors of PAQR4 could provide a feasible path for a as providing the building blocks for S1P production. Consistent with novel class of targeted therapies for breast cancer, either as a stand- the work by Zhang and colleagues (29, 40, 41), we find that down- alone treatment or in combination with chemotherapies known to regulation of PAQR4 reduces cancer cell growth. Their studies sug- alter ceramide levels. gested that PAQR4 controls cellular growth through stabilization of cyclin-dependent kinase 4 (CDK4) and hence cell-cycle state. We were Disclosure of Potential Conflicts of Interest not able to experimentally observe cell-cycle arrests in PAQR4-deplet- No potential conflicts of interest were disclosed. ed cells. Instead, through a combination of structural homology modeling, enzymatic, and lipidomics approaches, we propose that Authors’ Contributions PAQR4 functions as a ceramidase. Consistent with the previous Conception and design: P. Panahandeh, P.E. Scherer, K. Teigen, N. Halberg findings, we also observe reduced CDK4 expression in PAQR4 knock- Development of methodology: L. Pedersen, P. Panahandeh, M.I. Siraji, A. Molven, K. Teigen, N. Halberg down cells (Supplementary Fig. S6A). However, this is phenocopied by Acquisition of data (provided animals, acquired and managed patients, provided incubation of breast (Supplementary Fig. S6B) and lung (42) cancer facilities, etc.): L. Pedersen, P. Panahandeh, S. Knappskog, P.E. Lønning, R. Gordillo, cells with C6-ceramide, suggesting that the reduced CDK4 abundance K. Teigen, N. Halberg in PAQR4-depleted cells is an indirect consequence of increased Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ceramide levels. computational analysis): L. Pedersen, P. Panahandeh, R. Gordillo, P.E. Scherer, The de novo synthesis of ceramides is initiated in the endoplasmic K. Teigen, N. Halberg Writing, review, and/or revision of the manuscript: L. Pedersen, P. Panahandeh, reticulum (ER) by condensation of serine and fatty acids by the serine S. Knappskog, P.E. Lønning, R. Gordillo, P.E. Scherer, A. Molven, K. Teigen, palmitoyltransferase complex (43). Ceramides are then converted N. Halberg enzymatically into different classes of sphingolipids in the ER, cis-, Administrative, technical, or material support (i.e., reporting or organizing data, and medial-Golgi network that subsequently are integrated in organ- constructing databases): P. Panahandeh, S. Knappskog elle membranes (1). In this study, we show that PAQR4 is localized to Study supervision: P. Panahandeh, A. Molven, K. Teigen, N. Halberg the Golgi apparatus where it degrades ceramides into sphingosine. Although PAQR4 is homologous with other PAQR family members, Acknowledgments in particular, PAQR1-3, PAQR4 appear to be selectively induced in N. Halberg is supported by the Trond Mohn Foundation Starting Grant. We thank James Lorens and Claudio Alacorn for comments on previous versions of the breast cancer tissue compared with patient-matched nontumor tissue. manuscript. We thank the VCU Lipidomics/Metabolomics Core, the NIH-NCI We speculate that the selective advantage of PAQR4 is related to its Cancer Center Support Grant P30 CA016059 to the VCU Massey Cancer Center, placement in the Golgi. Other members, PAQR1 and 2 (commonly as well as a shared resource grant (S10RR031535) from the NIH for assistance with known as AdipoR1 and 2), are placed in the plasma membrane, where lipidomics analysis. We also acknowledge the Flow Cytometry Core Facility, Depart- they serve as receptors for the antidiabetic hormone adiponec- ment of Clinical Science, and the Molecular Imaging Center, Department of Bio- tin (44, 45). By intervening early in the sphingolipid pathway, we medicine, University of Bergen. N. Halberg is supported by the Trond Mohn Foundation Starting Grant. suggest that the tumor cells gain a selective advantage by being able to reduce apoptotic ceramide species while at the same time creating the The costs of publication of this article were defrayed in part by the payment of page intermediates required for S1P synthesis. We did not detect PAQR4 in charges. This article must therefore be hereby marked advertisement in accordance the plasma membrane, but it is tempting to speculate that PAQR4 with 18 U.S.C. Section 1734 solely to indicate this fact. activity could be regulated by an intracellular ligand. Despite the local placement in the Golgi apparatus, we find evidence Received October 13, 2019; revised March 2, 2020; accepted April 9, 2020; of global cellular accumulation of ceramides. In particular, the release published first April 14, 2020.

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2174 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

Golgi-Localized PAQR4 Mediates Antiapoptotic Ceramidase Activity in Breast Cancer

Line Pedersen, Pouda Panahandeh, Muntequa I. Siraji, et al.

Cancer Res 2020;80:2163-2174. Published OnlineFirst April 14, 2020.

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